iGEM in Tsinghua

iGEM in Tsinghua

The Department of Biological Sciences and Biotechnology,

Tsinghua University,China

2007.11.3

Why do we come for iGEM?

We have a common dream:

To build BioMachines as powerful as electronic and mechanical mac

hines.

Even the simplest machine has two parts:

• Power generator

-The original force to

drive the system.

• Wire

-information propagation

What did we make?

• An oscillator: RAP (AC power) Recoverable Auto-Pulse generator (RAP)

by using a negative feedback loop.

• A communicator: CelCuit (wire) Cell-cell communication system by using

conjugating DNA as protein carrier.

Part 1. RAPPart 1. RAP

• Oscillator is a basic element in engineering.

- It’s importance in electronic engineering is

well known.

- It’s also widely required for genetic

machine and natural organism (circadian

rhythm).

• Only one successful genetic oscillator published up to now.

(Nature 403:335-338, 2000)

We use another strategy

• Negative feedback loop consisting of two components.

Principle so easy!Never constructed…

So, why not try it?

• T7 RNA polymerase (T7 RNAP) is used as the driver and Lac operon repressor (LacI) is used as the brake.

• Both protein are tagged with Umd to implement fast degradation.

Implementation

Repressor:

Amplifier:

Modeling

• We model the mRNA levels and protein levels respectively.

(In the following description, we use lower-case of the first letter to indicate mRNA levels whereas up-case to indicate protein levels. )

• mRNA level is governed by:

1. spontaneous degradation (minus)

2. transcription (plus)

7 _

d( ) ln 2round[R( 7) ]

d T lac plasmidmRNA

lacIlacI rTranscribe T n

t

7

d( 7) ln 27

d T PmRNA

tt rTranscribe activatedNum rTranscribe freeNum

t

First order reaction Binding of proteins to DNA according to M-C equation

• M-C equation

[ ]R([ ])

[ ]

h

hd

PP

K P

Protein concentrationOccupation

ratio

Dissociation constant

Hill coefficient

• Protein levels are governed by:

1. spontaneous degradation (minus);

2. translation (plus)

d( ) ln 2

d LacI

LacILacI rTranslate lacI

t

7

d( 7) ln 27 7

d T

TT rTranslate t

t

First order reaction

Stochastic Model

• To make our model more realistic, we modify our model to a stochastic one. In this model, all the parameters, except copy numbers of plasmids, are assumed to obey to a norm distribution and a ‘sigma’ value (which indicates the peak width) is set for each parameter.

Parameter Estimation

• Copy number of the repressor plasmid: n_lac_plasmid=30 copy

• Copy number of the amplifier plasmid: n_t7_plasmid=5 copy

• Transcription rate of T7 polymerase: r_transcribe_T7=300 mRNA/min

• Transcription rate of host polymerase: r_transcribe_P=30 mRNA/min

Transcription rate of host polymerase is adapted form (Michael B. Elowitz, Nature, 2000 )

Transcription rate of T7 polymerase is about 10-20 folds of host polymerase.

• Translation rate: r_translate=5.0 proteins/min

Adapted from (Michael B. Elowitz Nature, 2000 )

• Half life time of mRNA: τ(mRNA)=3min

• Half life time of LacI: τ(LacI)=10min

• Half life time of T7: τ(T7)=10min

• Half life time of EGFP: τ(EGFP)=40min

Modified from (Michael B. Elowitz, Nature, 2000 )

• Dissociation constant of LacI: K(LacI)=20 dimers per cell

• Dissociation constant of T7 polymerase: K(T7)=20 monomers per cell

K(LacI) is adapted from (Michael B. Elowitz Nature, 2000 )

According to (William R. McClure, PNAS, 1980), the Kd of T7 binding to double strand DNA is comparable to that of LacI.

Simulation Results

• Numerically simulated in MATLAB by using ODE toolbox.

When Translation Rate Fluctuates

What makes our RAP different?

• RAP is more stable than the repressilator. It should be emphasized here that it is unnecessary for t

he driver to degrade faster than the brake

τ(T7)=40min

• In a synchronized oscillator model proposed by coupling oscillator to quorum sensing system, RAP, which is of an relaxation type, converges more rapidly than repressilator, which is an phase oscillator. (PNAS 99(2):679-84, 2002 )

Proceedings

• We finished almost all the molecular cloning.

