Difference between revisions of "Team:UCopenhagen/Interdependency"
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<p class="lead"> Both AroG and TrpE (Figure 1) are regulated by the concentration of the amino acids produced. This feedback signalling reduces the tryptophan concentration that we can achieve with WT <i>E.coli</i> <i>aroG</i> and <i>trpE</i> genes, as simply overexpressing them would lead to inhibition due to increased production. | <p class="lead"> Both AroG and TrpE (Figure 1) are regulated by the concentration of the amino acids produced. This feedback signalling reduces the tryptophan concentration that we can achieve with WT <i>E.coli</i> <i>aroG</i> and <i>trpE</i> genes, as simply overexpressing them would lead to inhibition due to increased production. | ||
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<li><i>aroG</i>: <a href="http://parts.igem.org/Part:BBa_K2455000">BBa_K2455000</a></li> | <li><i>aroG</i>: <a href="http://parts.igem.org/Part:BBa_K2455000">BBa_K2455000</a></li> | ||
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| + | <p class="lead">The last gene, <i>yddG</i> is an aromatic amino acid exporter and is responsible for the secretion of both tryptophan, tyrosine and phenylalanine. <br><br> | ||
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| + | Gu et al. (2012) have shown that the over-expression of YddG in <i>E.coli</i> increases the accumulation of tryptophan in the growth medium. This is likely due to the decrease in intracellular concentration, thus bypassing the feedback sensitive regulatory steps in tryptophan biosynthesis. We have designed a codon-optimised version of <i>yddG</i> that we have submittd as Biobrick <a href="http://parts.igem.org/Part:BBa_K2455004">BBa_K2455004</a>. <br><br> | ||
| + | <u>Constructs</u> with each gene, or two of the genes together, will be evaluated in order to examine both the combined and isolated effects of the genes. | ||
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Revision as of 12:03, 1 November 2017
Introduction
The first mechanism that we have decided to investigate is the interdependency between two cell types. In order to have a stable relationship in which the host-endosymbiont relationship is 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 coexistence of the symbiotic pair, whilst also ensuring mutual demise in case of the host’s or the symbiont’s perishment. 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. We are working to establish a dependency between free living cells, as a groundwork for a later endosymbiotic relationship. Natural endosymbiotic relationships have been found between fungi and bacteria in mycorrhiza strains (Bianciotti 2000), and we will be working with two cells from these two phylogenetic kingdoms in our project; a tryptophan auxotrophic yeast AM94 and E. coli.
Introduction
Background: Synthetic yeast media, suitable for growth of tryptophan auxotroph yeast, contains 76mg tryptophan pr. liter (Sigma-Aldrich, 2017). An accumulation of 1.7g tryptophan pr. liter was achieved by Gu et al. (2012) with a modified E.coli strain, which is sufficient for yeast growth. The production of tryptophan is regulated by negative feedback mechanisms that inhibits tryptophan synthesis in the presence of tryptophan or related amino acids.
Our goal is to produce and export enough tryptophan from E.coli to complement growth of an auxotroph yeast strain, grown in media without tryptophan. Additionally, we are interested in the number of endosymbionts necessary per host. This is included in our modelling.
Circuits and biobricks: In our circuit, we are overexpressing the three genes AroG, TrpE and YddG, which will also become our biobricks. We have made our selection based on the papers by Gu et al. (2012) and Wang et al. (2013).
TrpE belongs to the tryptophan operon and has been overexpressed frequently in L-tryptophan producing E. coli strains.
AroG is the first enzyme of the shikimate pathway, thereby determining the carbon flow towards tryptophan synthesis.
Both AroG and TrpE (Figure 1) are regulated by the concentration of the amino acids produced. This feedback signalling reduces the tryptophan concentration that we can achieve with WT E.coli aroG and trpE genes, as simply overexpressing them would lead to inhibition due to increased production.
We have made use of known mutant feedback resistant alleles for trpE and aroG to overcome this regulation (Gu et al., 2012). In the proteins TrpE, a mutation in a methionine to threonine at position 293 is required, and for AroG, the proline at position 150 is changed to leucine.
By making these point mutations, the feedback inhibition should be released, and overexpression would lead to elevated tryptophan production. We submitted the feedback resistant alleles as biobricks:
- trpE: BBa_K2455001
- aroG: BBa_K2455000
The last gene, yddG is an aromatic amino acid exporter and is responsible for the secretion of both tryptophan, tyrosine and phenylalanine.
Gu et al. (2012) have shown that the over-expression of YddG in E.coli increases the accumulation of tryptophan in the growth medium. This is likely due to the decrease in intracellular concentration, thus bypassing the feedback sensitive regulatory steps in tryptophan biosynthesis. We have designed a codon-optimised version of yddG that we have submittd as Biobrick BBa_K2455004.
Constructs with each gene, or two of the genes together, will be evaluated in order to examine both the combined and isolated effects of the genes.
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.
