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MOLECULAR GENETICS OF PROKARYOTES
MBIO 4600
LAB MANUAL
2014
Lab manual is available as a pdf file on the website.
2
Molecular Genetics of Prokaryotes MBIO 4600 SCHEDULE
Lab Location: 201 and 204 Buller starting at 2:30 pm
TITLE
WEEK
#
2014
DATE
Lab
Data
Experiment Duea
Report
Dueb
No lab.
1
Lab 1: The lac System: Review of Basic Genetics
Techniques
2
Sept 16
Sept 30
Lab 2: Conjugation
3
Sept 23
Oct 14
Lab 3: Transformation: Plasmid DNA Isolation and
Analysis.
4
Sept 30
Lab 3: Transformation: Competent Cell Preparation
5
Oct 7
Lab 3: Transformation: Xgal detection system
6
Oct 14
Lab 4: Transduction: P1 Generalized Transduction
7
Oct 21
8
Oct 28
9
Nov 4
12
Nov 25
Lab 5: Transposition: Tn5 Transposition via carrier
pRK602 plasmid
Lab 5: Transposition: Tn10 Transposition via carrier
λ1098Tn10Tc
Lab exam
a
Oct 28
Oct 24
(Friday)
Nov 4
Nov 7
(Friday)
Nov 14
(Friday)
due by 2:30 pm day requested. Honesty Declaration does not need to be attached data handed-in.
due by 4:30 pm day requested - completed Honesty Declaration must be attached
b
3
TABLE OF CONTENTS
Lab #
Description
Schedule
1
2
3
4
5
Page
2
General Instructions
4
Lab standard operation procedures
8
WHMIS
11
Experiments
The lac System: Review of Basic Genetics Techniques
Conjugation
F factor transfer
Hfr factor transfer
Plasmid Isolation and Transformation
Plasmid DNA preparation
Plasmid DNA concentration determination using
NanoDrop Spectrophotometer
Plasmid DNA Restriction Enzyme Digestion and Agarose
Gel Electrophoresis
Preparation of competent E. coli cells
Transformation: Xgal detection system
Transduction
P1 Generalized Transduction
Transposition
λ1098 Lysate Preparation
λ1098 Titration
Tn10 Transposition with λ1098
Appendix
Media
Solution Components and Function
Pipetman Operation
Refrigerated Centrifuge Operation
Colony Counter Operation
Determination of Viable Cells
Outlier plate counts
Sample lab exam
14
22
28
41
50
56
57
59
60
63
64
66
67
4
GENERAL INSTRUCTIONS
Lab Instructor:
Dr. L. Cameron
Demonstrators:
Justin Hawkin & TBA
Lab Location: 201 and 204 Buller Bldg.
Office: 414B
WEBSITE: http://umanitoba.ca/science/micro300400labs/
OR via University of Manitoba Department of Microbiology webpage select Undergraduate
programs left column, select Laboratory Information from drop down menu,
Information available at the website: reference links, changes/corrections, additional
information, data, marks
REGULATIONS
1. Lab attendance is compulsory. Please inform the instructor if you are unable to attend a lab.
2. Read SOP before coming to lab. Students must wear a lab coat. Bring a permanent marker.
3. Food or beverage is not permitted in the lab.
4. Students work in pairs.
5. The lab is opened Monday to Friday from 7:30 am to 5:00 - 5:30 pm. Check the lab schedule
posted on lab door for lab availability times as many of MBIO 4600 labs are continued
throughout the week.
6. Emails: subject must contain course number and subject, e.g. 4600 lab 1 report. If no subject
given, email is deleted. Emails replies occur only during working hours. Email must include
student name. If you have a lot of questions, please come to see me. Effective Sept 1, 2013 all
email communication must be via an official University of Manitoba email account, see details
at University governing documents website, Electron Communication With Students. Staff are
not permitted to reply to emails sent from email accounts other than an official University of
Manitoba email account.
EVALUATION
Before handing in your report review report to ensure that all information is included. When
printing Excel spreadsheets make sure you have selected all information before printing. If you
are using text boxes, they must be completely within the selected area.
1. All reports must have an Honesty Declaration attached at end of report - available as a
pdf file on lab website.
2. The lab is worth 20% of the final course mark, 8% for lab reports and 12% for lab
examination. There are no marks given for data handed in, but marks will be subtracted
for data not handed in.
3. You must pass the lab to pass the course (10/20%).
4. The lab reports due dates are listed in the schedule.
5. Lab reports are to be handed in as stated in schedule by 4:30 pm of that day. ONLY
hand in lab reports through slotted filing cabinet drawer located on the 300 level of
Buller bldg. in the hallway across from room 302 entrance. Instructor and TAs do not
accept lab reports. Marked lab reports will be returned to students the next week. If
handing in lab late, 10% of mark will be subtracted for each class day late. A late report
will not be accepted after one week past due date. If applicable, also hand in data,
release forms, assignments through slot of filing cabinet. Data may be emailed. All
reports, assignments, and quizzes not collected by the student are destroyed six months
after end of term via confidential shredding.
5
6. Lab report marks are final unless an obvious error in addition of marks has been made.
However, if a student feels they have a legitimate complaint, please direct attention to
lab instructor.
7. The lab exam will be held during normal lab time, starting at 2:30 pm (refer to schedule
for date). The lab exam is 1.5 h. 1-2 weeks prior to exam date the exam schedule is
posted on lab door and lab website.
8. Approximately two weeks prior to the lab exam, a brief outline of lab exam format and
information content will be available on the lab website (includes location). There is a
sample lab exam in the lab manual appendix.
9. You must notify the lab instructor no later than two school days after the missed lab. A
Doctor’s certificate is required for a missed lab exam. All deferrals will write the lab
exam at a scheduled time set by the instructor. Failure to comply will result in a zero on
your lab exam.
10. Plagiarism (copying another student’s lab report (present or previous year) or
copying published literature without citing is a violation of University regulations.
Refer to the STUDENT DISCIPLINE BY-LAW in your student handbook (rule
book) for action taken for plagiarism.
LAB REPORT PRESENTATION
[Before handing in your report review report to ensure that all information is included. When
printing Excel spreadsheets make sure you have selected all information before printing. If you
are using text boxes, they must be completely within the selected area or they do not print.]
1.
2.
3.
4.
5.
6.
7.
8.
All reports (not data) must have an Honesty Declaration attached - available as a pdf file
on lab website.
Lab reports must be typed. Up to 10% of the mark subtracted for reports not typed.
Number pages.
On the front page of the report state (This does not need to be a separate page or added if
already provided in report format.):
Date
Course number
Experiment number
Group #
Individual or Group name(s).
Lab report information is to be presented exactly as requested. Must type information
into Word and or Excel lab report format available on lab website
http://umanitoba.ca/science/micro300400labs/60_460.htm .
Lab report may be done as an individual effort or a group effort by the students in each
group that carried out the experiment. Student in two different groups cannot submit a
group report together. One report or more reports may be handed in per group. The
decision on the number of reports per group is totally dependent on members of the
group. This decision may be changed any time during the term. Therefore for each lab
report the group has the option to hand in one or more reports exclusive of what has
been done before or after that particular report. Indicate on the cover page of the report if
the report is a group report or an individual report. If handing in an individual report
also include lab partner’s name.
Always include a sample of each type of calculation in your lab report.
If a group’s data is not workable, borrow data from another group and reference. Non
workable refers to data that cannot be plotted, used for calculations or required analysis.
It does not necessarily mean the expected data.
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9.
Cite reference in text of lab report and record full reference at end of lab report. When
should you cite and reference. The following is a good definition of plagiarism that
explains when you should cite a reference. “The unacknowledged use of another
person’s work, in the form of original ideas, strategies, and research, as well as
another person’s writing, in the form of sentences, phases and innovative
terminology.” (Spatt1, 1983, p.438) This is done by using bracketed reference number
that you used when listing references at end of lab report or by bracketing first authors
name and date. Quote text unless you paraphrase completely in your own words. But
remember, quotes should only be a small part (~5%) of your work. If you are using the
name year system, list the references alphabetically. Some examples are as follows
(McMillan2 1997):
Binder V. Hendriksen C, Kreiner S. 1985. Prognosis in Crohn’s disease - - based on
results from regional patient group from county of Copenhagen. Gut 26:146-50.
Danforth DN, editor. 1982. Obstetrics and gynecology. 4th ed. Philadelphia: Harper
and Row. 1316 p.
Petter JJ. 1965. The lemurs of Madagascar. In: DeVore I, editor. Primate behavior:
field studies of monkeys and apes. New York: Holt, Rinehart and Winston. p 2920319.
If journal article assessed on the internet, site as journal. However, if available only on
the web, reference as follows:
Kingsolver JC, Srygley RB. Experimental analyses of body size, flight and survival in
pierid butterflies. Evol. Ecol. Res. [serial online] 2000;2:593-612. Available from:
Colgate University online catalog. Accessed 2000 Oct 3.
10.
Personal or Professional Electronic sources2:
Cite in-text by putting the following in parentheses, author’s last name or file name (if
no author’s name is available) and publication date or the date of access (if no
publication date is available).
At the end of report list: author or organization, publication date or date last revised,
title of Web site,URL site, and the date accessed.
Cameron, L. MBIO 4440 Systems Microbiology Lab Information
http://umanitoba.ca/faculties/science/microbiology/staff/cameron/60_344.htm Accessed 2012, April 17.
Table presentation
Report tables are provided in Word or Excel format following format rules by McMillan
(1997)2.
· Table number and title (legend) presented above the table body.
· Number tables using Arabic numbers, even if only one table in a report.
· Include enough information in title to completely describe table, eliminating the
necessity to search elsewhere in the lab report to understand information presented in
table. Table title starts with an incomplete sentence. Additional complete sentences may
be included to adequately describe the table (this also applies to figures).
1
2
Spatt, B. (1983). Writing from Sources. New York: St. Martin’s Press.
McMillan V.E. 1997. Writing Papers in the Biological Sciences. 2nd ed. Boston: Bedford Books: 1997.
197 p. and McMillan, V.E. 2001. Writing Papers in the Biological Sciences. 3rd ed. Boston: Bedford Books. 123 p.
7
· If abbreviations are used in table, indicate what abbreviations mean as a footnote. Other
footnotes may be required to clarify material in the table.
· Like information should be in columns making it easier to view the table.
· Column or Row headings should be complete and self explanatory. A heading is a
separate entity from the title. It cannot be assumed information given in the title is
adequate for a heading.
· Group related column headings under larger headings.
· Tables should be properly set up with a straight edge. Horizontal lines must be included
but it not necessary to always include vertical lines.
· Make the table as concise as possible but include all necessary information. For
example, any constant experimental conditions that would change the data presented.
· If information is the same for each column or row do not include but treat as a footnote.
·
·
·
·
·
·
Table Data Entry
Align decimal points (ideal but not required for reports).
If a number value is less than 1 always include zero before the decimal.
Do not include the unit of measurement in column data if present in column or row
heading.
Omit percentage sign in column or row data.
If a whole number is large use scientific format (e.g. bacterial titre).
Any acronyms used in column data should be described as a table footnote.
Figure presentation (graphs, diagrams, photographs, films)
· All figure graphs must be computed generated using Excel (detailed procedure given
in the lab)
· Figures are to be numbered separate from tables, using arabic numbers. Include figure
number even if only one figure, eg. Figure 1.
· Following the figure number a figure title should be presented below graph. The figure
title, like the table, starts with an incomplete sentence describing the graph. For
example, do not repeat just the labels of the x- and y-axis but present in a descriptive
manner. Additional sentences should be included if additional information is required to
completely describe figure, for example, abbreviations explanation, any constant
experimental conditions, etc.
· All diagrams, photographs, and films are figures and should be completely labelled.
· For graphs: Usually there is one independent variable plotted and one or more
dependent variables plotted. The dependent variable is a function of the independent
variable. It is accepted practise to plot the independent variable on the x-axis and the
dependent variable on the y-axis. For example the measurement of absorbance
(dependent) with increasing concentration of protein (independent). The size of the
graph should fit the plot(s). The overall size of graph should not be too large but should
not be so small that information is obscured. Graph axes must be completely labelled
(always include units). If more than one plot include legend.
· For other figures. Do not write on figure data area, i.e. gel lane, film surface, etc. Use
small symbols or arrows to indicate what you want and explain in figure title. If an
agarose gel, small symbols may be inserted between lanes. If a standard ladder is
included, label sizes. Make sure all relevant lanes are labelled. Make sure all essential
information is included in the figure title such that when you look at the figure you
understand the experiment that was performed and what the data means.
8
Note: When writing your lab reports you are frequently requested to present both a table and a
figure for a given set of data, similar to keeping a research lab journal. This is not the accepted
practice for papers published in journals or books. Usually either a table or a figure is presented
for a given set of data and depending on nature of data; it may only be summarized in the text.
How do you make a choice of data presentation? The aim is to effectively and efficiently
demonstrate what you want to show, for example, correlations, comparisons, pattern, trends,
etc.
LAB STANDARD OPERATIONS PROCEDURE (SOP)
Safety information relevant to the Microbiology department is available at
http://umanitoba.ca/faculties/science/departments/microbiology/general/1601.html
Each lab contains a Safety Station (hand wash sink area). Pertinent safety information
and supplies are stored in the drawers/cupboards.
PERSONAL SAFETY:
•You must wear a buttoned lab coat. If you forget your lab coat, lab coats are available in the
lab - must sign out and sign in when returning.
Lab Coat Laundry instructions: wash separately from other clothes with detergent and bleach.
When taking lab coat home for washing, carry in plastic bag separate from all other personal
effects, i.e. not in your back pack.
•No personal effects (this includes outer clothing and back packs) are permitted in the lab, only
essential lab supplies. There are cupboards available in the hall across from the lab for outer
clothing and backpacks. Cupboards may be locked but locks must be removed each time after
using. Please do not leave any of your belongings on the hallway floor.
•Long hair must be tied back. Keep your hands away from your hair.
• Wash hands with antibacterial soap (SWISH contains sodium lauryl sulfate (SDS) a detergent,
coco diethanolamide, coco amido betaine, and copolymer of acrylamide) for minimum of 30 sec
before leaving the lab. Use the hand wash sink located at safety station. Use your wrists (wrist
levers for your safety) to turn the water on and off, not your hands.
• Remove gloves using finger of opposite hand to peel off other glove by inserting at wrist,
rolling off glove. Repeat with other hand. Dispose of gloves in Petri plate containers.
•No eating or drinking in the lab.
•Never mouth pipette. Always use a pro-pipette.
•Cover any cuts with a bandage (first aid kit at Safety Station, hand wash sink area).
• Students must wear shoes with closed toes and heels.
•MSDS book located in Safety Station drawer. Other safety information is also located at the
Safety Station. A flashlight is available at the Safety Station.
The Spill Kit is located at the Safety Station (below sink).
LAB ENVIRONMENT:
•Wash bench area before and after with BDD (Backdown Detergent Disinfectant containing
nonyl phenoxy polyethoxy ethanol, alkyl-aryl ammonium chloride and ethyl benzyl ammonium
chlorides). This is especially important since fermentations students use strong acids and bases.
Never assume that the lab bench has been cleaned by the previous students.
•Know location of exits, fire extinguisher, eye wash, full body shower.
• Know how to operate equipment before use. DO NOT use equipment unless you know
exactly how to operate the equipment. The demonstrator is always available to assist.