• We confirmed that T7 RNAP we cloned is functional by luciferase test.

• We have no time to put repressor and amplifier together before we leave for the Jamboree…

Parts Registered for RAP

AAV-110LLA-110LVA-110AAV-111LLA-111LVA-111AAV-112LLA-112LVA-112

pACYC-LuxpEB-AclANcoI-pEB-SC101pEB-T7XT7BLUEpT7Blue-XisT7XpEB-EGFPLpET15b-lacI(-)

Summary of RAP Q&A

• What is RAP proposed to? -automatic oscillation• How is RAP constructed? -negative feedback loop by T7 RNAP and LacI• What are the advantages of RAP over existin

g oscillator? -stability -converge rapid when synchronized

Part 2. CelCuitPart 2. CelCuit

• Cell-cell communication is a very important issue in synthetic biology.

• It is widely required for synchronized oscillator, populational computation, control of population size, artificial organogenesis and so on.

• Drawbacks of current cell-cell communication system-quorum sensing system (small diffusion molecule as messenger)

1. unrecoverable

2. difficult to achieve multiple independent

signals

Why are they so important?

Focus shifts from small chemical to CONJUGATION

• Two strategies have been tried to implement information transference by using conjugation.

Recovery

problem

Muti-signal

problem

New problem

“relaxase-fusion”

Solved

Solved

Too weak

“plasmid-encoding”

Unsolved

Solved

Plasmid compatibility

CelCuit: Using DNA to Carry Proteins

Step 1

Step 2

FLP recombinase

SSB

EGFP

SSB binding site

Terminator

Step 3

movie

Which Executor?

• The pool of executor is very large:

transcription factors, two-hybrid system, …

• In this case, we chose FLP recombinase for its high efficiency.

Which Carrier (SSB)?

species High amount

Functions in monomer

Specific binding site

Known to be transferred during

conjugation

SSB1

(E. Coli) + + - -TraD

(E. Coli) ? - ? +M. jannasch

ii - + - -S. cerevisia

e - - - -POT1

(mammals)depends + + -

• We also tried other modes:

- Lamda cI protein as the carrier

- “relaxase-fusion” strategy: Mob-FLP

fusion protein

Implementation

Proceedings

• We finish all the molecular cloning of CelCuit except SSB part.

• We confirm that the FLP recombinase we cloned is functional by using LacZ reporter.

• Because the cloning of SSB is not finished yet, we are still not sure whether the protein transference during conjugation does occur.

Parts Registered for CelCuit

pLZHinDIII2pLZCI3FLP-p15apEASY-p15apBL-BluntpKD-pBLpKD-pBL-LambdapEB-FLPpEB-Lambda

pEB-Lambda Amp-pBL-FLPpEB-C1pEB-MobpBL-LinkerpBL-FLPMobpGEM-FLP

• We are all exciting for what we are doing.

We think the CelCuit, once finished, will certainly revise the methodology of gene machine in population in many cases!

Three Application Examples

• Pattern Formation (Nature. 2005 Apr 28;434(7037):1130-4 )

Pattern formation by reciprocal interaction between nearby cells.

• Bacterial neural network (U.C Berkley’s dream several years ago)

Neurons in one layer innervates those in their downstream layer in an all-connection mode.

• Cell Circuit (ambitious concept, isn’t it?)

If we could set a cell array…

Insulate cells which are near from each other.

Summary of CelCuit Q&A

• What is CelCuit proposed to?

-transfer proteins between cells• How is CelCuit constructed?

-fuse signal proteins (FLP, T7 RNAP…) to SSB • What are the advantages of CelCuit over exis

ting communicator?

-recoverable

-large signal pool

These two projects are done by:

Jiang Ming, Li Keyu, Dong Peng, Zhao Xinyu, Chen Chen, Shi Zhenyu, Liu Yexing, Zhou Lijun, Yu Zhou, Liu Zhe, Song Xindong

Prof. Chen Guoqiang

Dr. Xu Feng

Acknowledgement

• We thank Dr. Xie Liping for her encouragement.

• We thank the students who took practical synthetic biology summer course for their technique help.

• This work is supported by the International Competition Funds of Tsinghua University.

Thank iGEM for giving us such an unforgettable experience!

Thank you for your attention!