IGEM:Tsinghua/2007/Projects/RAP

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<h1>Introduction</h1>
<h1>Introduction</h1>
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Revision as of 08:59, 6 August 2007

Contents

Introduction

Synthetic oscillator in a single operon that simulates the natural oscillations.

Referring to the expression oscillators, the Elowitz oscillator is the first and the only typically successful one so far, in which three operons express three different transcriptional repressors, lacI, lambda C1 and tetR, respectively. The three transcriptional repressors in the Elowitz oscillators inhibit one another to generate oscillations. However, this is a relatively complex system, and protein accumulation has been observed at the single-cell scale during the oscillating cycles. While the natural oscillations usually works in pulses including a stage when the signal is reset to zero. For example, in the process of the active potential, the cardiac cycles and the muscle contraction, the neural/electric signal is triggered at the state of "zero", and then amplified until a feedback inhibiting mechanism is reinforced to blocked the amplification. At last, as the inhibition dominates, the signal falls to the state of "zero", which can be thought as the "reset". To understand and simulate the natural oscillations, we propose a method to allow the oscillator being reset to "zero" after each cycle and find that it is an still simpler method to generate oscillations. A fast-degrading DNA polymerase and a fast-degrading transcriptional repressor are engaged in this system. This oscillation works in four stages: (1)Triggering: the RNA polymerase gene is expressed by constructive promoters. (2)Amplication: the RNA transcripts the gene coding itself. (3) Inhibiting: the RNA transcripts a transcriptional repressor, which has longer degrading half-life than the RNA polymerase, that blocks the transcription of the RNA polymerase gene. (4) Resetting: the transcription of the RNA polymerase is blocked and until the transcriptional repressor degrades to a concentration below a specific level.

And we found that this oscillator can be constructed in a single operon.

Fast-degrading T7 RNA polymerase.

Fast-degrading transcriptional repressors are available, such as the lacI, lambda C1 and tetR, because those protein are not conservative in their C-terminus and ready to be fused with a degrading marker. While the fast-degrading RNA polymerase is the key in this project. Most RNA polymerases consist of multiple subunits, except for T odd number RNA polymerase, SP6 RNA polymerase and so on. Those RNA polymerases are very conservative at their C-terminus and can not be engineered to fuse a C-terminus degrading marker. Fortunately their N-terminus are not that conservative and can be engineered to get some N-terminus fusions. Some degrading marker of E. coli can be identified on the N-terminus, such as the degrading marker recognized by Lon and other heat shock proteases. UmuD degrades quickly in E. coli and its N-terminal 30 amino residues were reported to work as a N-terminal degrading marker. Therefore, we try to construct a fast

degrading T7 RNA polymerase with UmuD N-terminal degrading marker.

Model and simulation

Ongoing.

Strains & Plasmids

Strains

Strain Source
E. coli TOP10 Transgen biotech company, Beijing.
E. coli MC4100 GCSC of Yale
E. coli JM109 Prof. Chen's lab collection
E. coli BL21(DE3) Transgen biotech company, Beijing.

Plasmids

Plasmid Source
pEASY-Blunt Transgen biotech company, Beijing.
pET15b Prof. Chen's lab collection
pBluescript II SK(-) Prof. Chen's lab collection
pMD18-T Takara, Japan.
pBHR91 Prof. Chen's lab collection
pZS1-lTLrLLtCL Prof. Michael B. Elowitz
pJBA110 Prof. Michael B. Elowitz
pJBA111 Prof. Michael B. Elowitz
pJBA112 Prof. Michael B. Elowitz


Materials

Reagents

Reagents Source
Mouse-anti-T7 RNA Polymerase monoclonal antibodies. Novagen

 


Flowchart

Image:RAPv1.4.gif


Experiment Records

Thread 1: RAP repressor.

Number Date Inherit Chart Index Notes Results Plan Expected Time Operator
              Lijun Zhou  

Thread 2: RAP Amplifier.

Number Date Inherit Chart Index Notes Results Plan Expected Time Operator
1 6-23 - 2-1 PCR clone the pSC101 ori from pBHR91. Success Extract of the PCR product. 6-23 Zhenyu Shi
2 6-23 1 2-2

Extract the PCR product and insert the product into pEASY-B cloning

vector. Transformation with blue/white screening.
- Pick clones. 6-24 Zhenyu Shi
3 6-24 2 2-2 Get the white clones and perform PCR screening. Three clones were found correct. Have the correct clones sequenced. 6-25 Zhenyu Shi
4 6-25 3 2-2 Send the correct clones for sequencing. - Wait for the sequencing results. - Zhenyu Shi
5 6-29 4 2-2 The sequencing result was analyzed. The sequence is correct. Synthesize the RAP promoter and insert the RAP promoter into the vector. 6-29 Zhenyu Shi
6 6-29 5 2-3 Synthesize the RAP promoter. - Wait for the synthesis result. - Zhenyu Shi

Thread 3: Fast-degrading T7 RNA polymerase.

