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    <div class="mainheader"> ZEAXANTHIN STRAIN </div>
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      <div style="padding-bottom: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 <i>E. coli</i> strain and make performing the remaining steps easier.
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      <div class="miniheader"> Resulting Zeaxanthin Producing <i>E. coli</i> Strain </div>
      <div style="padding-bottom:3%">We created a zeaxanthin producing <i>E. coli</i> strain using lambda red recombineering (figure 1), with the whole pathway from FPP to zeaxanthin integrated into the chromosome. All of the steps were confirmed with PCR, gel electrophoresis and sequencing. We were able to extract and purify the expensive yellow zeaxanthin compound from our strain (figure 3). After creating the zeaxanthin strain, we combined it with the plasmid containing the extended crocin pathway which gave us an <i>E. coli</i> strain including the entire production pathway from FPP to crocin. 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>

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      <img src="https://static.igem.org/mediawiki/2017/1/18/Uppsala-ZeaPlate.png" style="display: block; margin: auto; width:40%; height: auto;">
        <figcaption class="figure-caption figtext" style="padding-bottom: 2%; text-align:center;"> Figure 1. Top: Wild-type <i>E. coli</i>. Bottom: Zeaxanthin producing <i>E. coli</i> strain with 5 genes inserted into the chromosome.</figcaption>
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      <div class="miniheader"> How we did it </div>
      <div style="padding-bottom:3%"> Zeaxanthin has previously been expressed in <i>E. coli</i> by iGEM teams using a plasmid. We decided to integrate this pathway into the chromosome using the <a href="https://2017.igem.org/Team:Uppsala/Experiments">Lambda red recombineering method</a>. 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 <i>E. coli</i> 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 <i>cat-sacB</i> 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: <i>crtE</i>, <i>crtB</i>, <i>crtI</i>, <i>crtY</i> and <i>crtZ</i>. All these genes have been previously BioBricked into an operon for <i>E. coli</i>. Due to the time limitations of the project we decided to synthesize two constructs each containing two of the genes – <i>crtEB</i> and <i>crtZY</i>. 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 <i>crtI</i> gene was amplified from a <a href="http://parts.igem.org/Part:BBa_K323125">BioBrick</a> (iGEM Slovenia 2010), without the zinc finger domain, with the primers that contained the homologous sites.</div>

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      <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.
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      <div style="padding-bottom:3%"> The <i>E. coli</i> 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 <i>cat-sacB</i> selection cassette (based on Uppsalas own BioBrick <a href="http://parts.igem.org/Part:BBa_K864150">BBa_K864150</a>) carrying chloramphenicol resistance gene and <i>Bacillus subtilis</i> levansucrase <i>sacB</i> 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 <i>∆gsp::cat-sacB /pSIM5-tet</i></div>
       <div style="padding-bottom:2%"> 2. Eco <i>∆bglGFB::cat-sacB /pSIM6</i></div>
       <div style="padding-bottom:3%"> 3. Eco <i>∆IS150::CP25-cat-sacB /pSIM5-tet</i></div>

       <div style="padding-bottom:3%"> Lambda red recombineering was performed in three steps. For more detailed information, please, see the protocol for <a href="https://2017.igem.org/Team:Uppsala/Experiments">Lambda red</a> and the <a href="https://2017.igem.org/Team:Uppsala/Notebook">Lab Notebook</a>.
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      <div class="miniheader"> Step 1 </div>
      <div style="padding-bottom:3%"> Phytoene dehydrogenase gene <i>crtI</i> was amplified with PCR from zeaxanthin BioBrick and the homologies were added with the primers. Lambda red was performed to replace <i>cat-sacB-T0</i> selection cassette with <i>crtI</i>, and the cells were plated on sucrose agar plates to select for transformants that had successfully integrated the <i>crtI</i> gene.</div>
      <div style="padding-bottom:3%"> Eco <i>∆IS150::CP25-cat-sacB-T0 /pSIM5-tet</i> + <i>crtI</i> (PCR product) →  Lambda Red (<i>sucR</i>) →  Eco <i>∆IS150::CP25-crtI /pSIM5-tet</i></div>
      <div style="padding-bottom:3%"> A construct with beta-carotene hydroxylase gene <i>crtZ</i> and lycopene cyclase gene <i>crtY</i> was inserted with lambda red into another <i>E. coli</i> strain. </div>
      <div style="padding-bottom:3%">Eco <i>∆gsp::cat-sacB /pSIM5-tet</i> + <i>crtZY</i> (PCR product) → Lambda Red (<i>sucR</i>) →  Eco <i>∆gsp::pFAB70-crtZY /pSIM5-tet</i> </div>

      <div class="miniheader"> Step 2 </div>
      <div style="padding-bottom:3%"> The trimethoprim resistance gene <i>dhfr</i> (dihydrofolate reductase) was inserted with lambda red into the strain that now contained <i>crtZY</i> genes. The genes we placed very close to each other to enable the following transduction with P1 bacteriophage.</div>
      <div style="padding-bottom:3%"> Eco <i>∆gsp::pFAB70-crtZY /pSIM5-tet</i> + <i>dhfr</i> (PCR product) →  Lambda Red (<i>tmpR</i>) →  Eco <i>∆gsp::pFAB70-crtZY gspC::dhfr</i></div>
      <div style="padding-bottom:3%"> Transduction with P1 bacteriophage was done to transfer <i>cat-sacB-T0</i> selection cassette from a donor strain into the strain containing <i>crtI</i> gene.</div>
      <div style="padding-bottom:3%"> