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Team members
Results & Modeling
Lin, Yi-Ting _Department of Molecular Biology and Human Genetics, MBHG
Shan, Zhe_MBHG
Hsieh, Siang-En _MBHG
Chen, Shih-Jie _MBHG
Chen, Yu-Ting _MBHG
Lien, Pei-Yi_MBHG
Hsu, Hao_Department of Medical Informatics
We constructed our phagemid with a RFP as reporter gene (shown in Fig.1) and use this to monitor our experiments.
The experimental result show phagemid-carrying phage is successfully released (shown in Fig.2 and Fig.3). Then we want
to figure out two efficiency question in our experiment ： How to make the most amount of phagemid-carrying phage
released, and how to make their infection rate achieve 100%.(Shown as Model A and Model B)
Introduction
This year, TCU_Taiwan provide a new tool dealing with bacterial antibiotic resistance ---- the
Trogene Horse. Our Trogene Horse can produce one-generation phage carrying CRISPR system
targeting bacterial antibiotic resistance gene.
To achieve this, we use a special phagemid for phage display. The phagemid contains a
additional phage replication origin. When helper phage (a phage whose own ori is knocked out)
infection takes place, phagemid can be packaged as genome for progeny. Since we put an antibiotic
resistance gene targeting CRISPR system on our phagemid, this phage display can spread our
CRISPR among bacterium nearby and remove their antibiotic resistance efficiently.
Trogene Horse is a new concept combining phage therapy with CRISPR, and this
combination can solve both safety concern(phage therapy) and efficiency problem(CRISPR).
We believe our project can deal with bacterial resistance problems both
in agriculture and medicine.
Filamentous phage life cycle
Figure 1. Shows that
construction of pBluescript
with reporter gene RFP.
System Design
Figure 3. Shows that use
ten fold serial dilution
before dropping on the
plate for six duplicate.
Minimized
crRNA
terminator
pRha
promoter
Tet-off
promoter
×1010
T1
terminator
pfu/ml
tracrRNA
Figure 2. Shows that use
phage titer determination
experiment on agar plate.
Phagemid
pBluescript II
SK(-)
M13KO7
Helper phage
Incubating time
Second structure
of package signal
Filamentous phage can only infect bacteria with F pili. Its life cycle described in following steps:
Adsorption: In this step, M13 and M13KO7 are the same. Both of their pilus attachment protein
gp3 and gp6 will help them bind on F pilus, then the phage will move into host cell by F pilus and
take off its coat protein.
Replication: After infection, both kind of phage’s ssDNA genome will act like a template and use
host bacteria’s DNA polymerase to generate replicative form(RF). So the RF can use host’s enzyme
to express its genes such as coat protein.
Assembly: Then the RF will make gp2 first, this protein can make a nick on intergenic(IG) region of
positive strand. A normal M13 phage contains IG region on its f1 ori. But a M13KO7’s f1 ori has
been mutated, so gp2 will not work. However, if a phagemid pBluescript, which carries an additional
f1 ori, is transformed into the bacteria, gp2 can make a nick on phagemid’s positive strand. The
positive strand will then be pulled out and combined with gp5 dimers to stabilize itself. Meanwhile,
RF will make coat proteins and other construction proteins, they will move along to host bacteria’s
membrane first. Next, gp7 and gp9 will recognize packaging signal around IG (shown in following
image) and start packaging. At the same time, gp8 and other proteins will replace gp5 dimers.
Finally, a progeny phage is made and released from host bacteria’s membrane.
infection time
Model A
Result from Model A shows that if we incubate bacterium 30 minutes
after adding M13KO7 to infect them, and then add kanamycin and
incubate for additional 14hr. We will get the best release with amounts
of phagemid-carrying phage: 4 x 1010 pfu/ml.
DNA replication, Arthur Kornberg, Tania A. Baker, 1991
Shows the intergenic(IG) region of Ff phage.
Vertical elements are hairpins in the
secondary structure of the viral strand.
Team achievements
Built and documented biobrick to registry.
Experimentally validate Biobricks of our team design and construct
were as expected.
Use helper phage and phagemid to solve safety problems because
those phages cannot have next generation.
 We improve the function of BBa_K1218011 from 2013 Stanford-Brown .
 Cooperation with NCTU-Formosa Team on modeling.
Human practice
Boost synthetic biology to stores near TzuChi university
Introduce basic concept of synthetic biology
Held iGEM talk at Senior High Schools
Promote iGEM and synthetic biology to local senior high school
students
Attend iGEM conference in NCTU
Present our project
Communicate ideas with students from Taiwan and China
A small iGEM presentation at TzuChi university
Introduce the history and format of iGEM
Make the 2015 iGEM team get prepared
Question Survey
Designed Several question based on synthetic biology
Video conference with NCTU-Formosa
Discussing about modeling part
Video conference with NYMU_Taipei
Share our project and cheer up each other
Acknowledgments
Woon, Peng-Yeong, Ching, Yung-Hao, Ingrid Liu, Yen JH.
Chen, J.H., Lin, Guang-Huey, Lin, NT., Lin, Ming-Der
Model B
Result from Model B shows that as long as the
MOI is higher than 6 pfu/cfu, the infection rate can
achieve almost 100%.