Number Date Inherit Chart Index Notes Results Plan Expected Time Operator
1 7-11 - 3-1

PCR clone the T7 RNA polymerase gene from a previously construct vector

containing T7 RNA polymerase gene.
Success Extract of the PCR product. 7-11

Die Hu, Tian Fang,

Zhenyu Shi
2 7-11 1 3-1

Extract the PCR product and insert the product into pEASY-B cloning

vector. Transformation with blue/white screening.
- Pick clones. 7-12

Die Hu, Tian Fang,

Zhenyu Shi
3 7-12 2 3-1 Pick the white clones and perform PCR screening. Three clones were found correct. Have the correct clones sequenced. 7-13

Die Hu, Tian Fang,

Zhenyu Shi
4 7-13 3 3-1 2 clones were sent for sequencing. - Wait for the sequencing results. -

Die Hu, Tian Fang,

Zhenyu Shi
5 7-19 4 3-1 The sequencing result was analyzed.

All the clones have the same mutations. The template may contains

mutations.
Try a different template to clone the T7 RNAP gene. 7-19

Die Hu, Tian Fang,

Zhenyu Shi
6 7-20 5 3-1 Clone the T7 RNA polymerase gene from E. coli BL21(DE3) culture. Failed. Extract the genome DNA from E. coli BL21(DE3). 7-23

Die Hu, Tian Fang,

Zhenyu Shi
7 7-23 6 3-1 Extract the E. coli BL21(DE3) genome DNA. Success. PCR clone the T7 RNA polymerase from the E. coli BL21(DE3) genome DNA. 7-23

Die Hu, Tian Fang,

Zhenyu Shi
8 7-23 7 3-1 PCR clone the T7 RNA polymerase from the E. coli BL21(DE3) genome DNA. Success Extract of the PCR product. 7-23

Die Hu, Tian Fang,

Zhenyu Shi
9 7-23 8 3-1

Extract the PCR product and insert the product into pEASY-B cloning

vector. Transformation with blue/white screening.
- Pick clones. 7-24

Die Hu, Tian Fang,

Zhenyu Shi
10 7-24 9 3-1 Get the white clones and perform PCR screening. Three clones were found correct. Have the correct clones sequenced. 7-25

Die Hu, Tian Fang,

Zhenyu Shi
11 7-25 10 3-1 Send the correct clones for sequencing. - Wait for the sequencing results. - Zhenyu Shi, Die Hu, Tian Fang
12 7-29 11 3-1 The sequencing result was analyzed. There were two mutations at the restriction site in the primer. Synthesize another pair of primers for cloning. 7-29

Chun Li, Qin Zhou,

Zhenyu Shi
13 7-29 12 3-1 Synthesize another pair of primers for cloning. - Wait for the primer and perform PCR cloning again. -

Chun Li, Qin Zhou,

Zhenyu Shi
14 8-2 13 3-1 PCR clone the T7 RNA polymerase from the E. coli BL21(DE3) genome DNA. Success Extract of the PCR product. 8-2

Chun Li, Qin Zhou,

Zhenyu Shi
15 8-2 14 3-1

Extract the PCR product and insert the product into pEASY-B cloning

vector. Transformation with blue/white screening.
- Pick clones. 8-3

Chun Li, Qin Zhou,

Zhenyu Shi
16 8-3 15 3-1 Get the white clones and perform PCR screening. Three clones were found correct. Have the correct clones sequenced. 8-5

Chun Li, Qin Zhou,

Zhenyu Shi
17 8-5 16 3-1 6 clones were sent for sequencing. - Wait for the sequencing results. -

Chun Li, Qin Zhou,

Zhenyu Shi
                 

Project Staff

Dry Experiment

Project ProposalZhenyu Shi, Peng Dong
Mathametical SimulationXinyu Zhao

Wet Experiment

Experiment Design & ManagementZhenyu Shi
Experiment OperationChen Chen, Lijun Zhou, Peng Dong, Xinyu Zhao, Yexing Liu, Zhenyu Shi, Zhou Yu.
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