•Leave your bench area clean. All equipment and supplies should be returned to original
location.
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DISPOSAL:
Treat animal tissue as a biohazard.
•All biohazard disposable containers must be labelled with a biohazard label. After autoclaving
the biohazard label is removed.
•All biohazards must be autoclaved. Biohazards include any surface that has come in contact
with bacteria. The autoclave is monitored weekly to ensure all organisms are destroyed. The bag
from the Petri plate container is removed, untied, opened and place in a large tray on the
autoclave trolley. After autoclaving, the biohazard sticker is removed and the bag is tied and
placed in a black plastic bag before disposing. Individual bags from plastic lined buckets are
carefully removed and placed in a larger autoclave bag (labelled biohazard) and autoclave
procedure performed as above. The tied bag is placed in a corrugated cardboard box pre-labelled
as broken glass, taped shut for disposing by caretaker.
•Petri plate container (large plastic lined buchet located on the floor): Discard all non-sharp
biologically contaminated items in the Petri plate container. This includes agar culture plates,
disposable gloves, disposable gloves and bacteria contaminated paper towels.
•Plastic lined bucket3: Located on your work bench. Any ‘pointy’ item must be disposed in
plastic lined bucket (not Petri Plate container), this includes pipetman tips, disposable cuvets,
sticks, toothpicks, slides, Pasteur pipettes, disposable 1 ml and 10 ml pipettes, broken glassware,
brittle plastic objects, metal objectsa (not needles or blades), etc. This includes all items that are
‘pointy’ regardless of biological contact.
•Biological Spills: Put on glove. Immediately wash container or test tube rack with BDD
disinfectant. Put stack of paper towels on top of spill, pour disinfectant around and over. Do not
press down. Collect soaked paper towels in Petri plate container. If spill includes broken glass or
any sharp item put in plastic lined basin. A spill container is located in Safety Station cupboard
that contains a spill absorbent pad (use instead of paper towels). This method of spill cleanup is
only suitable for level 1 nonhazardous bacteria (E.coli with known strain number).
•Glassware (unbroken): Remove tape and pen markings (use alcohol) from glassware before
placing on discard trolley. Used glassware that has not contained bacteria should be rinsed and
placed on the discard trolley. Rinsed test tubes (no biological contact) should be placed in tray
provided on the discard trolley.
•Chemical hazardous material: Read the MSDS information available in lab or online at
http://ccinfoweb.ccohs.ca/msds/search.html . Organic solvents must be disposed of in organic
solvent container. The lab TA will instruct proper disposal methods for labs that contain
hazardous materials. These containers are disposed of through the university safety office. Never
pour solvents down the sink. Use extreme care with flammable solvents. Alcohol used to flame
spread rod should never be positioned within 40 cm of flame. Never put a very hot spread rod
into a beaker of alcohol. The alcohol may catch fire.
Handle caustic (acids and bases) solutions with care. Never discard an acid or base greater than
one molar down the sink. Discard in labelled glass containers provided. Use lots of water when
discarding caustic solutions (< 1M). These materials are disposed of through the university
safety office.
3
due to the multi-use nature of the teaching lab, all ‘pointy’ items will be treated the same as similar items
contaminated with microorganisms.
10
•Biohazard sharps disposal: Dispose of all sharps (needles, syringe tops, razors, scalpel blades)
in specified container (red or yellow). Dispose of syringe with needle attached - do not take
apart. Do not replace the needle cap before disposing (high frequency of accidents occur when
replacing cap). Sharp’s containers are autoclaved before disposing. You must dispose of the
syringe top in the biohazard sharps container even if not used for biologicals as it is a perceived
hazard by the general public.
•General garbage disposal: Nothing ‘pointy’ should be disposed in the general waste basket.
Nothing that has come into contact with biological material should be disposed in general waste
container. No liquids, the caretaker does not know what the liquid is!
LABORATORY BIOSAFETY GUIDE
In this lab you use only Level 1 bacteria risk group. However, level 2 bacteria may be used by
other labs in this room. Follow standard operation procedures, SOP (see above).
The University of Manitoba Biosafety Guide and Health Canada Laboratory Biosafety
Guidelines booklets are available in your lab.
UM Biosafety Guide:
http://umanitoba.ca/admin/human_resources/ehso/media/BiosafetyGuideMarch05.pdf
Canadian Biosafety Standards and Guidelines:
http://canadianbiosafetystandards.collaboration.gc.ca/
MSDS (infectious agents): http://www.phac-aspc.gc.ca/msds-ftss/index-eng.php
There is no listing of level 1 agents in the guidelines or MSDS pamphlets
Risk group 1 bacteria are low individual and community risk and are unlikely to cause disease
in healthy workers.
Risk group 2 bacteria are moderate individual risk and limited community risk. Bacteria in this
group can cause human or animal disease but are unlikely to infect healthy laboratory workers.
Effective treatment is available. Risk of spreading is limited.
CONTAINMENT LEVEL 1 (UM biosafety guide)
•
microbiology lab with washable walls, countertops and hand wash sink
•
established safe laboratory practices (hand washing and disinfection of countertops)
•
general WHMIS safety training
•
UM lab registration
CONTAINMENT LEVEL 2 (UM biosafety guide)
•
all of level 1 specifications
•
biosafety permit
•
biological safety cabinet (not required)
•
biohazard sigage
•
a written standard operations procedure
•
MSDS for the infectious agent
11
WHMIS
The Workplace Hazardous Materials Information System (WHMIS) is a system for safe
management of hazardous materials. WHMIS is legislated by both the federal and provincial
governments.
Under WHMIS legislation, laboratories are considered to be a workplace, and students are
workers. By law, all workers must be familiar with the basic elements of the WHMIS system.
The WHMIS program includes:
1. Cautionary labels on containers of controlled products. Consumer products, explosives,
cosmetics, drugs and foods, radioactive materials, and pest control products are regulated
separately, under different legislation.
2. Provision of a Material Safety Data Sheet (MSDS) for each controlled product.
3. A worker education program
1. A. SUPPLIER LABELS
Controlled products must have a label of prescribed design which includes the following
information:
PRODUCT IDENTIFIER - trade name or chemical name
SUPPLIER IDENTIFIER - supplier's name and address
MSDS REFERENCE - usually, "See MSDS supplied"
HAZARD SYMBOL - (see illustration on next page)
RISK PHRASES - describes nature of hazards
PRECAUTIONARY MEASURES
FIRST AID MEASURES
B. WORKPLACE LABELS
All material dispensed in a workplace container must be labelled with the Product Name,
Precautionary Measures (simplified) and Reference to Availability of MSDS.
2. MSDS
Material Safety Data Sheets (MSDS) are available for each lab. Refer to binder located in each
lab. Also main binders are located in the Microbiology preparation room, 307/309 Buller.
MSDS are also available on the internet. The MSDS will provide: relevant technical
information on the substance, chemical hazard data, control measures, accident prevention
information, handling, storage and disposal procedures, and emergency procedures to follow in
the event of an accident.
3. SAFETY
The Laboratory Supervisor will provide information on the location and use of safety equipment,
and emergency procedures.
12
13
14
LAB 1 THE lac SYSTEM: Review of basic bacterial genetics techniques
INTRODUCTION
The following figure is a brief reminder of the Lac Operon, for additional required lac Operon
information refer to any basic molecular biology book.
Lac Operon Mutations
E. coli CSH100 has IQP- mutations in the lac region. IQ repressor mutation results in a ‘super
repressor’ as the cell contains ~10 times the amount of repressor. P- promoter mutation, cis-dominant,
reduces RNA polymerase binding to P resulting in reduced transcription of the structural lac genes. E.
coli CSH101 has I-Z- mutations in the lac region. I- phenotype is due to the lacI gene product either
defective or absent, resulting in constitutive transcription of the lac operon. However, this E. coli strain
also as a Z- mutation, resulting in the absence of β-galactasidase structural product. E. coli CSH140 has
only the I- phenotype.
Selective Media
MacConkey agar contains the carbon sources lactose, peptone and poly peptone and the
dye neutral red. Neutral red indicator dye is red below pH 6.8. If the E. coli strain can ferment lactose
(process initiated by β-galactasidase that breaks down lactose to glucose and galactose), resulting in acid
products that produce deep pink to red colonies. Lac regulatory mutations that do not completely inhibit
production of β-galactasidase may produce intermediate pink colonies. However, this is time dependent
and difficult to see. A more precise method to detect various lac mutations is to use Xgal agar medium.
Xgal is a direct histochemical stain, 5-bromo-4-chloro-3-indolyl-β-D-galactoside, which turns to
blue shades when cleaved by varying amounts of β-galactosidase. Basal levels (presence of
repressor) of functional β-galactosidase can be detected giving a pale blue color even in the
presence of glucose. IPTG (isoproproyl-β-D-thiogalactoside) is an inducer of the Lac operon. It
is not cleaved by β-galactosidase. IPTG binds the repressor reducing its affinity for the operator.
Note: The lac operon is also positively regulated by cAMP receptor protein (CRP) which is
required to bind to the promoter to initiate transcription. The CRP is only active in the presence
of cAMP. The level of cAMP is inversely proportional to the level of glucose. IPTG overrides
15
glucose repression. Even in the absence of IPTG and the presence of glucose, the lac operon is
not completely repressed by glucose. The reason for this is not known.
Bacteria manipulation Techniques
In a molecular genetics lab it is essential that pure culture methods by practised. Aseptic
transfer technique involves avoiding any contact of the pure culture, sterile medium and sterile
surfaces with contaminating microorganisms. This is accomplished by bench area cleaned with
BDD, use of sterile sticks or sterile Pipetman tips for culture transfer and streaking and the work
performed quickly and efficiently to minimize the time of exposure during which contamination
of the culture or laboratory worker can occur. In this lab pure cultures of microorganisms are
isolated by either streak plate or spread plate. All the methods involve separation of single
bacterium on solid (agar) media where it grows into a colony (clone). Individual colonies
represent a single microorganism type. In the streak plate method a loopful of bacterial cells is
streaked across the surface of nutrient agar plate. The method of streaking established a dilution
gradient so that single colonies develop. In the spread plate method 0.1 ml of microbial
suspension is placed on the centre of an agar plate and spread over the surface of the agar using a
sterile glass rod. Usually culture dilutions are plated to obtain an appropriate dilution permitting
separated single colonies. The pick plate method of replication transfer is very reliable allowing
numerous replica agar plates. The agar plates used to screen the bacteria may be agar plates
containing an indicator (e.g. Xgal), or defined medium with varied nutrients or antibiotics (e.g.
ampicillin and tetracycline) as selective markers.
For short term storage (2-4 weeks) colonies of most bacterial strains may be maintained
at 4oC on the surface of agar media. Also liquid cultures may be maintained at 4oC for short
term storage. For long term storage, bacteria are maintained in 15-50% glycerol or 8%
dimethylsulfoxide (DMS0) at -80oC.
REFERENCES used for introduction.
Sambrook J, & Russell D. 2001. Molecular Cloning: A Laboratory Manual. 3rd edition. New York: Cold Spring
Harbor Laboratory Press. Three volumes.
Miller, J.H. 1992. A Short Course in Bacterial Genetics. New York: Cold Spring Harbor Laboratory Press. 456 p.
Lewen, B. 2004 Genes VIII. Chapter 10 The Operon (with respect only to the Lac Operon).
16
Genetic Nomenclature
Example:
Strain Number
Mating Type
Genotype
Important Properties
E. coli CSH101
F+
F'lacproA+,B+
(carries I-Z- mutations in
the lac region) ara Δ(gptlac)5
LacDonates F'lacproA+,B+
episome
CSH125
F-
ara leu lacY purE gal supE try
his argG malA rpsL xyl mtl ilv
metA
Ara- Leu- Lac- Pur- GalTrp- His- Arg- Mal- Strr
Xyl- Mtl- Ilv- Met- Su+
• Each E. coli has a strain number.
• Genotype lists contains altered genes, usually defective, or some type of resistance which can
be recognized by abbreviation for gene written in italics and lowercase letters. If a gene is not
listed, it is assumed it is functional (wild type) or wild type genes may be written with uppercase letters and italics.
• Important properties are usually phenotypic expression of genotype. Not necessarily all
phenotypes are listed just those pertaining to a particular experiment. Wild type genes are not
included in the phenotype list, for example if an E. coli strain has the ability to utilize lactose; it
is not included in the phenotype list.
There are several types of altered genes used in this lab. The following are examples of each type.
Genotype
Phenotype
Type of Mutant
Explanation
ara,
Ara-
carbon source
E. coli strain cannot use arabinose as a carbon
source.
thiA
Thi-
nucleotide source
Thiamine must be supplied in medium as the E.
coli strain cannot synthesize thiamine
(auxotroph).
bioB
Bio-
vitamin source
Biotin must be supplied in medium as the E. coli
strain cannot synthesize biotin (auxotroph).
metA
Met-
amino acid
Methionine must be supplied in medium as the E.
coli strain cannot synthesize methionine
(auxotroph).
rpsL
Smr
antibiotic resistance
E. coli strain resistant to antibiotic, streptomycin.
It is assumed that a strain is sensitive to the
antibiotic unless resistance stated.
Δ(gpt-lac)5
Lac-, Pro-
deletion
Delta symbol means deletion of genes listed in
brackets. Starts with gpt gene located at 5.5 min
on E. coli map ending with lac genes at 7.8-7.9
min. Included in this large deletion are proA and
proB genes.
17
18
E. coli STRAIN LIST
STRAIN
NUMBER
MATING
TYPE
GENOTYPE
IMPORTANT
PROPERTIES
CSH100
F+
F'lacproA+,B+
(carries IQP- mutations in the lac region) ara
Δ(gpt-lac)5
I +P - Z +
CSH101
F+
F'lacproA+,B+
(carriesI-Z- mutations in the lac region) ara
Δ(gpt-lac)5
I - P +Z -
CSH140
F+
F'lacproA+,B+
(carriesI- mutations in the lac region)
Δ(gpt-lac)5
I - P +Z +
F'lacproA+,B+
I +P +Z +
ara Δ(gpt-lac)5 supE rpsE
Notes: Genes listed after F’ are carried on the F’ episome. Under important properties + (positive) or –
(negative) refers to phenotypic expression dependent on type of gene mutation, see introduction.
CSH141
F+
PROCEDURE
STUDENTS WORK IN PAIRS FOR THIS LAB AND ALL SUBSEQUENT LABS.
Comment: Remember to treat all bacteria as possible pathogens. Read general instructions
and introduction before starting this lab. USE GOOD ASEPTIC TECHNIQUE.
Week 2
Come prepared to be tested on Pipetman Operation – see appendix for information on
Pipetmen available in the MBIO 4600 lab.
Come prepared to demonstrate ability to use a colony counter – see Review of Colony
Counter.
Part I: Multiple single colony isolation and application of selective indicator plates.
1.
2.
Prior to start of lab, each E. coli strain was inoculated in 5 ml LB broth and incubated
with rotation at 37oC
overnight.
Take four plates, LB agar,
MacConkey, Xgal glucose
and Xgal glucose + IPTG.
Divide plates into four
sections and streak each E.
coli strain (CSH100,
CSH101, CSH140 &
CSH141) on one section.
Use sterile sticks in metal
capped test tubes (not
toothpicks) or sterile loops to
streak plates. The easiest
method
19
is to first label and quarter plates. Line plates up orientating the same. Starting with
first E. coli strain streak the first streak covering ¼ of appropriate section, dip stick in
culture again and repeat first streak on next plate, then remaining plates (all done with
one stick). Take another stick doing 2nd set of streak on each plate type (this may be
done with 1 stick, if confident in technique or 4 sticks). Refer to diagram for streaking
method. Repeat process for remaining three strains.
3.
Incubate agar plates upside down in plastic carton at 37oC overnight. Record results as
requested in data sheet. It is important to incubate only overnight (18-24 h) before
recording results as color production is time dependent with even Z- strains eventually
turning pale blue. If you cannot read your plates the next day, come the next day and put the
plates in the student cold box which will slow down color production but not stop it – record
results as soon as possible.
4.
Next day use the LB agar plate for pick plate procedure.
Day 2
Part II: Pick plate technique review
1.
From the LB plate containing the four E. coli strains, CSH100, CSH101, CSH140, &
CSH141, pick plate each strain onto a quarter section of a glucose M9 minimal media
plate and a lactose M9 minimal media plate. Use 6 colonies of each strain. You
require only one plate of each medium.
· Use the duplicate pick plate grid at the end of this lab’s introduction.
· Place the two agar plates (glucose M9 minimal media plate & lactose M9 minimal
media) on the diagram containing the two circles, each divided into four sections
with six pick marks. The agar plates must be orientated for quick identification of a
particular colony or quick comparison of duplicate agar plates by placing a small
mark on the bottom of the agar plates as shown by arrow on gridded circles.
Colony one:
· First lightly touching the sterile tooth pick (use a separate tooth pick for each
colony) to a pure colony
· next streak a line about 0.5 to 1.0 cm long (depends on space available) in number
1 grid or line of the first plate
· then without retouching the original colony streak in number 1 grid or line of the
second plate
Colony two, etc:
· the next colony is picked to number 2 grid/line
· the process repeated until required number of colonies (this experiment is 6) have
been 'picked'.
· make sure the surface of LB plate is gently streaked so as not to cut through the
agar.
2.
Incubate plates in plastic container at 37oC for 1-2 days. Record results as requested
in lab report. Record as growth if there is a good amount of growth. If only faint
growth, it is most likely due to carry over of nutrients from LB or internal nutrients –
record as no growth.
20
Molecular Genetics of Prokaryotes MBIO 4600
LAB 1 REPORT THE lac SYSTEM: Review of basic bacterial genetics techniques
Save format and open in Word before typing requested information; keep question format.
Date:
Group #:
Group or Individual Report:
Student Name(s):
Data Presentation and Analysis
6
1.
Record requested indicator plate information in the following table.
Table 1. E. coli Lac Operon phenotype data using indicator plates. Incubation Time: MUST BE 18-24 h at 37oC
Indicator Pate E. coli colony COLORb
E. coli strain
MacConkey (lactose)
X-gal M9 glucose
X-gal M9 glucose + IPTG
record as translucent or pink
record as translucent, light
record as same or darker
blue, medium blue or dark
than X-gal M9 glucosec
blue
group data
expecteda
group data
expecteda
group data
expected
color
color
CSH100 (IQP-Z+)
CSH101 (I-P+Z-)
CSH140 (I-P+Z+)
CSH141 (I+P+Z+)
a
expected = record theoretical color for E.coli strain genotype given the experimental conditions used in the
MBIO 4600 lab
b
Do not include detailed description of colony characteristics. Translucent equivalent to same color as medium.
c
darker means any blue degree greater than colony color on X-gal M9 glucose, e.g., if light blue on Xgal M9 glucose but blue on X-gal M9 glucose + IPTG then record as darker.
1
2. Record requested pick plate information in the following table.
Table 3. E. coli strain pick plate results demonstrating lactose utilization.
E. coli strain
Number of picksb that showed growth
State whether resultsc
(maximum = 6)
expected (yes or no ONLY
required)
M9a glucose
M9 lactose
Q - +
CSH100 (I P Z )
CSH101 (I-P+Z-)
CSH140 (I-P+Z+)
CSH141 (I+P+Z+)
a
see MBIO 4600 lab manual appendix, all essential nutrients including required amino acids have been added for
growth of each strain – growth only dependent on ability to utilize the carbon source.
b
6 colonies picked for each strain on each plate type
c
If only one, possibly two picks grow consider this the same as zero. Possibly due to either experimental
technique, carry over, or spontaneous mutation. Growth at 37oC for 2 days.
21
Question
1
1. Given the following E. coli pick plate results, state E.coli mutant #3 strain phenotype. Line
after strain number indicates growth and no line after strain number indicates no growth.
M9 glucose lysine agar
M9 lactose lysine agar
M9 lactose agar
M9 glucose agar
E. coli mutant phenotype for pick #3 (Answer must be phenotype symbol nomenclature.):
__
8
22
LAB 2 CONJUGATION
Introduction
Conjugation is the transfer of DNA between bacterial cells. One of the earliest forms
of conjugation discovered is F factor mediated conjugation. The F factor or F plasmid exists
as a 100 kb free circular plasmid. The F plasmid has its own origin of replication (ori V) and
origin of transfer (ori T). Each bacterium (F+) can only have one copy of the F plasmid. The
recipient in F plasmid conjugation must be F- (no F plasmid). The F factor contains transfer
genes (tra, mob) that are involved in phili formation, establishment and maintenance of
contact between two bacteria, initiation and transfer of DNA. oriT (start of transfer) on the F
factor is nicked by one of the tra genes. The generated 5' end starts the transfer of single
stranded DNA to the recipient bacterium. Concomitantly a complementary DNA strand is
synthesized in both the donor and recipient (rolling circle). The F+ donor strain, E. coli
CSH101 used in this lab has the genotype F'lacproA+,B+ . The mutant Z gene requires only a
T –> G basepair reversion to change amber stop codon TAG to glutamic acid codon GAG,
which is the amino acid required for active β-galactosidase (located in the active site)7. The F
plasmid also contains functional proline genes since there is a deletion of both lac and proline
genes on the E. coli chromosome to permit phenotypic observation of bp reversion on F
plasmid. The F- recipient E. coli CSH114 strain contains mutT mutator locus. The mutT strain
is specific for bp transversions A:T –> C:G at a higher than spontaneous mutation frequency
inserting a G instead of T. This complements the E. coli CSH101 donor permitting phenotypic
observation of bp change by papillae formation. The conjugant, now contains the F plasmid
with bp mutation in lac Z gene, recipient strains also have the lac gene deleted on the
chromosome. The conjugant colony starts out
completely white on MacConkey. After extended
incubation time, bp reversions (cells that now have a
functional lac Z gene, β-galactosidase) arise in the
colony that are observed phenotypically as red dots in
the colony. The colony may have any number of red
dots, each indication a bp reversion to functional βgalactosidase.
7
Cupples, C.G. & Miller, J.H. 1989. A set of lacZ mutations in Escherichia coli that allow rapid
detection of each of the six base substitutions. PNAS 86:5345-5349.
23
In addition to F plasmid transfer, a typical
modern conjugation will be performed using the
donor strain E. coli DH5α (pUFR-GFP). The
plasmid pUFR-GFP is transferred to the recipient
strain, E. coli CSH125, via conjugation. Plasmid
pUFR-GFP is a broad-host-range (Ori IncW)
plasmid that has the green fluorescent protein (GFP)
inserted in the polylinker. The presence of green
fluorescent protein in the recipient, E. coli CSH125,
confirms conjugation by observing cell fluorescence
over UV light.
E. coli STRAIN LIST
Strain
Mating
Type
Genotype
Some Phenotype Properties
E. coli CSH101
F+
F'lacproA+,B+
(carries I-Z- mutations in the lac
region) ara Δ(gpt-lac)5
LacReverts to Lac+ via a specific
base pair substitution in lacZ.
Donates F'lacproA+,B+ episome
E. coli CSH114
Fdonor
ara Δ(gpt-lac)5 rpsL mutT
Pro- Lac- Smr MutT
gfp
Ampr GFP
recipient
ara leu lacY purE gal supE try
his argG malA rpsL xyl mtl ilv
metA
Smr
E. coli DH5α (pUFR-GFP)1
E. coli CSH125
Sm, streptomycin; Amp, ampicillin; GFP, green fluorescent protein.
Unless otherwise stated, E. coli strains are sensitive to all antibiotics.
When performing conjugation experiments it is important to select against parents by plating
on appropriate media.
1
provided by Aniel Moya Torres (Dr. K. Brassinga’s lab)
24
Week 3
Part I: F plasmid conjugation
Day 1
1.
An overnight culture of donor E. coli strain CSH101 was subcultured 1:50 in 5 ml LB
medium and an overnight culture of recipient CSH114 was subculture 1:10 in 5 ml LB
medium. Both donor and recipient E. coli were incubated for 3 h at 37oC with rotation.
STUDENT LAB STARTS HERE
2.
Prepare mating mixture by mixing 0.2 ml of donor strain E. coli CSH101 with 0.2 ml
recipient strain E. coli CSH114 in an Eppendorf tube. Incubate in 37oC waterbath for
1 hour.
3.
Using a disposable plastic loop, streak mating mixture on a glucose M9 glucose +
streptomycin (M9glucoseSm) agar plate. Also streak the donor and recipient on ONE
M9glucoseSm agar plate divided into 2 sections (negative controls).
4.
Incubate at 37oC for two days.
Day 3
5.
Papillae observation: After two days, select a single conjugant (E. coli
CSH101xCSH114) and streak each on a MacConkey agar plate. Repeat with a second
colony and another MacConkey plate. Use a streak technique that will give you well
isolated single colonies.
6.
Incubate at 37oC. You should start to see papillae after 3 or 4 days. Label plate with
your group number (not sure, check bulletin board in lab). Once papillae have formed
store plates in student cold box.
Day 8 (next lab day Week 4)
7.
At start of lab hand in one MacConkey papillae plate (select best plate with single
colonies) to designated tray on lab bench. Make sure plate is labelled with your group
number (see lab bulletin board for group number if forgotten). During the lab the TA
will photograph the best demonstration of papillae formation (red dots in white colony)
for each group. Papillae jpeg files will be posted on lab website for lab report.
Part II: Conjugation
Day 1
Conjugation parents: E. coli DH5α (pUFR-GFP) was grown overnight at 37oC with rotation in
LBAmp (100 μg/ml). E. coli CHS125 was grown overnight at 37oC with rotation in
LBSm (100 μg/ml).
1.
2.
3.
4.
5.
Set up the conjugation mixture by combining in an Eppendorf tube: 0.2 ml donor, E.
coli DH5α (pUFR-GFP) and 1.0 ml recipient, E. coli CSH125.
Mix by inverting several times and immediately micro centrifuge for 1 min at room
temperature. Why immediately?
Use P1000 to remove supernatant. Add 1 ml sterile saline and resuspend cells using
P1000 by pipetting up and down.
Micro centrifuge for 1 min. Remove supernatant. Add 100 μl saline and resuspend.
Carefully spot the 100 μl suspension in the centre of a non-selective LB plate. Keep the
spot as small as possible.
25
6.
7.
Incubate upright at 37oC overnight.
Negative Controls: Using a marker divide the bottom of a selection plate, LBSmAmp,
into two sections. Use a sterile disposable loop to streak plate each parent in a section.
Allow to dry. Incubate upside down overnight at 37oC. Check for growth the next day.
The expected result is no growth or only a few scattered colonies (considered no
growth). Record results and discard control plate.
Day 2
8.
Place 1 ml saline on conjugation mating growth. Using a disposable sterile loop mix
until growth is suspended. Tilt plate to allow suspension to run to side. Take up
suspension several times with Pipetman to obtain a homogenous suspension. Then
transfer the entire amount to a sterile Eppendorf tube (fine if recovery is less). Streak
one LBSmAmp agar plate using 50 µl suspension and a second LBSmAmp agar plate
using 50 µl suspension (located in Student Cold Box). The disposable loops hold 10 µl
so procedure as usual. To streak 50 µl suspension place 50 µl on plate where starting to
streak then use a loop to streak the 50 µl droplet over ¼ of plate, proceed as standard
streak plate method. Incubate at 37oC for 2 days. Store E. coli CHS125 (pUFR-GFP)
conjugate LBSmAmp plates in student cold box (4oC) until next lab.
Day 8 (Week 4)
When using the UV transilluminator NEVER TURN ON THE UV LIGHT WITHOUT
THE LID DOWN.
9.
Transfer 50 µl sterile dH2O to a glass slide. Using a sterile loop transfer 4 or 5
generous loopful of culture to saline on slide. Mix, okay if still particles. Place the
slide on UV transilluminator. Close lid (UV protector). Face shields are also supplied.
Presence of green fluorescence indicates the pUFR-GFP plasmid is now in the E. coli
CHS125 (pUFR-GFP) conjugate colony. If no fluorescence yet, use conjugation plate
that has been stored in the cold box for 3 weeks to check presence of pUFR-GFP
plasmid in E. coli CSH125. Record results.
Comment: Fluorescent may be faint as the quantity of green fluorescent protein after
this short amount of time is still low. It may take several weeks storage in the cold for
obvious flourescent colonies to appear.
26
LAB 2 REPORT
(Use Word Format available on lab website. Save format and open in Word before typing
requested information; keep report format.)
Date:
Group #:
Group or Individual Report:
Student Name(s):
1.5
Part 1 F plasmid Conjugation
1. a) Record requested information in the following table.
Table 1. Initial M9glucose + streptomycin streak plate results for
individual E. coli strains and mating mixture.
Degree of growth.
E. coli strain
Expected degree
Recorda as - or -/+
of growth.
or +
Recorda as - or -/+
or +
CSH101
CSH114
mating mixture
(CSH101 x CSH114)
a
- no growth, +/- few colonies (considered as no growth), + growth
1.5
b) Record requested information in the following table. Refer to strain list and M9 glucose
medium composition.
Table 2. Prevention of donor and recipient E. coli strain growth on conjugate
selection medium, M9glucose + streptomycin.
E. coli strain
Onea media component
(added or not added) that
prevents growth of donor
or recipients. Also state
whether added or not.
E.coli strain genotype and
phenotype that prevents
growth of donor or recipient.
genotype
phenotype
CSH101 donor
CSH114 recipient
marks subtracted if give more than one media component added or not added. Also applies to
genotype and phenotype.
a
27
1
1.5
2. Present one labelled figure of F plasmid conjugant (E coli CSH101 x E. coli CSH114)
papillae results on MacConkey (lactose) streak plate. Label colony and papillae. If not printed
in color indicate papillae color. Copy and paste jpg files of colony with papillae available on
lab website into report Word document HERE and if necessary resize to fit 1/3 – ½ page. In
the event that your group does not have papillae, borrow another group’s jpg file and
reference. Refer to general lab report instructions for figure presentation. Figure may be
labelled with pen but title must be typed.
Part II Conjugation
3. Record requested information in the following table.
Table 3. LBSmAmp initial streak plate results for individual parent E. coli strains and
conjugant.
E. coli strain
Degree of growtha
Expected
Degree of growtha
E.coli strain
phenotype that
prevents growth
of donor or
recipient
DH5α (pUFR-GFP)
CSH125
conjugant
CSH125(pUFR-GFP)
a
- no growth, +/- few colonies (considered as no growth), + growth
0.5
__
7
N/A
4. State whether green fluorescent conjugant, E. coli CSH125(pUFR-GFP) cells present when
viewed on a slide over UV light. If it is necessary to use 3 week old E. coli CSH125(pUFRGFP) colony cells supplied in lab, make a note of this in your answer.
28
LAB 3 PLASMID ISOLATION AND TRANFORMATION
INTRODUCTION
Plasmid Isolation
Plasmid DNA isolation starts with the growth of host bacteria with amplification of plasmid.
Normally there are ten to two hundred plasmids (relaxed - not connected to chromosomal
replication) per bacterial cell. The bacteria containing the plasmid grow in LB medium plus
antibiotic that selects for plasmid (antibiotic resistant gene located on plasmid). pBluescript
has the AMPr gene, bla that codes for TEM β-lactamase which confers ampicillin resistance.
In this lab, the QIAGEN plasmid kit is used to isolate plasmid DNA. Mild alkali (up to pH
12.5) treatment breaks most of the hydrogen bonds in DNA and degrades chromosomal DNA.
Closed circular plasmids regain their native configuration when returned to neutral pH while
larger linear chromosomal DNA fragments remain in the denature state trapped in the cell
debris. The cell debris is precipitated and the supernatant containing the plasmid applied to a
silica gel membrane that binds the plasmid. The membrane bound plasmid is washed to
remove contaminants then eluded as pure plasmid prep. The yield of plasmid DNA is
dependent on the plasmid copy number, plasmid type, bacterial strain, and growth conditions.
Preparation and Transformation of Competent E. coli cells
Preparation of competent E. coli cells requires treatment with calcium, magnesium and
temperature near 0oC, all of which promote uptake of DNA during subsequent transformation.
Mandel and Higa9 (1970) first demonstrated that the uptake of lambda DNA by E. coli is
enhanced by treatment of E. coli cells with calcium chloride under cold conditions followed
by short heat shock treatment at 43oC. During transformation heat shock at 43oC facilitates
complete DNA molecule uptake by the cells. Just prior to plating on LB Ampicillin agar, LB
broth is added and the cells are incubated at 37oC for 1 hr without shaking. During this time
the plasmid DNA becomes established in the cell. Establishment is a difficult process as there
must be correct mini-chromatid (protein-DNA structure) formation. Plasmids cannot exist as
naked DNA. Replication is essential for establishment of the plasmid. Also it is essential that
plasmid antibiotic resistant gene begins to express prior to exposure to antibiotic to increase
the chance of survival once plated on LB-antibiotic agar plate. A similar procedure was used
by Cohen et al10(1972) to transform bacteria with plasmid. This simple method, similar to
what we use in the lab generates 105 to 106 transformed colonies/μg plasmid DNA11.
Approximately 50 ng plasmid per 100 μl competent cells should be added to obtain efficient
transformation but the volume of plasmid should not be greater than 5% of the competent
cells. Both these rules may be broken to some degree and still obtain fairly good
transformation.
9
Mandel M, Higa A. 1070. Calcium-dependent bacteriophage DNA infection. J. Mol. Biol. 53: 159-162.
10
Cohen SN, Chang ACY, Hsu L. 1972. Nonchromosomal antibiotic resistance in bacteria: Genetic
transformation of Escherichia coli by R factor DNA. PNAS 69: 2110-2114.
11
Sambrook J, & Russell D. 2001. Molecular Cloning: A Laboratory Manual. 3rd edition. New York:
Cold Spring Harbor Laboratory Press p 1.109-1.111.
29
Xgal Detection system12
pBluescript® ~3 kb plasmid (see diagram) contains only the promoter region and the
first 146 amino acids (5’end starting with first methionine) of E. coli β-galactosidase lacZ
gene (α-donor fragment).
The E. coli chromosomal lac operon is removed, Δ(argF-lacZYA) but carries
prophage Φ80 lacZÎM15 which contains lacZ gene but with deletion of the α-donor fragment.
A o or α-acceptor lacZ fragment is generated starting at the next methionine. Neither
fragment (α-donor or α-acceptor) is active without the other. However, they associate to form
a functional β-galactosidase by α-complementation.
The chromogenic substrate, Xgal (5-bromo–4-chloro-3-indolyl-β-D-galactosidase) is
converted to an insoluble blue compound by β-galactosidase . So when E. coli
DH5α(pBluescript) is plated on media containing Xgal/IPTG the colonies turn blue.
IPTG (isopropyl β-D-thiogalactoside), a non-fermentable lactose analog removes the
repressor, ie fully activates lacZ gene. However, in most plasmid host E. coli strains the level
of repressor is low making the need for IPTG optional.
A multi-cloning site (MCS) is inserted in the coding sequence of lacZ gene of
pBluescript. The opening reading frame is maintained and the few additional amino acids
does not interfere with a functional β-galactosidase. The MCS consists of numerous restriction
enzyme sites that permit insertion of desired gene. None of the MCS restriction sites are found
elsewhere in the plasmid. In this lab the 1.4 kb rhaK gene is cloned into pBluescript via the
BamHI/HindIII restriction sites, designated pMR106. When E. coli DH5α(pMR106) is plated
on media containing Xgal/ IPTG the colonies are white due to inactive β-galactosidase.
12
Sambrook, J, Russell, DW. 2001. Chapter 13 Mutagenesis. In Molecular Cloning A Laboratory
Manual 3rd edition. Cold Spring Harbor: CSHL Press p. 1.116 - 1.150
30
31
E. coli STRAIN LIST
STRAIN NUMBER
GENOTYPE
IMPORTANT
PROPERTIES
DH5α
Φ80dlacZΔM15 Δ(argF-lacZYA) U169 recA1
endA hsdR17 supE44 thi-1 gyrA96 relA1
DH5α(pBluescript)
as above for DH5α with pBluescript plasmid
contributing bla (TEM β-lactamase) and functional βgal gene
Ampr Lac+
DH5α(pMR106)
as above for DH5α with pBluescript plasmid
contributing bla (TEM β-lactamase) and R.
Ampr Lac-
Lac- Amps
leguminosarum rhaK gene (1408 bp) insert via
BamHI/ HindIII restriction enzymes in multi-cloning
site
Rha = rhamose C-source
PROCEDURE
Week 4
This is a long lab, come prepared.
Part I Plasmid DNA preparation (QIAGEN13 kit method)
Please take care when carrying out this experiment procedures. You will be graded on your
ability to isolate and restriction digest plasmid (combined with lab report).
E. coli cultures were grown overnight with rotation at 37oC in 5 ml LB-AMP broth.
Each group carries out the following procedure for E. coli DH5α(pBluescipt) and E. coli
DH5α (pMR106).
1. Cell Harvesting: Aseptically transfer 3 ml
E. coli culture into two Eppendorf tubes (each
tube only holds 1.5 ml). Micro centrifuge at
room temperature for 1 min (12,000 x g).
Remove supernatant using a Pipetman, make
sure the entire supernatant is removed
including any liquid on inside surface of tube.
Micro centrifuges available in the lab:
All centrifuges may be used for short spins,
however, only the larger models may be used for the
10 min spin as requires 12,000 x g (RCF, relative
centrifugal force).
Legend micro centrifuge has variable speed
maximum speed dependent on model (newest).
Eppendorf micro centrifuge variable speed up to
14,000 rpm (12,700 x g) - check that set at
maximum.
Mini centrifuges (4 only in room 201) - constant
speed of 6600 rpm (2200 x g) use only for mixing
reactions.
2. Condensing two tubes to one tube and
Cell Suspension: Add 250 μl Buffer P1 to
ONLY one pellet tube and completely
resuspending the cells using the pipetman.
Transfer the resuspended pellet to the second
pellet tube and again completely suspend
cells. The solution must be homogeneous or very little plasmid will be extracted. P1 buffer
may have a blue dye added to help you determine complete cell lysis (P2 buffer) and
neutralization (N3 buffer) of the cells. The solutions turns a homogeneous blue color after
13
QIAprep® Miniprep Handbook. QIAGEN March 2003.
32
correct mixing of cell lysis solution (P2). After correct mixing of neutralization solution (N3)
there should be no blue color remaining.
3. Cell Lysis: Add 250 μl Buffer P2 and mix gently by inverting the tube 4 -6 times. Do not
vortex.
4. Neutralization: Add 350 μl Buffer N3 and mix immediately by inverting the tube gently 4
-6 times. Do not vortex. The mixture should become cloudy. Micro centrifuge for 10 min
(12,000 x g) at room temperature. A white pellet forms.
5. Spin column loading: Label spin column. The spin column is supplied housed in a round
bottomed tube. Decant the supernatant into the spin column. Decant by quickly tipping your
tube over the spin column with top edges touching. Do not remove remainder of supernatant
with pipetman. It is important that none of the precipitate is transferred to the spin column.
Microcentrifuge for 1 min (12,000 x g). Discard the flow-through (liquid in round bottom
tube).
6. First Wash: Return the spin column to the round bottom tube. Add 500 μl Buffer PB to the
spin column. Micro centrifuge for 1 min. Discard the flow-through. This step is required for
to destroy nucleases in nuclease rich bacteria.
7. Second Wash Return the spin column to the round bottom tube. Add 750 μl Buffer PE to
the spin column. Micro centrifuge for 1 min. Discard the flow-through. Micro centrifuge
again for 1 min to remove residual wash buffer. Discard round bottom tube.
8. Plasmid Elution: Cut the top off an Eppendorf tube. Then place the spin column into the
tube. Add 50 μl EB buffer directly to the centre of the spin spin column. Incubate at room
temperature for 1 min. Micro centrifuge for 1 min. Discard spin column in plastic lined
bucket on the discard trolley. Replace cap on tube.
9. Clearly label tube with group # ON LID TOP and on the side with plasmid name, your
names or initials, lab day and room #. Store plasmid DNA samples on ice during lab. DO
NOT DISCARD PLASMID PREPS as needed for next week’s lab. At the end of the lab store
plasmid DNA samples in designated tray located in -20oC freezer in each lab.
33
Part II Plasmid DNA concentration determination using NanoDrop Spectrophotometer
[Thermo Fisher]
Carry out this procedure during wait times of gel electrophoresis.
(On/off switch, on power bar. Wait until Home screen appears.)
Cautions: Use only a Pipetman to add liquid to pedestal. Never use a squirt bottle.
Use only Kim wipes, never Kleenex or paper towel to clean pedestals.
Never drop liquid on black area around bottom pedestal (silver).
1. Defaults to DNA on the Home
screen. Or use the up and down
arrowhead buttons to box DNA.
Press Select button.
2. Defaults to dsDNA. Press Select
button.
3. Open arm of NanoDrop (right
side). Pipette 2 µl sterile distilled
water on bottom pedestal. Close
arm. The sample is automatically
drawn between the upper and
bottom pedestals, forming a liquid
column. Press Blank.
4. Requests confirm blank. Raise
arm and wipe upper and bottom
pedestal using Kim wipe (never use
any other tissue to wipe pedestal).
Again pipette 2 µl sterile distilled
water on bottom pedestal. Close
arm. Press Blank. Once blanked the
Blank is stable for 30 min.
5. Open arm and wipe upper and
bottom pedestal with a Kim wipe.
Pipette 2 µl DNA sample on bottom
pedestal. Close arm. Press Measure.
6. Screen displays concentration information. Concentration is displayed as ng/µl – record this
value in data table. Also displayed is the A260/A280 ratio – record this value in data table
(should be close to 1.8 indicating a pure sample). DNA peak is 260 nm while protein peak is
280 nm.
7. Open arm. Use Kim wipe to wipe upper and bottom pedestal.
8. Now ready for second sample. Students do not need to clean pedestal with water between
samples.
9. Last student or TA. Pipette 3 µl sterile dH2O onto bottom pedestal. Lower arm. Let it sit for
2 min. Raise arm and Kim wipe the upper and bottom pedestals. Turn switch on power bar to
off NanoDrop spectrophotometer.
Data Table
plasmid DNA sample
pBluescript
DNA concentration (ng/µl)
pMR106
-make sure you know how to convert to µg/µl.
A260/A280 ratio
34
Part III: Casting Agarose Gel using Sub-Cell® GT Mini agarose gel electrophoresis
system (BIO-RAD)
Each group sets up their own gel electrophoresis system.
1. Remove lid and place the gel electrophoresis bottom container on a level surface.
2. Place the UV transparent
plastic gel tray on centre
support with upper notches
close to negative (black)
electrode.
3. Place the black gates
securely into slots as shown in
the figure.
4. Set the comb into tray upper
notches.
5. Obtain a tube containing 25
ml 0.7% agarose prepared in
1x TAE buffer from the 55oC
waterbath. Immediately pour
the gel into centre tray area between gates. When pouring, avoid bubble formation. If your get
bubbles, remove with Pipetman or move to edge of tray. Allow the gel to solidify for 20 min.
During this time set up restriction enzyme digestion.
Part IV: Plasmid DNA Restriction Enzyme Digestion
1. Set up the following restriction enzyme digestion for each of your plasmid preps,
pBluescript and pMR106. Use a separate tip for each addition. After removing specified
amount of each plasmid for RE digestion store plasmid DNAs in -20oC freezer in student
sample box for next week’s transformation experiment. Do not discard.
Addition to Eppendorf tube
Amount added (μl)
plasmid DNA
4
10x FastDigest® buffer
2
ddH2O (double distilled water)
13
1 μl
FastDigest® HindIII
Mix reaction by doing a quick spin (5-15 sec) in the micro centrifuge.
2.
Incubate for 10 min in a 37oC waterbath. Vortex EZ-Vision™One2 6x loading buffer
Eppendorf before adding 4 μl to each restriction enzyme digest. Do a quick spin to mix
loading buffer into reaction.
2
EZ-Vision™One is a 6x loading buffer containing fluorescent dye, 15% Ficoll and magenta tracking dye,
amaranth (10 bp). EZ-Vision™ is a fluorescent dye for visualization of DNA bands by UV illumination of
agarose gels. It is non-mutagenic and non-toxic. EZ-Vision™ complexes tightly to DNA and co-migrates. This
eliminates the use of the carcinogen, ethidium bromide. 15% Ficoll increases the density of sample, thus it sinks
35
Part V: Gel electrophoresis
1. After gel has solidified and just before gel electrophoresis carefully remove comb followed
by removal of gates. Rinse gates and comb with distilled
water (white handle tap) and place on paper towel on your
bench to dry.
2. Pour 1x TAE buffer into gel unit filling to just below max
level (indicated on side of gel container). Take care when
filling to ensure the buffer goes into the wells. The agarose
gel should be covered by ~2 mm buffer.
3. In the first lane well add 8 μl 1 kb Plus DNA standard
ladder prepared with EZ Vision loading buffer. Load 6 and
12 µl of each sample restriction enzyme digestion. Each
sample is loaded using an Eppendorf micropipet by holding
the pipetman just above the well and releasing sample such
that is sinks to the bottom of well. Good idea to steady your
hand with the other hand.
4. Carefully place the lid on gel holder to avoid spilling the
buffer and ensuring that the electrode contact is complete. The Lid only fits on one way –
electrode contacts black to black (negative) and red to red (positive). The negatively charged
DNA will migrate to the positive electrode (red).
CAUTION WHEN ATTACHING GEL ELECTROPHORESIS UNIT TO POWER PACK –
always turn off power pack before attaching or removing gel unit. Make sure the power pack
is turned back on if other gel units are still attached.
5. With the assistance of the TA attach red electrode to red outlet and black electrode to black
outlet on power pack. Turn on power supply and set at constant voltage (~100 volts).
Electrophoresis for 35 min.
Part VI: Gel Doc XR+ photography of agarose gel.
to bottom of agarose gel well while loading through electrophoresis buffer. when 10bp tracking dye is near the
bottom of the gel it is time to shut off the electrophroesis.
36
1. Remove gel tray containing agarose gel and place in a small container. Rinse gel
electrophoresis unit using distilled water tap (white handle) and invert on paper towel to dry
on your bench.
2. Take gel to Gel Doc XR+ (located in room 204 or 302 – confirm with TA). With the
assistance of the TA take a picture of your gel. Pull out drawer, transfer gel from gel tray and
centre on UV screen. Close drawer completely. Use Image lab Software and red interference
filter (filter 1) to photograph (tif files) agarose gel containing electrophoresed fluorescent
stained plasmid DNA samples. Group jpg files will be available on the website as soon as
possible. For best EZ-Vision stained results must remove gel from gel tray for photographing.
3. Discard gel in Petri plate container since contains DNA. Rinse gel tray with distilled water
and place on paper towel to dry on your bench. Leave entire distilled water rinsed gel system
unit on your bench drying.
Week 5
Part VII: Preparation of competent E. coli DH5α cells
1. 5 ml LB broth inoculated 1/50 with overnight culture
of E. coli DH5α. Rapid rotated at 37oC for 31/2 h.
Abs550 nm should be 0.4-0.5. It is important a good
oxygen supply is present to ensure log phase growth of
E. coli subsequently competence. Each group receives
two 5 ml log phase E. coli DH5α cultures.
When removing supernatant the pellet
should be on the upper side such that it is
always visible to you as you remove
supernatant. When removing supernatant
with the Pipetman keep tube slightly
tilted so the tip does not touch the pellet.
Never allow the remaining supernatant
to run back into the pellet.
STUDENT LAB STARTS HERE
2. Combine two 5 ml E. coli DH5α cultures in one centrifuge tube. Centrifuge at 10,000 rpm
for 5 min at 4oC. Use a P-1000 to remove the supernatant. Discard supernatant. Gently
resuspend the cells in 4 ml sterile 0.1 M MgCl2 (dispensed in 4 ml aliquots, no need to
measure). Centrifuge at 10,000 rpm for 5 min at 4oC. Decant off supernatant, removing any
remaining droplets with a Pipetman. Gently resuspend cell pellet in 4 ml sterile 0.1 M CaCl2
(dispensed in 4 ml aliquots, no need to measure).
3. Incubate on ice for 30 min.
4. Again centrifuge at 10,000 rpm for 5 min at 4oC
Resuspension of pellet: You should
always use the gentlest method possible.
Froth has the potential to kill cells.
Resuspend cell by using Pipetman
bringing the liquid up and down on the
pellet, remember not to create froth.
Check that the pellet is completely
suspended.
5. Resuspend the cells in 0.5 ml sterile CaCl2 +
glycerol. Label four Eppendorf tubes with ONLY
your group # on the lid, no initials or names. On the
side of tube, label competent E. coli DH5α. Check
bulletin board to make sure you have the correct group
#. Dispense 100 μl in each Eppendorf tube. Incubated
on ice overnight in student cold box. A box of ice containing tube holders should already be in
the cold box.
6. Your competent cells will be transferred to the -80oC freezer.
37
Week 6
Part VIII: Transformation: Xgal Detection System
Day 1
1.
Thaw three 100 μl competent E. coli DH5α cells at room temperature and put on ice (if
not used immediately).
Set up the following transformation reactions.
plasmid name
competent E. coli
DH5α (μl)
volume of plasmid
pBluescipt
100
5 μl
pMR106
100
5 μl
Negative control - no plasmid
100
0
After using plasmid preps RETURN
Dilution and Plating Information:
GOOD PREPS TO FREEZER.
2.
Heat shock at 43oC for 1 min.
The general rule for all experiments is that 0.1 ml of
Put on ice for 3 min.
culture or dilution is spread plated on agar medium unless
3.
Add 1 ml prewarmed LB broth
otherwise specified.
(37oC) and incubate for 1 hour in Mix tube by vortexing after each transfer.
Use a P200 (labeled on top of Pipetman piston) to transfer
a 37oC waterbath.
0.1 ml (100 μl).
4.
For each plasmid transformation
Use a P1000 to transfer 0.9 ml (900 μl).
tube prepare 10-1 dilution in
It is extremely important that you do not turn the dial of the
saline by adding 100 μl
P200 above 0.2 ml/200 μl or P1000 above 1.0 ml/1000 μl
transformation mixture to 900 μl
as it causes permanent damage. Get assistance from the
saline in 5" sterile metal capped
demonstrator if you are not sure of pipetman operation.
test tube. Mix. Repeat 10-fold
Refer to the appendix for operation of pipetman.
dilution twice more until 10-3.
Use separate tips for each dilution and each different
Spread plate in duplicate 100 μl
dilution plating.
of each (undiluted, 10-1, 10-2 and
10-3 dilutions) on LB-AMP agar
Use spread plate technique to distribute the bacteria evenly
over the surface of the LB plate.
plates.
1) Aseptically transfer 0.1 ml of culture or dilution to
5.
For the negative control
centre surface of agar plate. Hold lid tilted above the plate
transformation tube plate only
when adding sample or spreading.
100 µl undiluted on one LB2) Use disposable sterile spreader to spread bacteria. Use
AMP agar plate.
the same sterile spreader for duplicate dilutions. Must use a
6.
Incubate plates at 37oC for 1
new sterile spreader for each dilution.
days.
3) Open lid keeping it tilted over the plate, move the plate
Day 2
around spreading bacteria evenly over agar surface. (Use
7.
Record transformation colony
turntable to rotate plate if available.)
data.
8.
Using only ONE LB-AMP-Xgal
IPTG agar plate, pick plate 6 tranformants of each plasmid type, pBluescript and
pMR106. You may use the same pick plate pattern from lab 1 (using only two of the
quadrants). Incubate at 37oC for 1-2 days. If expected color has not developed, transfer
LB-AMP-Xgal IPTG agar plate to Student Cold Box - enhances color. Check color
development after one or two days.
9.
Record pick plate results.
38
LAB 3 REPORT
(Use Word Format available on lab website. Save format and open in Word before typing
requested information; keep report format.)
Date:
Group #:
Group or Individual Report:
Student Name(s):
2
1. Include a completely labelled figure of agarose gel electrophoresis photograph of Hind III
digested pBluescript and pMR106 plasmid DNA. Insert your group’s agarose gel jpg
photograph available on lab website into Word doc. Complete figure should be no larger than
½ page (do not remove any lanes, show the complete gel picture). Use your group’s data
regardless of results. Refer to Lab Report Presentation section for figure set up.
1
2. Record requested information in the following table. Must be your group’s results.
Table 1. Plasmid concentration determination using Nanodrop spectrophotometry.
plasmid DNA
DNA
DNA concentration A260/A280 ratio
sample
concentration (µg/µl)
(ng/µl)
pBluescript
pMR106
1.5
3. Practical ability to isolate and restriction digest plasmid. Include this table in your report for
marker – the student does not enter any information.
Marking Criteria of Agarose Gel.
Acceptable indicated by check mark.
pBluescript
pMR106
Reasonable amount of plasmid present
Single DNA band for HindIII digest
0.5
4. How many bands are expected on the agarose gel when plasmid pMR106 is restriction
enzyme digest with KpnI? pMR106 has one KpnI site in the rhaK gene inserted in pBluescript.
Refer to pBluescript MCS (multi-cloning site) as rhaK is inserted in pBluescript using
restriction enzymes BamHI and HindIII.
Circle the correct answer:
TWO or THREE or
FOUR
39
4.5
5. Record requested information in the following table. Indicate in table with an asterisk or
highlight or boldface values used to calculate transformants/ml titre.
Table 2. Determination of pBluescript and pMR106 transformants/μg
plasmid for E. coli DH5α.
dilution
Plate counts for 0.1 ml
Transformatiom Mixture plated
on LB-AMP
pBluescript
pMR106
100
100
10-1
10-1
10-2
10-2
10-3
10-3
Negative control
100
Transformation titre (cfu/ml)
Total volume transformation mixture just
before plating (µl)
plasmid (µg/µl)
µg plasmid added to transformation mixture
transformants/μg plasmid
TNTC = too numerous to count. Indicate plates counts used to calculate all titres with asterisks or boldface
or highlight. If you do not have significant counts, use best data you have and state less than significant but
only data available. If no data, borrow from another group. See appendix titre calculation for more
information.
Show numerical calculations for either pBluescript or pMR106. Circle to indicate.
Calculations must follow table.
Tranformation Titre (cfu/ml):
Total volume transformation mixture just before plating:
µg plasmid added to transformation mixture:
Transformants/μg plasmid:
40
0.5
6. Record pick plate results in the following table.
Table 3. E. coli DH5α transformation with plasmids pBluescript and
pMR106 LB-AMP-Xgal-IPTG pick plate results.
Plasmid
# of colonies
picked showing
growtha
colony colorb
expected
colony colorb
pBluescript
pMR106
6 transformants picked
b
record as blue or white. The expected colony color for 6 colonies picked should be the same.
If differs, footnote.
a
__
10
41
LAB 4 TRANSDUCTION: P1 GENERALIZED TRANSDUCTION
Introduction
Generalized transduction14 of E. coli is mediated by phage P1. P1 phages contain a
110 kb, double stranded, linear, terminally redundant and circularly permutated DNA genome.
During the lytic cycle, some P1 phage accidentally package the host genomic DNA, the
resulting P1 particle is called a transducing particle. Although the transducing particle is
defective (cannot form lysogen or enter lytic cycle), it has the ability to inject host E. coli
DNA into recipient E. coli. The injected DNA can undergo homologous recombination with
host genome. Via transducing particles any gene on the E. coli chromosome may be
introduced into another E. coli strain at a frequency of 10-6 to 10-8 infected cells. Due to the
large size of DNA that can be packaged it is possible to transfer genes which are closely
linked on the E. coli chromosome (within 2.2 min), i.e., cotransduced. For instance, purB
(25.6 min)13 and galU (27.8 min)13 are 2% cotransducible. This means that if we transduce a
PurB- GalU- strain with a P1 lysate from a wild-type strain and select for PurB+, then 2% of
the PurB+ transductants will also be GalU+.
In preparing strains by P1 transduction, it is desirable to prevent non-defective P1
phage particles (majority of particles in P lysate) from lysogenizing or lysing the recipient
strain. This is because P1 lysogens restrict foreign DNA introduced during phage infection or
conjugation and continue to interfere with future genetic crosses. By using low multiplicities
of infection (only one phage/host cell) and by adding citrate to prevent further adsorption
(chelating agent that binds the divalent cation, calcium which is required for adsorption), we
can virtually eliminate this problem. Also, we can use a virulent mutant of P1 (P1vir) which is
unable to form lysogens.
In this lab we will use P1 generalized transduction to construct a new E. coli strain. P1
phage lysate carrying transducing particles of E. coli UM122 is used to transduce E. coli
JM96(cysH). First transduced E. coli JM96 is selected on LBTc for katF::Tn10. Colonies are
then picked onto defined medium with and without cysteine to select for CYSH cotransduced
with katF::Tn10. This results in a new E. coli strain construct which is resistant to tetracycline
and no longer auxotrophic for cysteine. Since a defined M9 medium is used to select for
CYSH all auxotrophic nutrients required by E. coli JM96 are added.
14
Madigan MT, Martinko, JM. 2006. Brock: Biology of Microorganisms. 11th edition. Pearson/Prentice
Hall: Upper Saddle River. p. 272
Sambrook J, & Russell D. 2001. Molecular Cloning: A Laboratory Manual. 3rd edition. New York: Cold Spring
Harbor Laboratory Press p. 4.35-4.4.36.
42
Table 1. Map position (min) of selected E. coli genes15.
gene
map position (min)
rpsL
74.8
katF::Tn10
61.9 (not on cited map)
cysH
62.2
argH
89.5
thiI
9.5
Phage Lysate
Prior to the start of this lab P1 phage lysate, was prepared by infecting host E. coli UM122
with P1 phage producing 100-200 progeny per host cell, with some of the phage being
transducing particles with E. coli UM122 DNA packaged. After infection, cells are plated in a
layer of soft agar on nutrient plate, incubated overnight at 37oC to allow confluent host lysis
over the surface of the plate due to overlapping plaques. The soft agar layer is scraped into a
test tube or centrifuge tube. Chloroform is added to further disrupt the cell wall. Cell debris
is then centrifuged, and the supernatant, which is the lysate, is stored in the cold. Lysates are
stored at 4oC in the presence of chloroform without decrease in titre for several years.
E. coli strain list
Strain number
Genotype
Tcr (one possibility), cannot
form lysogens
P1vir (UM122)
JM96
Important properties
cysH thr leu trp his argH rpsL thiI lac xyl
Cys-
gal mal supE44
UM122
HfrH thiI katF::Tn10
Tcr
Notes:
(i) katF::Tn10 means the transposon Tn10 (marker gene), which contains a tetracycline
resistance marker, is inserted in the KATF gene (catalase). The katF gene is no longer
functional, non-essential gene for survival.
(ii) Only defective genes are noted in genotype/important properties. It is assumed that any
gene not noted is wild type or not of particular interest for this lab. Also it is assumed that E.
coli strains are sensitive to antibiotic if resistant gene not present.
rpsL = streptomycin resistance
15
Berlyn MKB. 1998. Linkage map of Escherichia coli K-12, Edition 10: The Traditional Map. Micro
Mol. Biol. Rev. 62: 814-984. http://mmbr.asm.org/cgi/reprint/62/3/814 or search ASM journals, UM NETDOC
or Google Scholar.
43
PROCEDURE
CAUTION: When using a Pipetman to pipette phage you must wipe the Pipetman barrel at the tip end with
alcohol when you are finished using a particular phage. Just pour a small amount of 70% alcohol onto a
kleenex to wipe the tip. For the remainder of the labs you must always wipe the Pipetman barrel tip with
alcohol before using to pipette bacteria or a different phage. If you neglect to do this you may ruin your
experiment.
It is good practise to pipette slowly when pipetting phage to reduce aerosol.
Week 7
Day 1
1.
At start of experiment put 10 LBTc agar plates in
the 37oC incubator to pre-warm. Pre-warmed
plates allow slightly more time to spread soft agar
over surface of plate before hardening.
2.
An overnight cultures of E. coli JM96 has been
subcultured (1/50 dilution) into 5 ml LB + 5 mM
CaCl2 and grown for 4 hours (mid-log phase - 1 x
109 cells/ml) at 37oC with rotation.
Centrifugation is carried out at 4oC
to ensure stability of the biological
material. At 4oC bacterial cells
metabolic rate is slowed, less
chance of cellular damage if stress
applied (centrifugal force)
especially if high speed and or long
centrifugation times. In addition
centrifugation creates heat, to
prevent this the temperature is set
at a constant temperature.
3.
Transfer E. coli JM96 culture to sterile plastic
capped centrifuge tube and centrifuge at 10000
rpm for 5 min at 4oC. Decant off supernatant
removing any remaining droplets with a Pipetman. Gently resuspend pellet in 5 ml MC
buffer (0.1 M MgSO4, 5 mM CaCl2). Each tube of MC supplied by prep room contains 5
ml, no need to measure.
4.
Dilute P 1 phage by mixing 0.1 ml phage with 0.9 ml MC buffer (10-1) in a sterile 5" metal
capped test tube. Transfer 0.1 ml 10-1 dilution to another tube containing 0.9 ml MC buffer
and mix (10-2) dilution. Remember to use a separate Pipetman tip for each dilution and
when plating each dilution.
