Team:UCopenhagen/Notebook
Introduction
Why interdependency? In order to have a stable relationship where the host-endosymbiont relationship are maintained through generations, the host and endosymbionts must depend on each other for their continued survival.
Within the endosymbiotic relationship, we envision a resource based cross-network between host and symbiont. The exchange of vital metabolites necessary for proliferation would ensure the co-existence of the symbiotic pair while also ensuring mutual demise should either host or symbiont perish. Our goal would be that the pair would survive only if the condition of endosymbiosis is met. This is important, as our project is about laying the foundation for stable modular endosymbiosis.
In this subproject, we will use yeast as a substitute for the host, and E.coli as a substitute for an endosymbiont. This choice was done due to both organisms being readily available and since metabolic pathways in E.coli are well studied. Endosymbiosis between fungi and bacteria are occurring naturally in mycorrhiza (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC92331/), showing that endosymbiotic relationships between these two groups of organisms can occur.
Our aim is to make yeast depend on a metabolite produced by E.coli - an auxotrophic dependence. This will be achieved by engineering an E. coli strain to produce sufficient L-tryptophan to complement the lack of tryptophan synthesis in the yeast strain 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, the symbiont is thus dependent on the host for survival in these growth conditions.
Final Design
Goal: In synthetic yeast media there are 76 mg tryptophane pr liter (Sigma-Aldrich, 2017), so the goal is to produce and export similar amounts from E.coli. A strain by Gu et al. (2012) had few modifications and accumulated 1.7 g tryptophane per liter: Sufficient for yeast growth.
In addition to this, we have included tryptophane use and production in yeast and E.coli in the modelling to check how many endosymbionts would be necessary pr host.
Genes: Based on the papers by 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 feedback signalling reduces the tryptophane concentration we can achieve in a WT E.coli. We have made use of known (Gu et al., 2012) mutant feedback resistant alleles for trpE and aroG to overcome this regulation. For trpE, mutation in 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.
Codon-optimised, synthetically produced.
Experiments
Amplification of genes:
- Synthetically produce a codon-optimised version of yddG.
- Amplify trpE and aroG from WT E.coli MG1655
- Point mutations for feedback resistance in trpE and aroG: Using primers with overhangs containing point mutations, and splitting the gene in two, then combining the two parts when inserting in the expression vector.
- Created vector with combinations of one, two and three genes in the USER casette, using primers with overhangs.
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
Protein import. USER casette and His tag.
Vector design was performed in the protein import subproject, and the same vector was used for all cloning in the interdependency project.
Expression and production
- Expression checked with Western blotting: all genes HIS tagged.
- Tryptophane production by E.coli with one, two or three genes, and nuder different levels of inducing agents evaluated on HPLC
Co-growth of E.coli and yeast
- Find yeast minimal media where E.coli can grow
- Grow yeast in same media after E.coli has grown, in order to establish possibility for relationship.
- Grow tryptophane producing E.coli for different time periods, then remove them and grow auxotrophic yeast in the media containing the produced tryptophane.
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 (van Summeren-Wesenhagen and Marienhagen, 2014), 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 measured using OD600 measurements to evaluate the successful complementation of the yeast amino acid auxotrophy by E.coli tryptophan production.
Design process
In our design process, we have considered a wide range of possible gene combinations. Genes that when over-expressed would have the greatest impact were chosen. This is due to the time constraints set and simplicity. 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. We also thought of deleting endogenous trpR, but making such a deletion would make our project overly complicated due to the difficulty of making such a deletion is E.coli.
