Difference between revisions of "Team:UCopenhagen/Interdependency"

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                    <h2 class="section-heading">Introduction </h2>
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                    <p class="lead">Our team believes that establishing a stable platform for scientists to create naïve orthogonal living compartments, would allow for an unpredictable advancement in the field of synthetic biology. Our project will not attempt to create an endosymbiont, but instead investigate the mechanisms in free-living cells in a bottom-up approach to endosymbiosis.  <br><br>
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The endosymbiotic theory, formulated in the early years of the previous century, outlines that the organelles of the eukaryotic cell, such as the mitochondria, have their origin in free-living prokaryotes engulfed by bigger cells. These incorporated cells then co-evolved with their host conferring to it novel emergent properties which ultimately helped fuel the development of more complex multicellular biological systems such as plants and animals (Archibald, 2015). <br><br>
  
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We have identified three mechanisms we believe to be mandatory for the development of a stable endosymbiotic relationship, which we will be trying to replicate in free-living cells. First of all, in order for the relationship to be stable, the two organisms must be mutually dependent on each other; there must be a mutually beneficial interaction between host and symbiont. Secondly, there has to be some sort of control and synchronization of symbiont replication. If the symbiont were to be replicating freely we could end up with way too many or not enough symbionts in the host.  Finally, a common feature of the endosymbiotic organelles we have looked at, is the transfer of genes from the symbiont to the host. Because of this transfer, the gene and protein expression is taking place in the nucleus and the proteins and metabolites are transported to the organelle. This import of proteins is interesting not just for understanding endosymbiosis, but also for the potential applications in synthetic biology.<br><br>
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                    <p class="lead"> 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.
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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.  
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In this subproject, we will use yeast as a substitute for the host, and E.coli as a substitute for an endosymbiont.
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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.
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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.
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Based on these considerations, we decided to work on three distinct, but intertwined, projects pertaining to endosymbiosis, namely Interdependence, Number Control, and Protein import. We believe that by combining these three projects, a key step towards the understanding of endosymbiosis and its employment in synthetic biology will be obtained. </p>
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<p class="lead"><strong>Natural endosymbiosis (our model)</strong> Shortly: what is endosymbiosis?
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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.
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<strong> Synthetic endosymbiosis </strong> will have innumerable applications if developed to be the orthogonal system we envision. We have chosen to take a bottom-up approach to it, through the investigation of the following three essential mechanisms.  
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We have made a model of the tryptophane use and production in yeast and E.coli to check how many endosymbionts would be necessary.
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Add something about how our idea started?
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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.  
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<strong> Interdependence </strong> between host and endosymbiont is necessary in order for the endosymbiotic relationship to be stable and maintained throughout evolution or, more relevant for synthetic biology, through generations. The relationship should be beneficial and crucial for the host and the endosymbiont alike. Interdependency would thus decrease possible safety concerns: without the dependency relationship, the endosymbiotic relationship will not spread and be maintained in a wild population.
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trpE belongs to the tryptophan operon and has been over-expressed frequently in L-tryptophan producing E. coli strains.
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<strong>Number control</strong> addresses our concern that the endosymbiont will thrive too well in the host, and replicate uncontrollably - thus overwhelming the host system - or be lost in host replications. Thus we strive for a way to maintain a stable number of endosymbionts in the system. A control system that repress endosymbiont reproduction in high concentration/numbers, and allow replication when the concentration is decreased, would address this concern.
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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.  
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<strong>Protein import</strong> is a common trait observed in naturally occurring endosymbiotic relationships, such as mitochondria and chloroplasts. In these cases a down regulation of protein expression in the symbiont is observed, and the protein expression is instead moved to the cell. Proteins expressed in mitochondria and chloroplasts are transcribed in the nucleus, translated in the cytoplasm and subsequently transported across the cell membranes into the endosymbionts. This mechanism would tighten the relationship between host and symbiont, and would allow endosymbiotic manipulation of precursors produced in the host. </p>
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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.
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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.  
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<h4>Amplification of trpE and aroG from wild type</h4> 
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<p>We amplified yddG, trpE and aroG from wildtype E. coli MG1655, using 'method'  </p>
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<h3>Point mutations trpE and aroG</h3>
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<p>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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Vector design was performed in the protein import subproject, and the same vector was used for all cloning in the interdependency project.</p>
 
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<p>We checked expression of the genes using western blot, as all genes are HIS tagged in the vector (protocol)</p>
 
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<h3>Evaluation of E.coli tryptophan production by liquid chromatography (LC)</h3>
 
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<p>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.
 