5.
Set up transduction experiment as follows using 5 inch sterile metal capped test tubes:
mix 0.1 ml of E. coli JM96 cells with 0.1 ml of 100, 10-1 and 10-2 dilutions of the P1 lysate
in duplicate. Also prepare two control tubes one containing only 0.1 ml bacterial cells
and the other containing only 10 μl phage lysate stock. Incubate tubes in a 37oC
waterbath for 20 min. During this time label your LBTc agar plates (plates are at room
temperature).
6.
Add 0.2 ml 0.1 M Na citrate to each tube. Then working with one tube at a time, add
entire tube of melted F-top agar (3.5 ml) from a
*No need to mix as this happens when
55oC waterbath and immediately* pour on room
you swirl the top agar on the plate.
temperature LBTc agar plate, controlled swirl to
spread. Repeat for remaining transductions. Any signs of agar solidifying, stop swirling,
it’s too late.
7.
Incubate plates 1 day at 37oC.
44
Day 2
8.
Count colonies on plates and record data (data recording sheet follows).
9.
Pick between 100 and 120 isolated coloniesa
Colony Size:
growing on the LBTc agar plates (from any
The colony size may vary
dilution) onto M9glucose plus cysteine and
depending on whether the colony
M9glucose minus cysteine. If you have less than
is embedded in or on the surface of
100 colonies, pick as many as you have. Use the
the F top agar. Usually colonies on
grid supplied. Use only one toothpick for each
the surface are larger. Also colony
colony, first picking on M9glucose minus cysteine
size may vary dependent upon
and M9glucose plus cysteine. Discard toothpicks
where P1 transduction has
in plastic lined bucket on bench not in the Petri
occurred in the genome.
plate container (it is pointed). Put orientation mark
on the bottom of the plate to orientate picking.
After growth just put the plate on the grid lining up the orientation mark to see the number
for each space. Two plates of each type are available per group. If you do not have 100
colonies, pick as many single colonies as you have. All auxotrophic nutrients required by
E. coli JM96 are added to the M9 glucose medium.
10.
Incubate at 37oC for 2 days. Record data.
11.
Day 4 (Friday): Hand in a COPY of your P1 generalized transduction DATA SHEET to
the slotted located in the hallway across from 302 Buller – one copy only per group. May be
hand written. Include all requested information: group #, group names (in full), number of
picked colonies that grew on M9glucose plus cysteine and M9glucose minus cysteine. Data
must be handed in or emailed to [email protected] by 2:30 pm Friday. No
honesty declaration required for data submission. The data will be compiled and POSTED
ON THE WEBSITE in Excel format as soon as possible. Marks subtracted from report if
data is not submitted as requested.
45
TRANSDUCTION PLATE COUNT DATA SHEET
(only for recording your plate count data, use Excel report spreadsheet for report write-up)
Table 1. P1 transduced E. coli JM96 LBTc PLATE COUNT DATA.
a
Dilution plated
100
10-1
10-2
a
0.1 ml P1 dilution plated.
Colony plate count.
Plate 1
Plate 2
46
Molecular Genetics of Prokaryotes MBIO 4600
Lab 4 P1 Generalized transduction DATA SHEET - available as word document on lab
website
Only one copy/group required. No honesty declaration required for data submission.
Group #: _______________
Group names (in full): ________________________________________________________
# picked colonies that grew on M9glucose plus cysteine: _________________
# picked colonies that grew on M9glucose minus cysteine: __________________
Pick plate grid.
Remember to include orientation mark or label the bottom of
each plate as below. Remember to label plate type before
you pick plate.
plate 1
47
plate 2
orientate
1
2
6
7
8
9 10
65 66
67
68
69 70
14
15
16 17
71 72 73
74
75
31
32
33 34 35
36 37 38 39 40
41
42 43 44
4
5
11 12 13
27 28 29
30
45 46
3
47 48
52 53 54
62
49
55
88
89
90
96 97 98 99
91
64
92
76
77
93 94
100 101 102 103 1
50 51
56
59 60
87
63
57 58
61
119 120 121
plate 3
plate 4
orientate
62
64
2
6
7
8
9 10
65 66
67
68
69 70
14
15
16 17
71 72 73
74
75
31
32
33 34 35
36 37 38 39 40
41
42 43 44
4
5
11 12 13
27 28 29
45 46
30
47 48
52 53 54
55
59 60
3
63
1
49
56
61
87
88
89
90
96 97 98 99
91
92
76
77
93 94 9
100 101 102 103 1
50 51
57 58
119 120 121
48
LAB 4 REPORT
(Use Excel and Word Formats available on lab website. First save before opening in respective
program. Report must be typed.) Excel spreadsheet contains two worksheets, group and class –
select using tab at bottom.
Date:
Group #:
Group or Individual Report:
Student Name(s):
Data Presentation and Analysis
3.5
1. Include a completed group data Excel worksheet for tetracycline gene tranduction (P1(UM122)
into E. coli JM96 and sample calculations. Fit all information including sample calculation on one
portrait page.
1.5
2. Include a completed class data Excel worksheet including the frequency of cotranduction of
katF:: Tn10 and CYS H. Record group # or highlight your group’s data. Fit all information
including sample calculation on one portrait page.
Question (Use Word document available on lab website.)
3.0
1. A new E. coli stain Pro- Mlt- Tcr Ampr was created by P1 transduction using only the following
bacteria strains and P1. Refer to lab 5 introduction for additional information on transposons.
E. coli Strain Genotype
Phenotype
CD43
Arg- Mlt- Ampr
argY mltD::Tn10amp
SB21
Pro- Tc r GlyproW::Tn10 glyS
Map positions: mltD = mannitol, 81.3 min; proW= proline, 60.4 min; metC = methionine, 67.9 min;
glyS = glycine, 80.2 min; Ampr = ampicillin resistance; Tc = tetracycline resistance
(i) Circle the E. coli strain used to prepare the P1 lysate:
CD43 or SB21
(ii) Circle the E. coli strain transduced with P1 lysate:
CD43 or SB21
(iii) State the medium the transduction mixture is pour plated to select for transduced E. coli.
Take into consideration prep time and cost of medium, use the most economical medium.
(iv) In the following table list 4 pick plate media used to select and confirm the new strain.
Remember to also include type of medium and carbon source.
Pick Plate media used to select and confirm new strain new E. coli stain.
Presence or absence
Assume antibiotic resistance established - omit addition of antibiotic(s).
of growth (+ or -)
+
__
8
49
EXCEL INFORMATION
When taking the SUM of more than one column just hold down the CTRL key when selecting data.
Best to use a combination of SHIFT block and CTRL key.
Each spreadsheet should be printed to fit ONE PAGE, this includes all calculation and any requested
information. Print to fit ONE portrait page – Select Page Layout –Print Area – Set Print Area (click
and drag to select area) - Set Width to 1 page (default is automatic) and Height to 1 page (default is
automatic). ). When setting print area ensure that all text boxes and charts are completely within area
selected. Proof read printout.
PROOF READ page to ensure all information is included.
RIGHT CLICK is extremely helpful in EXCEL - especially for formatting. Right Click on
appropriate cell and select FORMAT to change Number, Alignment, Font, etc.
Numerical calculations may be done directly on the Excel spreadsheet or inserted Text box. With the
text box the calculation may be done first in Word and copy/pasted to inserted textbox. BE
CONCISE - this is a numerical calculation, do not include explanations. Resize text box and font to
fit.
50
LAB 5 TRANSPOSITION
INTRODUCTION
Transposons are important bacterial genetic tools that create non-leaky mutations.
Transposon movement into recipient genome is easily detected due to the presence of antibiotic
resistant gene. In this lab, two methods of transposition are performed, (1) suicide plasmid
(cannot replicate in recipient) carrying the Tn5 transposon and (2) λ1098 carrying the mini
Tn10Tc transposon (lambda cannot replicate or lysogenize under experimental conditions). The
resulting transposed bacteria are plated for antibiotic resistance marker and then scored on
indicator plates for transpositions in specific genes, i.e. lactose inactivation using Xgal as the
indication.
The suicide plasmid, E. coli MM294A/pRK602 with ColE1 replicon, is a derivative of
pRK600 that has a Tn5 transposon (neomycin resistance conferred). The recipient, streptomycin
resistant Sinorhizobium meliloti Rm1021, restricts replication of plasmids with the ColE1
replicon. Transposition of Tn5 transposon to Sinorhizobium meliloti Rm1021 is confirmed by
plating on agar plates containing neomycin and streptomcyin.
λ1098 has a number of alterations that allow transposition to occur with increased
efficiency and stability; (1) nonsense mutations in the replication genes (O and P), therefore
they cannot synthesize DNA in a Su- host, (2) carry a mutation in the λ cI gene, which prevents
repression at temperatures above 39oC and (3) mutations that inhibit λ integration. λ1098
phage17 carries the mini- Tn10Tc instead of Tn10. This also increases transposon efficiency and
stability by (1) increasing the activity of transposase, (2) positioning the transposase outside of
the segment to be transposed; once the antibiotic resistance gene is transposed it cannot be
transposed again, (3) removing segments of the IS10 elements thereby eliminating inversions
and deletions, and (4) allowing placement of different antibiotic markers (Kanr, Camr, Ampr,
and original Tcr) between shortened Tn10 ends. If different antibiotic marker, for example,
kanamycin resistant, genotype is written as Tn10kan. This strain has only kanamycin resistance
not tetracycline resistance. For λ1098 Tn10Tc transposition it is important to understand the
presence or absence of the SupE gene. λ1098 Tn10Tc lysate must be prepared in host E. coli
that contains the SupE gene (E. coli CSH110 Su+). SupE produces a suppressor tRNA which
suppresses the UAG Amber STOP condon, (nonsense mutation) by insert glutamine permitting
gene transcription. This allows λ1098 Tn10Tc replicate genes to be produced. λ1098 Tn10Tc
can now go through the lytic phase thus permitting preparation of phage lysate. E. coli CSH140
Su- (no supE gene) is used in the transposition experiment. Since λ1098 has nonsense mutations
(UAG codon) in the replication genes, λ1098 cannot replicate in E. coli. This is essential for the
transposition experiment as want only transposition of λ1098 Tn10Tc, not replication or
lysogeny. Lysogeny is prevented by incubating the transposition mixture at 39.5oC.
17
Way JC, Davis MA, Morisato D, Roberts, DE, Kleckner, N. 1984. New Tn10 derivatives for transposon
mutagenesis and construction of lacZ operon fusions by transposition. Gene. 32: 369-379.
51
Strain List
Strain
Genotype
Important
Properties/Phenotype
E coli CSH110
ara Δ(gpt-lac5) supE gyrA argEam metB rpoB
Su+
E. coli CSH140
F'lac+proA+,B+ (carries I- mutation in the lac
region) ara Δ(gpt-lac)5
Su- I-Z+
λ1098 Tn10Tc
mini- Tn10Tc
make clear plaques at
39.5oC on supE E. coli
strains
Sinorhizobium meliloti
Rm10213
SU47 Str-21
Smr
E. coli
MM294A(pRK602)3
pro-82-thi-1 hsdR17 supE4 Tn5
Camr Nmr
Cam, chloramphenicol (Tn5); Nm, neomycin (Tn5); Sm, streptomycin, hsdR1 host
specificity/DNA restriction
Reminder: strains are sensitive to antibiotics unless otherwise stated.
PROCEDURE
Week 8
Part 1 Tn5 Transposition via carrier pRK602 plasmid
1. Donor, E. coli MM294A/pRK602 was grown overnight with rotation at 37oC in LBCam
broth (5 µg/ml chloramphenicol). Recipient, Sinorhizobium meliloti Rm1021 was grown
overnight with rotation at 30oC in LBSm broth (200 µg/ml streptomycin).
STUDENT LAB STARTS HERE
2. Transfer 1 ml of E. coli MM294A/pRK602 to an Eppendorf tube. Transfer 1 ml
Sinorhizobium meliloti Rm1021 to another Eppendorf tube.
3. Micro centrifuge each for 1 min (max speed). For each, remove supernatant with P1000,
being careful not to disturb the pellet. Resuspend each pellet in 1 ml saline. Use P1000 to
resuspend by carefully drawing pellet up and down in tip. Again micro centrifuge for 1 min.
Remove supernatant.
4. To the tube containing E. coli MM294A/pRK602 pellet add 500 µl saline. Suspend pellet.
Transfer E. coli MM294A/pRK602 suspension to tube containing Sinorhizobium meliloti
Rm1021 pellet. Suspend pellet.
5. Carefully spot 100 μl bacterial suspension in the centre of a non-selective LB plate. Keep the
spot as small as possible.
3
supplied Dr. I. Oresnick’s lab
52
6. Incubate upright at 30oC overnight.
7. Negative Controls: Using a marker to divide the bottom of a selection plate, LBSmNm, into
two sections. Use a sterile disposable loop to streak plate each parent in a section. Allow to dry.
Incubate upside down at 30oC for 2 days. The expected result is no growth or only a few
scattered colonies (considered no growth). Record results and discard control plate.
8. Next day, Add 1 ml saline solution to bacterial spot and mix with disposable sterile loop.
Transfer suspension to sterile 5” metal capped test tube.
9. Dilute 10-1 (0.1 ml spot suspension + 0.9 ml saline).Spread plate 0.1 ml of undiluted and 10-1
dilution on LBSmNm in duplicate. Incubate at 30oC for 5 days –for this incubation only just
tape plates together not using containers due to limited space. Lab room 204 students need to
remove plates from incubator Tuesday morning – if necessary transfer to small 30oC in
lab room 201, next door. This incubator is being reset Tuesday pm to temperature required for
the next lab (not 30oC).
10. Monday: Pick plate up to 100 colonies using two M9glucoseXgalNm agar plates (use the
plate grid supplied in lab 4). If you have less, pick as many colonies as you have. Incubate 2-3
days at 30oC.
11. Count the total number of white colonies and total number of blue colonies (if any part of
the pick is blue consider this a blue colony). Record results on transposition data sheet. Hand in
with Week 9 results.
Week 9
Part II: Tn10Tc Transposition via carrier λ1098Tn10Tc
1. Prior to lab λ1098 Tn10Tc phage lysate was prepared by infecting host E. coli CSH110. After
infection, cells were plated in a layer of soft agar on LB plates to allow confluent host lysis over
the surface of the plate due to overlapping plaques (incubation overnight at 37oC). Remaining
steps same as P1 lysate preparation.
2. E. coli CSH140 was subcultured (1/50 dilution) into 5 ml LB and rotated for 4 h at 37oC (two
per group). The culture cell density is 3 x 108 cells/ml.
STUDENT LAB STARTS HERE
3. Each group takes two 5 ml E. coli CSH140 LB cultures. Pour both cultures into 1 centrifuge
tube. Centrifuge culture at 10000 rpm for 5 min at 4oC. Resuspend the pellet in 1 ml LB
containing 10 mM MgSO4.
a) First, remove 0.1 ml E. coli CSH140 and spread plate on one LB + Tc agar plate (negative
control).
b) For transduction the acceptable multiplicity of infection (m.o.i.) is 0.3 to10. Add 100 µl
λ1098 (titre = 4 x 1010 pfu/ml). Note: The volume of λ1098 should be no greater than 1/10 (100
μl) of the bacteria added.
53
4. Incubate in a 37oC waterbath for 15 min to allow phage adsorption. Add 1 ml LB and
continue incubating in the 37oC waterbath for 90 min to allow expression of antibiotic
resistance gene.
5. Spread plate 0.1 ml of 100, 10-1, 10-2, and 10-3 dilutions (prepared in total volume 1 ml saline)
in duplicate on LBTc agar plates.
6. Incubate overnight at 39.5oC. λ1098 has a temperature sensitive repressor. At temperatures
between 39oC and 42oC the repressor is inactivated preventing the formation of lysogens.
7. Record plate count data of tetracycline resistant colonies. These colonies are mini-Tn10
carriers.
8. Pick plate up to 100 colonies using two M9glucoseXgalTc agar plates. If you have less, pick
as many colonies as you have. Incubate 1 day at 37oC.
9. Count the total number of white colonies and total number of blue colonies.
10. Hand in a COPY of your group’s completed Transposition DATA SHEET (both plasmid
and phage transposition experiments) to the slotted filing cabinet located in the hallway across
from 302 Buller. May be hand written. Include all requested information. Hand in data or
emailed to [email protected] by 2:30 pm Friday. No Honesty Declaration
required for data submission. The data will be compiled and class data posted on the website
(Excel format) as soon as possible. Marks subtracted from report if data is not submitted as
requested.
λ1098 Tn10Tc transposition sample calculations
(find required information in procedure)
Total E. coli CSH140 in transposition mixture.
E. coli CSH140 titre = 3.0 x 108 bacteria/ml
10 ml cultured centrifuged, resuspended in 1ml magnesium sulfate
bacteria/ml x 10 ml = 3.0 x 109 bacteria/ml
remove 0.1 ml therefore 0.9 ml remain; 0.9 x 3 x 109 = 2.7 x 109 bacteria/ml
Sample calculations of MOI if add 100 µl (1/10 volume) λ1098Tn10Tc to E. coli CSH140 in
transposition mixture. MOI range must be between 0.3 and 10.
This is 1/10 volume since 0.9 ml E. coli CSH140 remains after centrifugation. There is removal
of 0.1 ml for negative control and 0.1 ml λ1098 added = total volume 1 ml.
λ1098Tn10Tc titre is 4.0 x 1010 pfu/ml: 0.1 ml added to transformation mixture = 4.0 x 109 pfu
added to 2.7 x 109 bacteria
moi = 4 x 109 pfu / 2.7 x 109 bacteria = 1.5
Sample calculation to determine VOLUME of phage stock to add if want a specific MOI,
e.g. 0.5
λ1098Tn10Tc titre is 4.0 x 1010 pfu/ml
There are 2.7 x 109 bacteria in the transposition mixture
Then 0.5 (moi) * 2.7 x 109 = 1.35 x 108 pfu needs to be added to transposition mixture.
1.35 x 108 pfu = 0.034 ml
4.0 x 1010 pfu/ml
54
Molecular Genetics of Prokaryotes MBIO 4600
Lab 5
Transposition Mutagenesis DATA SHEET - available as word document on
lab website
(only one copy per group is required)
No honesty declaration required for data submission
Group #: _______________
Group names (in full): ________________________________________________________
Transposition mutagenesis data
Transposon carrier
via carrier λ1098Tn10Tc
plated on M9glucoseXgalTc
via carrier pRK602 plasmid
plated on M9glucoseXgalNm
total # blue colonies
total # white colonies
55
LAB 5 REPORT
(Use Excel spreadsheet, contains two worksheets, see tab below each worksheet). See Excel
information (lab 4).Excel Format available on lab website. Save format and open in Excel
before typing requested information.)
Data Presentation and Analysis
4
1. Include a completed group Excel worksheet for λ1098 mini-Tn10Tc transposition data into E.
coli CSH140 and requested calculations; transposition titre, total transposition reaction volume
and transposition insertion frequency. Fit all requested information including numerical
calculations on one portrait page.
Transposon insertion frequency = total number of tetracycline resistant bacteria in the reaction mixturea
total number of phage added to the reaction mixture
When calculating the total number of tetracycline resistant bacteria in the reaction mixture you
need to know the total transposition reaction volume. This allows you to correlate to total
number of phage.
2
2. Include a completed class data Excel worksheet for transposition into the lac gene
experiments. Record all requested information and sample calculation. Highlight or state your
group number. Fit all requested information including numerical calculation and question on
one portrait page.
Frequency of insertion = total Lac- colonies
total viable colonies picked
Question (Inserted at the bottom of Excel class worksheet)
No explanations required.
1. Circle, underline or highlight the best E. coli strain for a λ1098 transposition into the lac
genome experiment as performed in the MBIO 4600 lab.
CSH114 ara Δ(gpt-lac)5 rpsL mutT
CSH111 ara gyrA argE metB rpoB
CSH112 ara leu lacY purE gal supE try his argG malA rpsL xyl mtl ilv metA
CSH123 relA1 spoT1 thiA::Tn10
__
7
56
APPENDIX
MEDIA
LB (Luria-Bertani) Medium: dissolve 10 g Bacto-tryptone, 5 g yeast extract, and 5 g NaCl in
800 ml distilled water. Adjust to pH 7.5 with NaOH and bring up volume to 1 litre with
distilled water. For agar plates add 15 agar/litre. Add 7 g agar/litre for top LB agar.
LB medium is a complex media that supplies all essential nutrients, C-source, N-source,
vitamins, minerals and trace metals. Yeast extract supplies all essential nutrients while tryptone
is mainly a N-source and to a lesser degree a C-source.
M9 Medium Agar plates: All plates are prepared with the final concentration per litre.
Autoclave agar and salts separately. Salts; 10.5 g K2HPO4, 4.5 g KH2PO4, 1.0 g (NH4)2SO4,
and sodium citrate.2H2O. 15 g/l agar. After autoclaving add cooled MgSO4.7H2O (add 1 ml
from a stock solution of 20g/100 ml) and approximately 1 μg/ml B1 (thiamine hydrochloride add 0.5 ml from a 1% stock solution). 10 ml carbon source is added from a 20% stock solution.
Some type of carbon source must be added, for example, the most common carbon source
added is glucose (assume if not stated). This is the basic M9 medium provided unless otherwise
stated in experiment.
If the medium requires additional nutrients, add 20 μg/ml L-amino acids or 40 μg/ml
D,L-amino acids (add 10 ml from a 4 mg/ml stock solution). Amino acid is not essential unless
the bacterium is an auxotroph for a particular amino acid. If the medium requires, add
nucleotide, i.e. if bacteria is a nucleotide auxotroph.
Xgal M9Glucose IPTG agar plates: Prepare M9 glucose agar. After autoclaving, add Xgal (5
bromo -4- chloro - 3 indolyl b D galactoside) in N,N-dimethylformamide at a final
concentration of 40 μg/ml and IPTG (Isopropyl β-DThiogalactoside) at a final concentration of
24 μg/ml.
M9 medium is a defined medium. All components are known. This allows you to select for
desired bacteria by adding or removing nutrient of interest.
F-top agar: per litre, 8 g Difco agar, 8 g NaCl.
MacConkey Agar plates: Prepared medium purchased from Difco Laboratories. Components:
peptone, poly peptone, lactose, bile salts, sodium chloride, agar, neutral red, crystal violet,
distilled water. pH 7.1
Stock solutions of Antibiotics:
Streptomycin (Sm): prepare 10 mg/ml stock solution in distilled water, filter sterilize. Add to
cooling agar at a final concentration of 100 μg/ml.
Tetracycline (Tc): prepare a 1 mg/ml stock solution in distilled water, filter sterilize. Add to
cooling agar at a final concentration of 10 μg/ml.
Ampicillin (Amp): prepare 10 mg/ml stock solution in distilled water, filter sterilize. Add to
cooling agar at a final concentration of 100 μg/ml.
57
SOLUTION COMPONENTS AND FUNCTION
Saline: dissolve 8.5 g NaCl in a total volume of 1 litre distilled water 0.85% saline is
physiological concentration, i.e., isotonic. Maintains the stability of bacteria, i.e., do not lyse.
Addition of Mg2+ or Ca2+ during phage lysate, titration or infection of host bacterium: enhance
adsorption of phage on host bacteria.
λ phage adsorb to trimeric maltoporin receptors18. Magnesium facilitates the reversible
attachment of the phage tail to the maltoporin receptor. Once attached the λ DNA is injected
into the host bacterium. Of interest neither injection or lytic phase occur at room temperature,
must be greater than 28oC.
P1 similar to λ phage require divalent cations for optimum adsorption. However, P1 adsorption
is facilitated by calcium and to a lesser degree, magnesium.
Magnesium or calcium is often added to LB medium during growth of host bacterium to
increase subsequent adsorption of phage. It is common practise to add vitamin free casamino
acids to medium to enhance phage growth.
Chloroform during preparation and storage of phage lysate: Chloroform lyses bacteria. During
lysate preparation host E. coli cells not yet lysed by infecting bacteriophage are lysed by
chloroform releasing remaining phage to give the maximum number of phage. Chloroform is
added during storage (as at 4oC) to prevent bacteria growth (lyse) especially resistant bacteria.
QIAGEN Plasmid DNA preparation:
Cell Suspension Buffer P1: 150 mM Tris-HCl, pH 8.0, 10 mM EDTA, 100 μg/ml RNase A
-centrifuge to remove media components, and resuspend in buffer to give a homogenous
suspension of bacteria cells that is appropriate for lysis of cells in the next step.
150 mM Tris-HCl, pH 8.0 optimum ionic and pH for stability of cells- DNA is more soluble at
pH 8.0.
10 mM EDTA - chelates divalent cations may be involved in initial destabilization of cell walls
but does not lyse the cells, available divalent cations removed from cell surface. Inhibits the
activity of nucleases (DNase).
100 μg/ml RNase A - degrades RNA, really required for next step, cell lysis, to degrade RNA
released from the cell. Usually added at 1 μg/ml or less.
Cell Lysis Solution P2:
0.2 M NaOH, 1% SDS
Lysis occurs under controlled conditions; the cell membrane should remain attached to the
genomic DNA so when the alkaline solution is neutralized with potassium acetate the cells
debris traps the genomic DNA and is precipitated out of solution
0.2 M NaOH - alkaline lysis of cells, also degrades DNA to single strands, both genomic and
plasmid DNA
1% SDS - dissolves cell membranes, lysis of cell, solubilizes phospholipids
Neutralizing Buffer N3: contains high concentration potassium acetate, pH 4.8 and
guanidine hydrochloride
High concentration of potassium acetate at pH 4.8 precipitates protein, neutralizes alkaline
conditions and adjusts the cleared lysate to high salt required for bind of DNA to the silica
membrane. As stated above the precipitating protein traps other cell debris including degraded
18
Sambrook, J, Russell, DW. 2001. Chapter 13 Mutagenesis. In Molecular Cloning A Laboratory Manual
3 edition. Cold Spring Harbor: CSHL Press p. 2.4
rd
58
genomic DNA. Genomic DNA cannot reanneal but the closed circular plasmid DNA can
reanneal as attached. The plasmid DNA is release in solution. This solution also contains
guanidine hydrochloride which denatures proteins, inhibits DNase activity and enhances
binding of the DNA to the silica membrane.
Spin column
- spin column contains silica based membranes that selectively bind plasmid DNA at high salts
and pH #7.5. Polar stationary phase, sieves - selectively retains (trapped) range of DNA wanted.
Wash Buffer PB: contains acetate, guanidine hydrochloride, EDTA and isopropanol
-second chance at destroying any remaining nuclease activity. DNA remains attached to spin
column, removes any contaminants soluble in isopropanol.
Wash Buffer PE (200 mM NaCl, 20 mM Tris-HCl, pH 7.5, dilute 1:1 with 95% EtOH)
removes impurities (nucleotides, proteins, etc), while not removing the DNA bound to the resin
200 mM NaCl - stablility of DNA, high salt DNA remains bound to spin column silica
membrane
20 mM Tris-HCl, pH 7.5 - optimum ionic strength and pH
5 mM EDTA - prevents degradation of DNA, chelates divalent cations and prevents nuclease
activity as requires divalent cations.
dilute 1:1 with 95% EtOH - solubilizes salts etc but not the DNA on resin
EB buffer (10 mM Tris-HCl, pH 8.5)
-elutes DNA from resin. It is important that the pH be alkali to efficiently remove the plasmid
DNA from the resin. Ensure stability of DNA. DNA is more soluble at pH 8.5 while DNA
remains stable. Tris base buffers at required pH. ddH2O is often used to elute the plasmid DNA
if required for PCR or sequencing, albeit with slightly reduced plasmid concentration.
59
PIPETMAN OPERATION (Accurate operation temperature 4oC to 40oC.)
Pipetmen that may be available in your lab are P2, P10, P20, P200 and P100. Look at the top of
the plunger to see the size of the pipetman.
P2 measures accurately from 0.2 μl to 2 μl.
P10 measures accurately from 1 μl to 10 μl.
P20 measures accurately from 2 μl to 20 μl.
P200 measures accurately from 20 μl to 200 μl.
P1000 measures accurately from 100 μl to 1000
μl.
Never turn the Pipetman above the
maximum volume - breaks the Pipetman.
Setting the Volume
The volume window for all sizes of Pipetmen
consists of three numbers stacked vertically read
top down. Numbers are black or red (decimal). The volume is set using the thumbwheel or the
push-button (easier when wearing gloves). If increasing volume value, need to go 1/3 turn
higher, then decrease to reach the desired volume. Caution, never turn above maximum volume
for each Pipetman.
Volume Display:
P2
P10
1
0
7
2
5
5
1.25 μl
7.5 μl
P20
1
2
5
12.5 μl
P200
1
0
0
100 μl
P1000
0
9
0
0.9 ml or 900 μl
Pipetting
Sterile tip boxes are available in two sizes, small for P2,
P10, P20 and P200 and large for P1000 (blue). Open,
remove tip and close lid. It is important to keep lid closed
when not removing a tip to ensure sterile tips.
1. Fit the tip to the Pipetman by pushing the tip holder
into the tip using a slight twisting motion.
2. Pre-rinse tip by pipetting up and down. Press the
push-button to the first stop (see diagram). Hold the
pipette vertically and immerse the tip in the liquid,
1 mm for P2 & P10, 2-3 mm for P20, 2-4 mm for
P200 & P1000.Wait 1 second. Remove. Check that
there are no droplets on the outside of tip. Visually
check volume to ensure expected volume.
3. Place the end of the tip against the inside wall of the
tube (10o to 40o angle). Press the push-button slowly and smoothly to first stop, wait 1
second, then press the bush-button to the second stop to expel any residual liquid from the
tip along the inside of tube. Release push-button smoothly and eject tip into plastic lined
waste bucket on bench top by pressing firmly on the tip-ejector button.
60
OPERATION OF REFRIGERATED CENTRIFUGES
Note: If procedure varies depending on centrifuge manufacturer a step by step operation
procedure is usually located on or nearby the centrifuge or the teaching assistant will help you.
HITACHI HIGH SPEED HIMAC REFRIGERATED CENTRIFUGE
1.
to select or change settings the CHECK button must first be pressed (light on). The light
stays on for 16 sec. When the light is off you can no longer select, change setting or carry
out any operation, just press check button again and continue.
2.
When the centrifuge is turned on and the CHECK button is not pressed. The centrifuge
displays real time parameters.
