Project

General introduction

Our project is based on the construction of a bacteria lemon that will look like lemon but made by only Escherichia coli modified to produce lemon scent and change colour from green to yellow. Thus, we will achieved our project by genetically modifying Escherichia coli to be free of any unpleasant odour mutating genes involved, by introducing genes for the production of lemon scent and amplifying this pathway and finally adding a yellow-bleu chromoprotein which its expression is controlled by tRNA suppressor for the colour changing.

This page is under Marie's responsibility

• Deadline: 08/oct.
  • General introduction of the project.
  • Introduction text of each part of the project
  • Sean's video (bettencourt)
• Deadline: 12/oct
  • Final review par Maher.

Inspirations

A - The E. coli odor free chassis

Escherichia coli stinks because of the tnaA gene which produces an enzyme that transforms the L-tryptophan into indole, responsible for the stench. If we want our lemon to smell like one, we have to delete this gene.

In the lab, we already had a strain in which the tnaA was replaced by a kanamycin resistance, but this strain was too modified to be used for our project. So we switched the tnaA sequence with the kanamycin resistance in our bacterium by phage transduction. After the recombination, we used a flipase to delete the kanamycin resistance. The remaining bacterium doesn't smell at all.

B - The Lemon Scent

The aim is to make three different populations of E.coli.

1. The first is transformed with a plasmid that contains the limonene synthase gene
2. The second is transformed with a plasmid that contains the β-pinene synthase gene
3. The third is transformed with a plasmid that contains the geraniol synthase gene and the CAD gene to increase citral A and B production by the bacteria

We could thus play with the density of these populations for a better lemon scent, like cooking, putting the right quantity of each ingredient.

Don't worry more explanation will be there soon (melanie)

The lemon fragrance contains mainly three different monoterpene components as citral (also called geraniol), limonene and pinene. These components come from the common precursor geranyldiphosphate (GPP). In E.coli, we would like to produce these monoterpene improving the GPP level by an additional plasmid containing a MVA-pathway from others species. Finally, we would cloned the enzyme required to generate monoterpene as limonene synthase, pinene synthase and geraniol synthase.

C - Lemon Appearance and Ripening

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.

D - The Lemon Shaping

The Workshop