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How to Assemble and Test
A Genetically Engineered Machine
In Six Weeks
iGEM Jamboree
November 3rd and 4th, 2007
Cambridge MA
Kiran, Krishna, Mukund, Navneet, Nilesh, Senthil,
Shashanka, Sugat, Sushant, Varun, Vini, Vivek
National Centre for Biological Sciences
Tata Institute of Fundamental Research
Bangalore, India
Countdown to iGEM
An all-undergraduate team, with members
from around the country, bringing together
skills from biology, physics, chemistry,
computer science, and engineering
Only 6 weeks to get something done!
-100
04.05 - 04.15
Constructs designed
-90
04.15 - 06.10
Constructs synthesized
-44
-40
-20
0
06.01
06.05 - 06.24
06.25 - 07.12
Team assembles
Preliminary measurements
Final measurements
07.14
07.15
Showtime!
Back to school
Nilesh
Shashanka
11.03
iGEM, Cambridge MA
Navneet
Vivek
Krishna
Vini
Mukund
Varun
Sushant
Senthil
Synthetic Biology:
Bottom-Up Engineering of Living Systems
Complex
devices
Simple
parts
Our Goal:
To Understand a Complex Device
By Characterizing its Simple Parts
Simple Parts:
Open Loops
Complex Device:
Closed Loops
Loop Theory
input
param
output
Open loop
p1
output
Open loop
characteristic
input
Equivalence
p1
state
param
Closed loop
Closed loop
response
state
parameter
 D Angeli, JD Ferrell & ED Sontag (2004)
Loop Theory
input
EQ
param
output
Open loop
p1
output
Open loop
characteristic
input
Equivalence
p1
state
param
Closed loop
Closed loop
response
state
parameter
 D Angeli, JD Ferrell & ED Sontag (2004)
Loop Theory
input
EQ
param
output
Open loop
p1
output
Open loop
characteristic
input
Equivalence
p1
state
param
Closed loop
Closed loop
response
state
parameter
 D Angeli, JD Ferrell & ED Sontag (2004)
Loop Theory
input
EQ
param
output
Open loop
p2
output
Open loop
characteristic
input
Equivalence
p2
state
param
Closed loop
Closed loop
response
state
parameter
 D Angeli, JD Ferrell & ED Sontag (2004)
Loop Theory
input
EQ
param
p3
output
Open loop
output
Open loop
characteristic
input
Equivalence
p3
state
param
Closed loop
Closed loop
response
state
parameter
 D Angeli, JD Ferrell & ED Sontag (2004)
The Parts-List
tet regulatory module
lac regulatory module
Inducer:
IPTG
Inducer:
aTc
lac repressor:
lacI
tet repressor:
tetR
lac promoter:
pL
tet promoter:
pT
lux regulatory module
fluorescent reporters
lux gene 1:
luxI
Inducer:
AI
cfp reporter:
C
lux gene 2:
luxR
yfp reporter:
Y
lux promoter:
pR
The Parts-List
lac regulatory module
tet regulatory module
IPTG
aTc
reporter
reporter
LacI
TetR
pLac
pTet
lux regulatory module
AI
LuxI
pX
LuxR
reporter
pR
The Main Constructs
Host Strain
Ecoli K12Z1
Sender
Closed-loop
receiver
Open-loop
receiver
p1.lacI::p2.tetR
pT.luxI.C
pR.luxR.Y
pL.luxR.Y::pR.C
K12Z1 generated b y S. Dab holkar from DH5αZ1 (Lutz & Bujard 1997)
Plasmid cloning b y C hromous Biotech
Genetic Implementation of Loops
Open loop
Closed loop
Equivalence
Experimental Protocol
S (sender)
R (receiver)
aTc
S
AI accumulates
~12h
Harvest at OD600 = 0.2
S
+R
S (sender)
-S
Imaging
R (receiver)
IPTG
R
Equilib ration
~12h
Harvest at OD600 < 0.1
R
Imaging
Fluorescence Imaging
cfp filter
yfp filter
LuxI.C
aTc
pT
LuxI.C
aTc (ng/ml)
IPTG
pL
yfp filter
LuxR.Y
cfp filter
LuxR.Y
IPTG (uM)
LuxI.C
LuxR.Y
Open-Loop Characteristic
IPTG (uM)
C
IPTG (uM)
aTc (ng/ml)
0
aTc (ng/ml)
6x7x2 = 84 full-day
experimental runs!
LuxI.C
LuxR.Y
Open-Loop Characteristic
IPTG (uM)
pR.C
pL.luxR.Y
aTc (ng/ml)
pT.luxI.C
Equivalences
C
IPTG
pL
LuxR.Y
IPTG
LuxR.Y
pL
C
C
IPTG
pL
LuxI.C
IPTG (uM)
LuxI.C
LuxR in Closed Loop
parameter
input
output
parameter
state
LuxR in Closed Loop
parameter
input
C
OL (50 aTc)
OL (0 aTc)
Open loop
characteristic
output
LuxR.Y
C
IPTG (uM)
parameter
0
state
aTc (ng/ml)
LuxR in Closed Loop
parameter
EQ
input
C
OL (50 aTc)
OL (0 aTc)
Open loop
characteristic
output
LuxR.Y
parameter
LuxR.Y
predicted
observed
Closed loop
response
state
Closed-loop data is preliminary, analyzed at midnight, July 14th!
LuxI.C
It worked!
Same Parts, Many Networks
LuxI in Closed Loop
input
parameter
output
parameter
state
LuxI in Closed Loop
parameter
C
OL (1000 IPTG)
OL (100 IPTG)
OL (10 IPTG)
Open loop
characteristic
output
parameter
LuxI.C
C
IPTG (uM)
input
0
state
aTc (ng/ml)
LuxI in Closed Loop
EQ
parameter
OL (1000 IPTG)
OL (100 IPTG)
C
input
OL (10 IPTG)
Open loop
characteristic
output
LuxI.C
parameter
LuxI.C
predicted
Closed loop
response
state
Experiments ongoing to validate these predictions!
LuxR.Y
Bistable response!
Pr
oo
fo
fp
rin
ci
pl
e!
Same Parts, Many Responses!
Monostable
Gradual response
Bistable
Sharp response
Same Parts, Many Responses!
Parts Datasheets
The Team
Instructor:
Mukund Thattai, Physics (NCBS Bangalore)
Advisors:
Sugat Dabholkar, Biology (graduate student, NCBS)
Navneet Rai, Biological Sciences (graduate student, IIT Bombay)
Nilesh Aghera, Biochemistry (graduate student, NCBS)
Vivek Raj, Biology (Technician, NCBS)
Kiran Hegde (Chromous Biotech)
Student members:
Krishna Ramkumar, Chemistry (IIT Bombay)
Varun Sreenivasan, Biological Sciences (St. Xavier’s Bombay)
Sushant More, Mathematics (IISER Pune)
Shashanka Kundu, Computer Science (St. Stephen’s Delhi)
Vini Gautam, Physics (Delhi University)
Senthil Kumar, Bioinformatics (PSG Coimbatore)
Supported by:
www.ncbs.res.in
Mukund conceived the experiments
Mukund & Sugat designed the constructs
Kiran synthesized the constructs at Chromous Biotech
The entire team designed the experimental protocols
Krishna & Varun performed the measurements
Sushant and Shashanka worked on computational modeling
Sushant, Shashanka, Vini & Senthil analyzed the data
The entire team updated the wiki
Mukund designed the parts data-sheets