Team:UCopenhagen/Interdependency
Introduction
Why interdependency? In order to have a stable relationship where the host-endosymbiont relationship are maintained through generations, the host and endosymbionts must be interdependent.
The host should benefit from allocating ressources to the symbiont, through more efficient metabolism (as the case of mitochondria) or by the production of metabolites the host is unable to produce itself. The dependency by the symbiont on the host is often in the form of protection from environment or predators.
In this subproject, we will use yeast as a substitute for the host, and E.coli as a substitute for an endosymbiont.
Our aim is to make yeast depend on a metabolite produced by E.coli, by engineering an E. coli strain to produce sufficient L-tryptophan to supplement a yeast auxotroph (not producing it’s own tryptophan) when grown in a media depleted for L-tryptophan.
We do not attempt to make E.coli depend on yeast at this stage, but in an endosymbiotic relationship the yeast can be grown in a media the E.coli could not survive on its own.
Design
Goal: In synthetic yeast media there are 76 mg tryptophane pr liter (Sigma-Aldrich, 2017), so the goal is to produce and export the same from E.coli. A strain from (Gu et al., 2012) had few modifications and accumulated 1.7 g tryptophane per liter: Sufficient for yeast growth, if the two can be combined.
We have made a model of the tryptophane use and production in yeast and E.coli to check how many endosymbionts would be necessary.
Genes: Based on the papers (Gu et al., 2012) and (Wang et al., 2013) three genes are overexpressed. Tryptophane production is regulated by feedback mechanisms, that we try to overcome.
trpE belongs to the tryptophan operon and has been over-expressed frequently in L-tryptophan producing E. coli strains.
aroG: the starting enzyme of the shikimate pathway, leading to synthesis of tryptophan. Being the first enzyme in the pathway, it determines the carbon flow towards tryptophan synthesis and thus the production.
Both aroG and trpE (figure) are regulated by the concentration of the tryptophane they produce, this reduce the concentration we can achieve. We have made use of known (Gu et al., 2012) mutant feedback resistant alleles for these genes to overcome this regulation. For trpE, a methionine to threonine at position 293 is required, and for aroG the proline at 150 is changed to leucine.
yddG: an aromatic amino acid exporter. yddG is responsible for the secretion of L-tryptophan, and the over-expression of this increase the accumulation (Gu et al 2012), likely due to bypassing the feedback sensitive regulatory steps in tryptophane biosynthesis by decreasing the intracellular concentration.
Experiments
Amplification of trpE and aroG from wild type
We amplified yddG, trpE and aroG from wildtype E. coli MG1655, using 'method'Point mutations trpE and aroG
To make the point mutations for trpE and aroG, two sets of vectors for each gene was designed (illustration). Overhangs in the end of the primers enable USER cassette insertion, while the primer overhangs in the center contain a point mutation. When the two parts are being amplified individually, the transformation into vector in expression host will be done with USER ligation.Vector design was performed in the protein import subproject, and the same vector was used for all cloning in the interdependency project.
Expression analysis
We checked expression of the genes using western blot, as all genes are HIS tagged in the vector (protocol)Evaluation of E.coli tryptophan production by liquid chromatography (LC)>/h3>
After ensuring expression of the genes, we use LC to evaluate the production of tryptophan by E.coli. We check the production from E.coli with different transformations (one, two or three vector insertions), as well as two different media: LB and YNB pH7.
These results will be part of the modelling
Growth of E.coli and yeast in same minimal yeast medium
E. coli strains MG1655 and BL21 were grown in several media in order to find a minimal yeast media where E.coli could survive. With inspiration from (reference), we decided to grow E.coli in the minimal yeast media YNB with the pH adjusted to 7 instead of 4 as original.
After ON growth of E.coli in YNB pH 7, the media was cleared of E.coli by spinning and filtration, after which it was inoculated with yeast (AM94), to ensure that the E.coli does not produce substances hindering yeast growth (protocol).
This experiment is a prerequisite for our next experiment.
Growth of tryptophan auxotrophic yeast in minimal medium subsequent to tryptophan producing E.coli
This experiment utilizes the same protocol as the previous (protocol), but now in YNB pH7 media without a tryptophan source, with tryptophan overproducing E.coli and with a tryptophan auxotrophic yeast strain.
This experiment is performed with single, double and triple transformations: That is, E.coli with trpE(fbr), aroG(fbr) and yddG alone or in combinations.
The growth of yeast is evaluated using OD600 measurements to evaluate the successful complementation of the the yeast amino acid autotrophy by E.coli tryptophan production.
Design process
In our design proces, we have considered a wide range of possible gene combinations. Initially, we considered simply overexpressing the tryptophane operon, but quickly realised this would be highly downregulated due to negative feedback regulation. We decided that an exporter would be beneficial by reducing the intracellular Trp concentration, which would release the feedback regulation.