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<br> These results will be part of the modelling </p>
 
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<h3>Growth of E.coli and yeast in same minimal yeast medium</h3>
 
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<p>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.
 
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<br> 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).
 
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<br> This experiment is a prerequisite for our next experiment. </p>
 
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<h3>Growth of tryptophan auxotrophic yeast in minimal medium subsequent to tryptophan producing E.coli</h3>
 
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<p>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.
 
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<br>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.
 
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<br>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.
 
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                    <p>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.
 
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Revision as of 17:06, 21 October 2017

P R O J E C T

Introduction

Our team believes that establishing a stable platform for scientists to create naïve orthogonal living compartments, would allow for an unpredictable advancement in the field of synthetic biology. Our project will not attempt to create an endosymbiont, but instead investigate the mechanisms in free-living cells in a bottom-up approach to endosymbiosis.

The endosymbiotic theory, formulated in the early years of the previous century, outlines that the organelles of the eukaryotic cell, such as the mitochondria, have their origin in free-living prokaryotes engulfed by bigger cells. These incorporated cells then co-evolved with their host conferring to it novel emergent properties which ultimately helped fuel the development of more complex multicellular biological systems such as plants and animals (Archibald, 2015).

We have identified three mechanisms we believe to be mandatory for the development of a stable endosymbiotic relationship, which we will be trying to replicate in free-living cells. First of all, in order for the relationship to be stable, the two organisms must be mutually dependent on each other; there must be a mutually beneficial interaction between host and symbiont. Secondly, there has to be some sort of control and synchronization of symbiont replication. If the symbiont were to be replicating freely we could end up with way too many or not enough symbionts in the host. Finally, a common feature of the endosymbiotic organelles we have looked at, is the transfer of genes from the symbiont to the host. Because of this transfer, the gene and protein expression is taking place in the nucleus and the proteins and metabolites are transported to the organelle. This import of proteins is interesting not just for understanding endosymbiosis, but also for the potential applications in synthetic biology.

Based on these considerations, we decided to work on three distinct, but intertwined, projects pertaining to endosymbiosis, namely Interdependence, Number Control, and Protein import. We believe that by combining these three projects, a key step towards the understanding of endosymbiosis and its employment in synthetic biology will be obtained.



Sub-projects

Natural endosymbiosis (our model) Shortly: what is endosymbiosis?

Synthetic endosymbiosis will have innumerable applications if developed to be the orthogonal system we envision. We have chosen to take a bottom-up approach to it, through the investigation of the following three essential mechanisms. Add something about how our idea started?

Interdependence between host and endosymbiont is necessary in order for the endosymbiotic relationship to be stable and maintained throughout evolution or, more relevant for synthetic biology, through generations. The relationship should be beneficial and crucial for the host and the endosymbiont alike. Interdependency would thus decrease possible safety concerns: without the dependency relationship, the endosymbiotic relationship will not spread and be maintained in a wild population.

Number control addresses our concern that the endosymbiont will thrive too well in the host, and replicate uncontrollably - thus overwhelming the host system - or be lost in host replications. Thus we strive for a way to maintain a stable number of endosymbionts in the system. A control system that repress endosymbiont reproduction in high concentration/numbers, and allow replication when the concentration is decreased, would address this concern.

Protein import is a common trait observed in naturally occurring endosymbiotic relationships, such as mitochondria and chloroplasts. In these cases a down regulation of protein expression in the symbiont is observed, and the protein expression is instead moved to the cell. Proteins expressed in mitochondria and chloroplasts are transcribed in the nucleus, translated in the cytoplasm and subsequently transported across the cell membranes into the endosymbionts. This mechanism would tighten the relationship between host and symbiont, and would allow endosymbiotic manipulation of precursors produced in the host.



Find Incell here: