Difference between revisions of "Team:Uppsala/Zea-Strain"

Line 62: Line 62:
 
     <div class= "col-xs-1"></div>
 
     <div class= "col-xs-1"></div>
 
     <div class= "col-xs-10">
 
     <div class= "col-xs-10">
−
       <div style="padding-bottom:3%; padding-top:3%"> Zeaxanthin is a powerful antioxidant and one of the most common carotenoids found in nature [1]. A high concentration of zeaxanthin can be found in macula lutea which gives it a characteristic yellow color. There are multiple studies that suggest its important role in eye health [2]. Due to its commercial value there were many successful attempts to produce synthetic zeaxanthin. The Edinburgh iGem team from 2007 constructed a biobrick containing an operon
+
       <div style="padding-bottom:3%; padding-top:3%"> In our project we chose to concentrate on the pathway that leads from farnesyl pyrophosphate (FPP) to crocin. The whole pathway consists of eight genes that code for eight enzymes which might make integrating all of the genes into a plasmid and keeping the plasmid in the bacteria more difficult. Dividing the pathway and integrating the part that leads from FPP to zeaxanthin into the chromosome would both give us a stable zeaxanthin-producing E.coli strain and make performing the remaining steps easier. </div>
−
with the genes necessary for zeaxanthin production (Fig.1) [3].</div>
+
 
 +
      <div class="miniheader"> Resulting Zeaxanthin Producing E. coli Strain </div>
 +
      <div style="padding-bottom:3%"> Our team has successfully integrated the whole zeaxanthin pathway into the chromosome using lambda red recombineering (figure 1) and extracted zeaxanthin (figure 3). This will hopefully give other iGEM teams more freedom to work with and build on carotenoid pathways and make zeaxanthin more affordable to use in experiments. </div>
 +
 
  
 
       <figure class="figure">
 
       <figure class="figure">
−
       <img src="https://static.igem.org/mediawiki/2017/d/d6/Uppsala-Zeaxanthin-Pathway.png" style="display: block; margin: auto; width:60%; height: auto; padding-bottom:3%">
+
       <img src="https://static.igem.org/mediawiki/2017/1/18/Uppsala-ZeaPlate.png" style="display: block; margin: auto; width:60%; height: auto; padding-bottom:3%">
−
         <figcaption class="figure-caption figtext" style="padding-bottom: 2%; text-align:center;"> Figure 1. Operon that was used by the Edinburgh iGEM team of 2007 with its respective gene products that are needed for the production for Zeaxanthin.</figcaption>
+
         <figcaption class="figure-caption figtext" style="padding-bottom: 2%; text-align:center;"> Figure 1. Top: Wild-type E. coli. Bottom: Zeaxanthin producing E. coli strain with 5 genes inserted into the chromosome.</figcaption>
 
       </figure>
 
       </figure>
 
   
 
   
  
−
       <div style="padding-bottom:3%"> Later in 2013 this operon was used by the iGem team in Uppsala for their project [4]. Zeaxanthin is also a precursor to crocin - the substance our team was working on. Using this operon could potentially result in a crocin producing E.coli strain. Our problem was the size of this operon that if combined with additional genes of the crocin pathway could make this new strain unstable. That is why the Lambda red recombineering method was chosen for integration of the zeaxanthin pathway into E.coli chromosome instead of a plasmid. Our aim was to create a stable zeaxanthin producing strain with no need for selective pressure and then integrate a plasmid containing the crocin pathway.</div>
+
       <div style="padding-bottom:3%"> We also combined this strain with the BioBrick that contains the crocin pathway. To see the combined result, click over to the <a href="https://2017.igem.org/Team:Uppsala/Results">Result page</a>. In the future it would be good to integrate the whole pathway from Farnesyl pyrophosphate (FPP) to crocin into the chromosome for a stable crocin producing strain that does not require antibiotic selection which would make it easier to use as for example food coloring. </div>
−
       <div style="padding-bottom:3%"> Lambda red recombineering is based on homologous recombination which is mediated by bacteriophage lambda proteins [5]. In case of recombination with double-stranded DNA three different proteins: Exo, Gam and Beta derived from lambda red phage are required. Protein Exo degrades double-stranded linear DNA while Gam protects it from endogenous nucleases. Protein Beta is a key protein in the recombination process. It protects single-stranded DNA which was created by Exo and facilitates annealing to a complementary strand [6]. Lambda red recombination usually requires only about 35 base pairs of homology on both sides to work.[picture with lambda red enzyme functions?]*</div>
+
      <div class="miniheader"> How we did it </div>
−
       <div style="padding-bottom:3%"> Transduction is a well-established method which is used to insert DNA into bacterial strains. There are two types of outcomes following a transduction either the inserted DNA integrates into the chromosome of the host cell which is called the lysogenic life cycle or the bacteriophage mediate the lysis of the bacterial cell after a brief replication in an extrachromosomal form. For our experiment, Enterobacteria phage P1(P1) were used to perform transductions to assemble our genes. P1 is a temperate phage that replicates as an episomal replicon in the bacterial host until it lysis the cell. During the P1 replication bacterial genes can be inserted into the empty phage particles. For these Lambda Red mediated insertions, each bacterial strain had a Cat-sacB gene cassette in their chromosome which would be displaced by one of the previously mentioned genes. To get only one strain containing all the Crt genes two transductions had to be performed. One transduction to insert a new Cat-sacB gene cassette into an CrtI containing E. coli strain and a second transduction which would insert CrtZY together with a Trimethoprim resistance into a CrtEBI containing strain.[Flow chart?]*</div>
+
      <div style="padding-bottom:3%"> Zeaxanthin has previously been expressed in E. coli by iGEM teams using a plasmid. We decided to integrate this pathway into the chromosome using the Lambda red recombineering method(Link Lambda red protocol). This would give our project several advantages such as releasing all the plasmid origins and cassettes which would make the insertion of the crocin pathway genes or any other genes of a pathway that originates from zeaxanthin easier. It would make the strain more stable because no constant selective pressure is needed and makes it possible to introduce larger constructs and longer pathways. This also means that there is no need for the use of antibiotics which makes the purification process easier, especially if the product is later used for nutritional purposes. And of course since the first step of our zeaxanthin pathway – farnesyl pyrophosphate – is endogenous to E.coli we would be able to express the whole pathway from farnesyl pyrophosphate to crocin with no need for costly intermediates. </div>
 +
       <div style="padding-bottom:3%"> If we use cat-sacB selection/counterselection in Lambda red recombineering (which we did) we get a scarless method that does not leave behind resistance markers. Lambda red is based on homologous recombination which is mediated by bacteriophage lambda proteins and usually requires only about 35 base pairs of homology on both sides of the inserted gene to work. </div>
 +
       <div style="padding-bottom:3%"> The pathway that leads from FPP to zeaxanthin (figure 2) includes five genes: crtE, crtB, crtI, crtY and crtZ. All these genes have been previously BioBricked into an operon for E. coli. Due to the time limitations of the project we decided to synthesize two constructs each containing two of the genes - crtEB and crtZY. For successful integration with Lambda red recombination the inserts should not exceed the size of 3000 bp and that is why we could not synthesize all of the genes as a single construct. Each construct also contained promoter, ribosome-binding sites and the necessary homologies for the future integration into the strains. The crtI gene was amplified from BioBrick (iGEM Slovenia 2010) with the primers that contained the homologies.</div>
 +
      <figure class="figure">
 +
      <img src="https://static.igem.org/mediawiki/2017/a/ab/Uppsala-ZeaPathway.png" style="display: block; margin: auto; width:60%; height: auto; padding-bottom:3%">
 +
        <figcaption class="figure-caption figtext" style="padding-bottom: 2%; text-align:center;"> Figure 2. The pathway from farnesyl pyrophospate to zeaxanthin.
 +
</figcaption>
 +
      </figure>
 +
      <div style="padding-bottom:3%"> The E. coli strains that were used for recombination contained a pSIM5-tet temperature sensitive plasmid with the lambda red system and tetracycline resistance. When temperature rises above 37°C the lambda red enzymes are expressed. They also had cat-sacB selection cassette (based on Uppsalas own BioBrick BBa_K864150(link this)) carrying chloramphenicol resistance gene and Bacillus subtilis levansucrase sacB gene that is lethal for gram-negative bacteria when expressed in presence of sucrose. </div>
 +
 
 +
      <div style="padding-bottom:3%"> Three different starting strains were used during the experiments. We also used three different promoters to avoid homology among these regions. The strain genotypes were:</div>
 +
 
 +
      <div style="padding-bottom:2%"> 1. Eco ∆gsp::cat-sacB /pSIM5-tet</div>
 +
      <div style="padding-bottom:2%"> 2. Eco ∆bglGFB::cat-sacB /pSIM6</div>
 +
      <div style="padding-bottom:3%"> 3. Eco ∆IS150::CP25-cat-sacB /pSIM5-tet</div>
 +
 
 +
      <div style="padding-bottom:3%"> Lambda red recombineering was performed in three steps. For more detailed information, please, see the protocol for Lambda red(link) and the Lab Notebook(link).
 +
</div>
 +
 
 
     </div>
 
     </div>
 
     <div class= "col-xs-1"></div>
 
     <div class= "col-xs-1"></div>

Revision as of 16:09, 31 October 2017

Zeaxanthin

PROJECT DESCRIPTION
ZEA-STRAIN
In our project we chose to concentrate on the pathway that leads from farnesyl pyrophosphate (FPP) to crocin. The whole pathway consists of eight genes that code for eight enzymes which might make integrating all of the genes into a plasmid and keeping the plasmid in the bacteria more difficult. Dividing the pathway and integrating the part that leads from FPP to zeaxanthin into the chromosome would both give us a stable zeaxanthin-producing E.coli strain and make performing the remaining steps easier.
Resulting Zeaxanthin Producing E. coli Strain
Our team has successfully integrated the whole zeaxanthin pathway into the chromosome using lambda red recombineering (figure 1) and extracted zeaxanthin (figure 3). This will hopefully give other iGEM teams more freedom to work with and build on carotenoid pathways and make zeaxanthin more affordable to use in experiments.
Figure 1. Top: Wild-type E. coli. Bottom: Zeaxanthin producing E. coli strain with 5 genes inserted into the chromosome.
We also combined this strain with the BioBrick that contains the crocin pathway. To see the combined result, click over to the Result page. In the future it would be good to integrate the whole pathway from Farnesyl pyrophosphate (FPP) to crocin into the chromosome for a stable crocin producing strain that does not require antibiotic selection which would make it easier to use as for example food coloring.
How we did it
Zeaxanthin has previously been expressed in E. coli by iGEM teams using a plasmid. We decided to integrate this pathway into the chromosome using the Lambda red recombineering method(Link Lambda red protocol). This would give our project several advantages such as releasing all the plasmid origins and cassettes which would make the insertion of the crocin pathway genes or any other genes of a pathway that originates from zeaxanthin easier. It would make the strain more stable because no constant selective pressure is needed and makes it possible to introduce larger constructs and longer pathways. This also means that there is no need for the use of antibiotics which makes the purification process easier, especially if the product is later used for nutritional purposes. And of course since the first step of our zeaxanthin pathway – farnesyl pyrophosphate – is endogenous to E.coli we would be able to express the whole pathway from farnesyl pyrophosphate to crocin with no need for costly intermediates.
If we use cat-sacB selection/counterselection in Lambda red recombineering (which we did) we get a scarless method that does not leave behind resistance markers. Lambda red is based on homologous recombination which is mediated by bacteriophage lambda proteins and usually requires only about 35 base pairs of homology on both sides of the inserted gene to work.
The pathway that leads from FPP to zeaxanthin (figure 2) includes five genes: crtE, crtB, crtI, crtY and crtZ. All these genes have been previously BioBricked into an operon for E. coli. Due to the time limitations of the project we decided to synthesize two constructs each containing two of the genes - crtEB and crtZY. For successful integration with Lambda red recombination the inserts should not exceed the size of 3000 bp and that is why we could not synthesize all of the genes as a single construct. Each construct also contained promoter, ribosome-binding sites and the necessary homologies for the future integration into the strains. The crtI gene was amplified from BioBrick (iGEM Slovenia 2010) with the primers that contained the homologies.
Figure 2. The pathway from farnesyl pyrophospate to zeaxanthin.
The E. coli strains that were used for recombination contained a pSIM5-tet temperature sensitive plasmid with the lambda red system and tetracycline resistance. When temperature rises above 37°C the lambda red enzymes are expressed. They also had cat-sacB selection cassette (based on Uppsalas own BioBrick BBa_K864150(link this)) carrying chloramphenicol resistance gene and Bacillus subtilis levansucrase sacB gene that is lethal for gram-negative bacteria when expressed in presence of sucrose.
Three different starting strains were used during the experiments. We also used three different promoters to avoid homology among these regions. The strain genotypes were:
1. Eco ∆gsp::cat-sacB /pSIM5-tet
2. Eco ∆bglGFB::cat-sacB /pSIM6
3. Eco ∆IS150::CP25-cat-sacB /pSIM5-tet
Lambda red recombineering was performed in three steps. For more detailed information, please, see the protocol for Lambda red(link) and the Lab Notebook(link).
References
(1) http://lpi.oregonstate.edu/ - micronutrient information center - dietary factors-carotenoids - 2017-09- 30
(2) Eisenhauer, B.; Natoli, S.; Liew, G.; Flood, V.M. Lutein and zeaxanthin-food sources, bioavailability and dietary variety in age-related macular degeneration protection. Nutrients 2017, 9, 120.
(3) https://2007.igem.org/ - edinburgh -yoghurt- design - 2017-09- 29
(4) https://2013.igem.org/ - project -metabolic engineering - zeaxanthin -2017- 09-27
(5) Ellis, H. M., D. Yu, T. DiTizio & D. L. Court, (2001) High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides. Proc. Natl. Acad. Sci. USA 98: 6742-6746.
(6) Mosberg JA, Lajoie MJ, Church GM. (2010) Lambda red recombineering in Escherichia coli occurs through a fully single-stranded intermediate. Genetics 186: 791–799.