OPERATION
Centrifuge tubes should be balanced by scale by adding or removing appropriate solution from
one of the tubes.
1.
Turn power switch on. The indicators on the control panel are illuminated. The door lock
is released.
2.
Open door. If required set the rotor gently in position and close door. Turn the rotor lightly
by hand to check that the rotor is correctly set. Remove the rotor lid and place balanced
tubes opposite each other in rotor. You cannot run the centrifuge with an odd number of
tubes. SCREW ON LID.
3.
Call up memory code number or enter parameters.
Call up pre-programmed memory code number: Press CHECK button, MEMORY button,
memory code number, and CALL button. Each memory code number consists of a
specified set of operation parameter (see sheet on centrifuge cover). See below for a list of
operation parameters and how to set and store operation parameters.
OR
Real time operation (enter original parameters): see setting of operation parameters below.
4.
After the parameters are set make sure the check light is still on. If not, press the
CHECK button.
5.
Press the START button. The rotor starts running. The start lamp begins flashing. The
timer starts to count down.
6.
The timer counts down to zero or press the STOP button. The rotor begins to decelerate.
The stop light begins flashing.
7.
The rotor stops. The stop light stops flashing. A buzzer sound occurs. The door lock is
released.
8.
Unscrew rotor lid and remove tubes. If required, use tweezers to help remove tubes. Wipe
out rotor if spills occur. DO NOT SCREW ON THE LID just place on top of the rotor.
9. Close centrifuge lid and turn off power.
61
Sorvall Legend X1R Centrifuge operation (room 302 and 304).
For Fiberlite F15-8x50cy rotor. (50 ml centrifuge tubes or conical tubes)
1. Turn on the power switch on the back left side. The centrifuge does a self check. When lid is
closed the display shows speed, time and temperature of the current conditions. Bottom line
shows the values for the last run. When lid is opened, the top line shows LID OPEN and the
bottom line shows values for the last run.
2. Press open key. Lid opens automatically – do not open manually.
3. If rotor needs to
be loaded, place
rotor over centre
spindle and let slide
down – rotor clicks
into place. Check
that it is secure by
trying to pull up on
the handle –must be
secure. Do this each
time before use to
ensure rotor in
placed correctly.
4. Press ACC DEC
to set acceleration
and braking – range
1 – 9, 1 being the
slowest and 9 the
fastest. Select
middle range.
62
5. Press SPEED key to set displays either rpm or rcf (g force, relative centrifugal force), want
rpm, toggle with toggle key to rpm. Enter speed using numeric pad. Press ENTER.
6. Press TIME key. Enter the time using numeric key pad. Press ENTER. (Pressing the TIME
again toggles between mm:ss and hold. [When centrifuge is running press toggle key between
time remaining and total time. Time starts once the speed is reached.
7. Press TEMP key, press toggle key for AIR or SAMPLE temperature, set to AIR. Enter
temperature, 4oC using numeric pad, press ENTER. If you want to precool the centrifuge, press
TEMP key for minimum of 3 sec to open the temperature selection menu. Display shows “Set
PreTemp”. Enter desired temperature and press enter. Display shows “Press start
4”. Press
the START key resulting in the rotor chamber being cooled to 4 oC. Press STOP key, display
shows current temperature.]
8. Balance tubes and put in rotor (remove and replace rotor lid by unscrewing or screwing)
opposite each other. Replace rotor lid.
9. Close the centrifuge lid by pressing down lightly in middle or both sides. One lock closes the
lid completely.
10. Press START key.
11. After the centrifuge the END key will illuminate. Press OPEN key, the lid opens
automatically. Unscrew lid and remove centrifuge tubes.
12. To remove the rotor, grab the rotor hand with both hands and press against the green
AutoLock™ key. At the same time pull the rotor straight upwards with both hands removing it
from the centrifuge spindle.
Note: If using the Bucket rotor (either 15 ml or 50 ml conical tubes)
must also input bucket information.
Press the BUCKET key. Press the BUCKET key repeated until the bucket being used is
displayed – the one we have is Cat. No. 75003655 for TX-400 Swing Bucket Rotor. Rmax 168
mm (believe this is the radius). Cannot find an instruction manual for this rotor. Press Enter.
Open radius imput menu. Enter a different radium assume 168 mm is maximum. Press Enter.
63
AUTOMATIC COLONY COUNTER
There are several makes of automatic colony counters in this department. All automatic colony
counters work on the same principle. The counter registers a count every time you touch the
colony with the counter probe as long as the L-shaped probe in inserted into the agar at the edge
of the plate. This completes the electrical circuit through the agar from the L-shaped probe to the
counter probe (needle shaped probe) touching the colony.
Operation
1.
2.
3.
4.
5.
6.
7.
Push or flip the power switch to turn on counter.
Press the button on the counter that resets the counter to zero.
Place agar culture plate on counter and remove cover.
Insert L-shaped probe into the agar at the edge of the plate.
Count colonies by touching each colony with the counter probe tip (needle shaped probe).
Remove plate, replace lid.
Remember to turn off power switch when you are finished counting.
Notes:
(i) Use a marker to divide the plate into sections or use the grid on the automatic colony counter
to facilitate counting.
(ii) The counter also comes with a magnifying glass but it is not required unless you are counting
very small closely spaced colonies.
64
Determination of Viable Cells
bacteria/ml [cfu/ml] or phage/ml [pfu/ml]
Significant Colony Counts
Traditionally the accepted colony count range is 30 to 300 or sometimes 25 – 2504. Although counts
greater than 300 are statistically significant, competition for nutrients between the closely spaced colonies
may result in not all viable cells showing visible colonies. The upper limit “should be set by
microbiologist based on knowledge and experience with the microbes under study”5. Since E. coli
colonies are small discrete colonies and when non-overlapping colonies greater than 300 are accurately
counted they are acceptable for titer determination. The lower limit of 30 is considered by many
microbiologists to be taken only as a recommendation – “it is foolish to disregard colony counts below 30
if they happen to be the only ones available.”6. For example, in cases of inhibition studies or genetic
studies the only colony counts available are often below 30 counts. Lower counts are biologically reliable
as there are no interaction between colonies preventing growth albeit concerns about statistically
reliability. If only counts below 30 are available for titer determination, select only the highest dilution
and footnote only counts available. For example, only counts available are duplicate counts at 10-5
dilution; 23 & 26 and 10-6 dilution; 4 &1. Use 10-5 dilution counts but not 10-6 dilution. For 3rd and 4th
year Microbiology lab when determining the titer of E. coli use all count ≥30 for accurately counted
colonies remembering that colonies greater than 300 must not overlap. If there are no counts ≥30, then
use only the greatest value counts.
.
Data for example calculations
Dilution plated
Number of colonies
Plate 1
Plate 2
10-2
TNTC
TNTC
10-3
320a
316
10-4
34
27
10-5
2
3
TNTC = too numerous to count
a
counts greater than 300 are acceptable as long as accurately counted and bacteria that produces small discreet
colonies. All counts recorded in your tables must be accurate counts or record as TNTC.
Terms
Plating factor = reciprocal of volume plated
Dilution factor = reciprocal of dilution for significant counts
Significant plate counts = the sum of the plate counts at significant dilution divided by number of
significant plates.
Accurate counts = number of non-overlapping colonies
4
FDA, On-going. Bacteriological Analytical Manual. Available at (http://www.cfsan.fda.gov/~ebam/bam-3/html).
Center for Biofilm Engineering. Hamilton MA & AE Parker. Enumerating viable cells by pooling counts for
several dilutions. Available at (http://www.biofilm.montana.edu/files/CBE/documents/KSA-SM-06.pdf).
6
Niemela, S. 1993. Statistical evaluation of results from quantitative microbiology examinations. NMKL Report no.
1, 2nd edition. Nordic Committee on Food Analysis. Ord & Form AB, Uppsala.
5
65
Titer Calculation
Titre must be in scientific format.
Do not average an average value as it incorporates error in your calculation (not statistically accurate)
especially if an odd number of significant plate counts. Use one of the following methods to calculate
bacteria titer.
Method 1: Bring all significant counts to the same dilution.
For this sample data, there are 34 counts at 10-4 dilution to bring to 10-3 just multiply by 10 = 340 counts
at 10-3.
Bacteria/ ml
=
significant plate counts x dilution factor
number of plates
x plating factor
(320 + 316 + 340)/3 x 1/10-3 x 1/10-1 = 3.25 x 106 bacteria/ml rounded to 3.3 x 106 cfu/ml
Since the smallest number of significant figures for plate counts is two, the answer is 3.3 x 106 bacteria/ml
or cfu/ml (colony forming units/ml).
Method 2: Calculate the titer for each significant plate count and average.
Bacteria/ml = significant plate count
x
dilution factor x plating factor
320 x 1/10-3 x 1/10-1 = 3.20 x 106 bacteria/ml
316 x 1/10-3 x 1/10-1 = 3.16 x 106 bacteria/ml
34 x 1/10-4 x 1/10-1 = 3.4 x 106 bacteria/ml
Average all values: (3.20 x 106 + 3.16 x 106 + 3.4 x 106)/3 = 3.25 x 106 bacteria/ml, since the smallest
number of significant figures for plate counts is two, the answer is 3.3 x 106 bacteria/ml or cfu/ml
Notes
(i) If the significant plate counts are all 3 digits, then the titre value should have 2 decimal places (total 3
digits).
(ii) If the titer is to be used for further calculations, do not round to significant figures until the final value.
(iii) Calculation of phage titre is identical to bacteria titre. Express in units of pfu/ml or phage/ml.
pfu = plaque forming units. pfu <30 may be used for rapid calculation method.
66
Outlier plate counts
When plating microorganisms, there should be a difference of 10-fold colony counts between 10fold dilutions. This is especially difficult to obtain for P1 dilutions.
What is an outlier? Any significant plate count (≥30) that does not follow expected pattern (10fold difference between dilution and duplicate samples similar) is an anomaly. The following is
an example of outlier data. For Plate 1 10-6 dilution and Plate 2 10-7 dilution do not include plate
count values in titer calculation. State outlier values as footnote in your data table.
Dilution plated
Number of colonies (0.1 ml of each
dilution plate
Plate 1
Plate 2
10-2
TNTC
TNTC
10-3
320
316
10-4
44
57
10-5
2
24
10-6
45
1
10-7
0
TNTC = too numerous to count
33
67
FINAL LAB EXAM: Microbiology MBIO 4600 MOLECULAR GENETICS OF PROKARYOTES
DATE: sample
TIME: 1.5 h
INSTRUCTOR: Dr. L. Cameron
PAGE: ___________
WRITE IN PEN ONLY.
CONCISELY ANSWER ALL QUESTIONS in spaces provided on exam paper.
Exam is longer than actual to demonstrate a wide selection of question types.
Also question type similar to lab report questions given on lab exam.
(spaces have been removed for sample exam)
2
1. Explain the following E. coli strain list and state phenotype.
E. coli Strain
Genotype
CSH143
F’lacproA+ ,B+;
ara, Î(gpt-lac)5, argE
pro = proline; ara = arabinose; argE = arginine
1
1
2. a) State the phenotype and most likely I and Z genotype of an E. coli strain that has pale blue
colonies on M9glucose Xgal and red colonies on MacConkey.
b) E. coli CSH100 genotype includes IQP- mutations in the lac region. Explain genotype at the
molecular level and state expected phenotypic expression on M9glucose Xgal.
6
3. State the chemical that corresponds to the following functions as relates to your MBIO 4600
lab.
a) movement of DNA during agarose gel electrophoresis _____________________
b) prevents the adsorption of P1 phage _____________________
c) maintains physiological ionic conditions ___________________
d) ensures β-galactosidase is produced at maximum levels _______________
e) preparation of competent E. coli ________________
f) added during phage lysate preparation to further disrupt host cells _________________
1
4. a) In your MBIO 4600 lab, F'lacproA+,B+ (carries I-Z- mutations in the lac region) ara Δ(gptlac)5 factor transfer (donor E. coli CSH101) allowed lac reversion at high frequency. Explain
why at the molecular level.
1
b) State two important features of pUFR-GFP that facilitate conjugation with E. coli CSH125.
1
c) Present a labelled papillae colony for a Lac- reversion. In your diagram make it obvious what
is the original color of the colony and selection medium.
1
5. The X-gal detection system permits detection of recombinant plasmids. Explain why it is
important that the host E. coli chromosome has both Δ(argF-lacZYA) and Φ80 lacZÎM15.
68
1.5
6. a) Plasmid DNA concentration was found to be 120 ng/µl with a A260A/280 0f 1.75. In the
MBIO4600 lab what instrument is used to determine concentration. Outline how sample is
placed in the instrument for concentration measurement. Convert concentration to µg/µl.
Comment on A260/A280 ratio. How is the instrument cleaned before turning off?
1
b) Determine # transformants/μg plasmid for the following data. 0.05 μg pBluescript added to
transformation mixture. Ampicillin colony titre 6.2 x 104 bacteria/ml. Transformation mixture
total volume plated = 1.2 ml.
answer: 1.5 x 106 transformants/μg plasmid
5
7. Explain the function of each of the following procedure steps/chemicals used in your
molecular genetics lab.
a) Plasmid DNA preparation solution containing 0.2 M NaOH, 1% SDS
b) incubation of λ1098 and CSH140 transposition mixture at 39.5oC
c) centrifugation of all molecular genetics lab E. coli cultures at 4oC
d) Plasmid DNA preparation solution containing high concentration potassium acetate, pH 4.8
and guanidine hydrochloride
e) neutral red in MacConkey agar
2
8. a) What is the advantage of P1 transduction when creating new E. coli strains?
b) What improved features does mini-Tn10 have compared to Tn10?
1.5
9. a) Diagram the Sub-Cell® GT Mini agarose gel electrophoresis system set up just before
pouring agarose gel.
1.5
b) Explain the three function of EZ-Vision.
1
10. a) Explain why plasmid, E. coli MM294A/pRK602 is a “suicide” plasmid with respect to the
transposition of Sinorhizobium meliloti Rm102.
1
b) What component of the transposition selection medium prevents the growth of recipient
Sinorhizobium meliloti Rm102 and donor E. coli MM294A/pRK602?
69
3
11. Experimental Data: Ten ml log phase culture of E. coli CSH140 (2 x 108 bacterial/ml) was
centrifuged and resuspended in 1 ml LB.Mg broth. 0.1 ml E. coli CSH140 removed to plate as
negataive control. λ1098 (stock titre 6.8 x 1010 phage/ml) added at a multiplicity of infection of
2. Mixture incubated at 37oC for 15 min. One ml LB broth added and incubated a further 90
min at 37oC. Dilutions of incubation mixture prepared and 0.1 ml dilutions plated on LB agar
plates containing tetracycline. The following plate count data was obtained for duplicate plating
at each dilution: 10-1: 250, 246, 10-2: 22, 28, and 10-3: 1, 0.
Class results for pick plating tetracycline resistant colonies onto glucose minimal medium
containing Xgal and tetracycline resulted in 4 white colonies and 3996 blue colonies.
(a) State bacteria/ml for titration data.
answer: 2.48 x 104 bacteria/ml
(b) Calculate volume of λ1098 phage stock added to E. coli CSH140 to give a multiplicity of 2.
answer: 52.9 μl
(c) Calculate the transposon insertion rate into E. coli using phage λ1098.
answer: 1.35 x 10-5
(d) Calculate transposon frequency insertion into the Lac gene.
answer: 0.001
- END -