IGEM:Tsinghua/2007/Projects/RAP

<title>RAP</title>

RAP

Introduction

Naturally oscillation works with pulses in a single operon.

Referring to the expression oscillators, the Elowitz oscillator is the first and the only successful one so far, in which three operons expression 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 on a single cell scale during the oscillating cycles. While the natural oscillations usually works pulses with a stage when the signal is reset to zero. For example, in processes of the active potential, the cardiac cycles and the muscle contraction, the neural signal is triggered with the state of "zero", and then amplify itself until an mechanism of inhibiting is reinforced to blocked the amplification. At last, the signal fall to the state of "zero". To understand the natural oscillations, herein 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.

Since 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 very problem 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. However, 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. The 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 A kindly gift from Prof. Michael B. Elowitz pJBA110 A kindly gift from Prof. Michael B. Elowitz pJBA111 A kindly gift from Prof. Michael B. Elowitz pJBA112 A kindly gift from Prof. Michael B. Elowitz

Flowchart

Eperiment Records

Thread 1: RAP Ampilifier.

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Thread 2: Fast-degrading T7 RNA polymerase.

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Thread 3: RAP repressor.

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