Introduction

The researches conducted so far have mostly been transforming Escherichia coli by changing external environments or introducing inductive factors. To the best of our knowledge, phenotypes of E. coli switch without any input from the exterior.

Until now, no study in iGEM competition has been done on changing autonomously phenotypes of E. coli before and after cell division.

Therefore, we have designed a system which automatically generates three phenotypes of E.coli every time a cell divides.

Specifically,in our system, if E. coli in which first generation of gfp is found divides, we cannot find gfp anymore but only rfp. If this second generation of E. coli divides then instead of rfp, cfp is expressed.

After another division in the fourth generation instead of cfp we find gfp again. In this system, the phenotype of E. coli differs from each other between the (3n)th, (3n+1)th, and (3n+2)th generation.

Figure

As we have created this genetic circuit，sigma factor, toehold switch and Pnrd promotors (link to the system) are chosen. In modeling we use them to simulate the system we made .(link to modeling) Modeling shows us whether the genetic circuit builded in thoughts is realizable.

In addition, after confirming the system runs on modeling, we ran an inspection on parts and devices in the experiment.

System

By designing a genetic device that shifts the dominantly expressed protein among GFP, RFP and CFP every cell cycle, our system can realize E. coli that in turn gives out green, red, blue (and green again) fluorescence. To understand how it works, let’s first take a look at the individual components we used. These include Toehold Switch, sigma factors, Pnrd, and PBAD.

Explanatory Notes

Genes are shown in the diagram below, where left represents the upstream.

Toehold Switch

Toehold Switch is a translational regulation system which takes advantage of the bonding between specific transcripts. Genes downstream of the Toehold Switch on a mRNA cannot be translated without the existence of specific trigger RNA.

Ribosomes cannot bind to toehold RBS under normal condition. Therefore, proteins cannot be made because mRNAs cannot be translated even when the gene is transcribed. For a ribosome to bind to toehold RBS, the existence of a specific short RNA named trigger RNA is necessary. [[]]

The previous gene which the toehold RBS stops transcription comes to the state below under the existence of the trigger RNA, and will be translated. mRNAs being translated are represented as yellow ones. [[]]

Sigma Factor

The promoter below (sigma promoter) works only under the existence of sigma-factor, which binds to and then activates the promoter. However, anti-sigma factors also bind to sigma factors, which competitively inhibit sigma factors from functioning. [[]]

The sigma factors are promoter recognition subunits of RNA polymerase. A sigma factor is associated with a part of promoters. A sigma factor recruits RNA polymerase to its corresponding promoter and initiates transcription. Sigma factors have great variety. Some of sigma factor-promoter pairs have one-to-one correspondence. Thus, if only sigma factors which have one-to-one correspondence are used, a transcription activating system in which a sigma factor activates the transcription only from corresponding promoter can be made.

Anti-sigma factors are proteins which are related to transcriptional control mechanism by sigma factors. An anti-sigma factor inhibits the binding between RNA polymerase and sigma factor. Consequently, anti-sigma factors repress the transcription from the promoters which sigma factors initiate. In the same way as sigma factors, anti-sigma factors have great variety and some anti-sigma factors prevent only a specific sigma factors from transcriptional control. Therefore a transcription control system (i.e. not only activating but also repressing) can be constructed by using specific sigma factors and anti-sigma factors which have one-to-one correspondence.[2](words underlined were grammatically modified from the original)

Multiple combinations of sigma factors, sigma promoters, and anti-sigma factors exist. Every factor we used functions exclusively within its combination and does not bind with factors of other combinations. In other words, they do not crosstalk. To make this easier to understand, factors and promoters of the same combination are presented with the same color. [[]]

Pnrd

Pnrd, another kind of a promoter, works only during cell division. [[]]

PBAD

We also installed a promoter called PBAD, which works only under the existence of arabinose, drawn as a pentagon in the chart. [[]] Now we’ve seen the 4 components and they will help us understand what our team wants to make. Let’s go to the journey to the gene constructions and systems.

Result

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Application

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