Endy:Dedicated systems/Transcription

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Contents

VM Transcription

Introduction

Implementing Dedicated Transcription
Implementing Dedicated Transcription

The T7 expression system developed by Studier and coworkers is essentially orthogonal from the E. coli transcription system. T7 RNAP does not recognize E. coli promoters and E. coli RNAP does not recognize T7 promoters.

Effects of producing dedicated transcription machinery in a cellular chassis

Growth curves for three colonies of BL21(DE3) in the presence or absence of maximally inducing levels of IPTG.
Growth curves for three colonies of BL21(DE3) in the presence or absence of maximally inducing levels of IPTG.

Three colonies of BL21(DE3) were grown in the presence or absence of 0.4mM IPTG. The IPTG induces the production of T7 RNAP. Results suggest that the presence of dedicated transcription machinery in BL21 does not noticeably affect the growth rate of the cellular chassis.

While this implies that the presence of a dedicated transcription system has negligible effect on a cells ability to supply a demand, it is possible that the added demand of these systems pushs the cellular chassis much closer to its maximum demand level before physiological changes become evident.

GFP production from T7 promoters of varying strength

Time courses of GFP accumulation per cell using promoters of different strengths
Time courses of GFP accumulation per cell using promoters of different strengths
  • I built GFP reporter systems that were driven by T7 promoters of various strengths, based on the in vitro data of Imburgio et al..
  • As expected, the initial accumulation rate of GFP is qualitatively in keeping with the in vitro promoter strengths. It is possible that there are saturation effects evident as the accumulation rate in the two strongest promoters is very similar. What was less expected was that the accumulation rate after 3 hours of induction dropped off significantly for the strong promoters and much more slowly for the weaker promoters.
  • The suggestion has been made that the transition to stationary phase affects the strength of the promoters. This seems unlikely, since the T7 RNAP, which is orthogonal to E. coli RNAP, should have no interaction with stationary phase E. coli sigma factors, nor is there any reason for there to be inhibition of the T7 RNAP during stationary phase. Another point that is important is that the four promoters all differ from the consensus sequence (as used E7104) by just one base. This makes it unlikely that one of them has a cryptic E. coli promoter or stationary phase sigma factor binding site.
  • I need to repeat this data again, and fit a trendline to the initial accumulation rates to see if they are quantitatively in keeping with the in vitro data. It might also be good to run the samples in a chemostat and see which has the highest level of GFP accumulation.



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