Difference between revisions of "Team:UCopenhagen/Interdependency"

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                     <h2 class="section-heading">Introduction </h2>
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                     <h2 class="section-heading">Final Design </h2>
 
                                         <p class="lead">
 
                                         <p class="lead">
 
<strong>Background</strong>: 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.
 
<strong>Background</strong>: 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.
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                     <h2 class="section-heading">Experiments</h2>
 
                     <h2 class="section-heading">Experiments</h2>
 
                     <p class="lead">
 
                     <p class="lead">
 +
 +
<h4>Overview</h4>
 +
 
<strong>Amplification of genes:</strong>
 
<strong>Amplification of genes:</strong>
 
<ul style="text-align:left; color:white;">
 
<ul style="text-align:left; color:white;">
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<li>Synthetically produce a codon-optimised version of yddG. </li>
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<li>Synthetically produce a codon-optimised version of <i>yddG</i></li>
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<li>Amplify trpE and aroG from WT E.coli MG1655</li>
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<li>Amplify <i>trpE</i> and <i>aroG</i> with point mutations</li>
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<li> 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. </li>
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<li> Multi-gene constructs inserted in vector</li>
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<li> Created vector with combinations of one, two and three genes in the USER casette, using primers with overhangs.</li>
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</ul>
 
</ul>
 
</p>
 
</p>
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<p class="lead">
 
<p class="lead">
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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.
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<strong>Serial growth of <i>E. coli</i> and yeast</strong>
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<br><br>
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<strong>Vector design</strong>
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<br><br>
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Protein import. USER casette and His tag. <br>
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Vector design was performed in the protein import subproject, and the same vector was used for all cloning in the interdependency project.
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<br><br>
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<strong>Expression and production</strong>
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<ul style="text-align:left; color:white;">
 
<ul style="text-align:left; color:white;">
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<li>Expression checked with Western blotting: all genes HIS tagged. </li>
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<li>Identify a minimal media supporting both <i>E. coli</i> and yeast growth </li>
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<li>Tryptophane production by E.coli with one, two or three genes, and nuder different levels of inducing agents evaluated on HPLC</li>
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<li>Analyse auxotrophic yeast growth in the minimal media both with and without tryptophan added </li>
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<li>Grow auxotrophic yeast in media only containing tryptophan produced by <i>E.coli</i> </li>
 
</ul>
 
</ul>
 
</p>
 
</p>
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<p class="lead">
 
<p class="lead">
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<strong>Co-growth of E.coli and yeast</strong>
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<strong>Gene expression and tryptophan production</strong>
 
<ul style="text-align:left; color:white;">
 
<ul style="text-align:left; color:white;">
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<li>Find yeast minimal media where E.coli can grow</li>
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<li>Western blotting to check expression of his-tagged proteins</li>
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<li>Grow yeast in same media after E.coli has grown, in order to establish possibility for relationship.</li>
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<li>HPLC-MS measurements of tryptophan production</li>
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<li>Grow tryptophane producing E.coli for different time periods, then remove them and grow auxotrophic yeast in the media containing the produced tryptophane. </li>
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</ul>
 
</ul>
 
</p>
 
</p>
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<h4>Verifications</h4>
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<p class="lead">
 
<p class="lead">
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<strong>Growth of E.coli and yeast in same minimal yeast medium</strong>
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 +
We have used <a href="https://static.igem.org/mediawiki/2017/c/cd/GelElectrophoresis_UCopenhagen.pdf">gel electrophoresis</a> across most of our experiments and sub-projects to continually monitor our progress.
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<br><br>
 
<br><br>
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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.
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<br>
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All <a href="https://static.igem.org/mediawiki/2017/9/94/Protocol8_UCopenhagen.pdf">transformations</a> in interdependency and protein import were done with USER <a href="https://static.igem.org/mediawiki/2017/2/25/Protocol7_UCopenhagen.pdf">cloning</a>and the cells were grown ON on <a href="https://static.igem.org/mediawiki/2017/6/62/Protocol14_UCopenhagen.pdf">LB</a> agar with the appropriate selection (Ampicillin or Chloramphenicol depending on the resistance marker). <a href="https://static.igem.org/mediawiki/2017/b/b0/Protocol16_UCopenhagen.pdf">Colony PCR</a>s were performed to pick colonies with the correct inserts for inoculation in liquid culture.  
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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).<br>
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This experiment is a prerequisite for our next experiment.
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<br><br>
 
<br><br>
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<strong>Growth of tryptophan auxotrophic yeast in minimal medium subsequent to tryptophan producing E.coli</strong>
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−
<br><br>
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Subsequently plasmid were <a href="https://static.igem.org/mediawiki/2017/c/c6/Protocol3_UCopenhagen.pdf">cured</a> from the cultures and <a href="https://static.igem.org/mediawiki/2017/2/2e/Protocol1_UCopenhagen.pdf">PCR</a> amplification and/or restriction <a href="https://static.igem.org/mediawiki/2017/a/ab/Protocol13_UCopenhagen.pdf">digestion</a>was used to check for correct insertion. Cells with appropriately sized inserts were sent to <a href="https://static.igem.org/mediawiki/2017/9/9f/Protocol17_UCopenhagen.pdf">sequencing</a> for sequence validation.  
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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.
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               </p>
 
               </p>
 
     </div>
 
     </div>

Revision as of 12:42, 1 November 2017

I N T E R D E P E N D E N C Y

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.

Final Design

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:

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.





Figure 1 AroG and TrpE location in aromatic amino acid biosynthesis: AroG initiates the shikimate pathway, and is inhibited by Phenylalanine. TrpE is the first protein in the trp operon and inhibited by tryptophan. The red crosses indicate feedback inhibition removed by the point mutations.

Experiments

Overview

Amplification of genes:
  • Synthetically produce a codon-optimised version of yddG
  • Amplify trpE and aroG with point mutations
  • Multi-gene constructs inserted in vector

Serial growth of E. coli and yeast

  • Identify a minimal media supporting both E. coli and yeast growth
  • Analyse auxotrophic yeast growth in the minimal media both with and without tryptophan added
  • Grow auxotrophic yeast in media only containing tryptophan produced by E.coli

Gene expression and tryptophan production

  • Western blotting to check expression of his-tagged proteins
  • HPLC-MS measurements of tryptophan production

Verifications

We have used gel electrophoresis across most of our experiments and sub-projects to continually monitor our progress.

All transformations in interdependency and protein import were done with USER cloningand the cells were grown ON on LB agar with the appropriate selection (Ampicillin or Chloramphenicol depending on the resistance marker). Colony PCRs were performed to pick colonies with the correct inserts for inoculation in liquid culture.

Subsequently plasmid were cured from the cultures and PCR amplification and/or restriction digestionwas used to check for correct insertion. Cells with appropriately sized inserts were sent to sequencing for sequence validation.

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.

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