Salicylate Inducible System

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This page is under Mathieu's responsibility

• Deadline: 08/oct.
  • Final text
• Deadline: 12/oct
  • Final review by Olivier

Introduction

As everyone knows, lemon is a fruit that can display a yellow color when it is ripped and a green color when it is not. Firstly, we chose to use chromoproteins to express these colors in E. coli. Chromoproteins are reflective proteins that contain a pigmented prosthetic group (for example, iron for the haemoglobin) and do not need to be excited to be seen. Although several chromoproteins have been synthesized in the past years, there is no known green chromoprotein yet synthesized in the iGEM. We aim to resolve this by fusing a yellow chromoprotein with a blue one that hopefully will display a green color. This construction will be referred as the green fusion chromoprotein.

Secondly, in order to make our lemon ripe like a real lemon, we decided to take advantage of the designing of the fusion protein by using a translational suppression system. Indeed, we plan to add amber codons within the linker separating the yellow and the blue chromoproteins. Therefore, the expression of a suppressor t-RNA will suppress amber codons allowing the translation of the green fusion chromoprotein. Conversely, the down regulation of the suppressor t-RNA through time will allow bacteria switch from green to yellow, thus simulating the ripening of a real lemon. This system will be referred and the color switch system.

Ancien abstract

In nature, a lemon is firstly green and becomes yellow after some month. We would like to follow this ripeness transforming our lemon from green to yellow. In order to make our lemon looks green and having reported that there is no green chromoprotein in the RFC, we would like to fuse a yellow chromoprotein with a blue one separated by a linker containing two amber stop codon. Thus, the expression of a tRNA suppressor would suppress amber stop codon and allow the translation of the yellow and blue fusion chromoprotein, hopefully resulting in a green chromoprotein. Then, in the absence of the tRNA suppressor, only the yellow chromoprotein would be translated, allowing our lemon to switch from green to yellow.

The aim of this section is to simulate appearance of a lemon and its ripening process by:

1. Making bacteria display a green color. This part required the design of a green chromoprotein.
2. Making bacteria change its color gradually from green to yellow through time. This part required the design of a color switch system.

Color Switch system design

The supD suppressor t-RNA

Given that the linker that separates both FwYellow and AeBlue chromoproteins is composed of serine and glycine amino-acid, we needed a t-RNA suppressor that could encode one of these amino-acids to not alter the properties of the linker. As we were looking for a t-RNA suppressor, we found that the iGEM Beijing 2009 team already worked on a translational suppression system. They worked with the supD suppressor t-RNA that encodes a serine making it an ideal candidate for our project. They placed supD under control of a salicylate inducible promoter Psal to suppress amber codon they had introduced in the T7 polymerase sequence. In turn, the T7 polymerase can express the GFP output gene. Their results show that supD does not induce bacteria lethality so such a system could be used [2].

Transcription factor sensible to salicylate

The nahR gene is involved in the degradation of the naphthalene pollutant in Pseudomonas putida. This gene encodes a transcriptional regulator that is induce by salicylate and thus bind nah or sal promoters. The BBa_K228004 Biobrick® contains the nahR gene under control of a constitutive promoter and the salicylate promoter (Psal). Thus, we plan to place supD under control of the Psal promoter.

Color switch mechanism

At the beginning, salicylate concentration is maximal into the agar media so that supD will be expressed and so the green fusion chromoprotein: bacteria will display a green color. However, as bacteria grow into agar, less salicylate will remain available into the media. Thus, the decrease of the nahR-salicylate complex amount within bacteria will lead to supD downregulation through time. In turn, decrease of supD amount will lead to less codon readthrought and so less translation of the green fusion protein and more translation of the yellow chromoprotein. As a result, bacteria will gradually change from green to yellow.

Results

Color switch system

As the fusion chromoprotein did not work, the colour switch system that we planned to do is no longer valid.

Alternative pathway

To resolve this issue, we planned to separately express both FwYellow and AeBlue in bacteria. We decided to place the FwYellow under control of a constitutive promoter whereas AeBlue will be place under the Psal inducible promoter, as mentioned above. As previously, the beginning, salicylate concentration is maximal into the agar media leading so both chromoprotein will be expressed. Assuming that the presence of both chromoprotein in the cytoplasm will display a green color, bacteria will be green. However, as bacteria grow into the agar media, the amount of nahR-salicylate complex will decrease through time. Thus, the expression of the AeBlue will decrease whereas the expression of FwYellow will remain equal. As a result, bacteria will gradually change from green to yellow.