IGEM:Tsinghua/2007/Projects/RAP

Introduction

Designed oscillator simulating the natural oscillations of pulses in a single operon.

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

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	Zhenyu Shi, Die Hu, Tian Fang
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	Zhenyu Shi, Die Hu, Tian Fang
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	Zhenyu Shi, Die Hu, Tian Fang
4	7-13	3	3-1	2 clones were sent for sequencing.	-	Wait for the sequencing results.	-	Zhenyu Shi, Die Hu, Tian Fang
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	Zhenyu Shi, Die Hu, Tian Fang
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	Zhenyu Shi, Die Hu, Tian Fang
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	Zhenyu Shi, Die Hu, Tian Fang
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	Zhenyu Shi, Die Hu, Tian Fang
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	Zhenyu Shi, Die Hu, Tian Fang
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	Zhenyu Shi, Die Hu, Tian Fang
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	Zhenyu Shi, Chun Li, Qin Zhou
13	7-29	12	3-1	Synthesize another pair of primers for cloning.	-	Wait for the primer and perform PCR cloning again.	-	Zhenyu Shi, Chun Li, Qin Zhou
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	Zhenyu Shi, Chun Li, Qin Zhou
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	Zhenyu Shi, Chun Li, Qin Zhou
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	Zhenyu Shi, Chun Li, Qin Zhou
17	8-5	16	3-1	6 clones were sent for sequencing.	-	Wait for the sequencing results.	-	Zhenyu Shi, Chun Li, Qin Zhou

Project Staff

Dry Experiment

Project Proposal Zhenyu Shi, Peng Dong Mathametical Simulation Xinyu Zhao

Wet Experiment

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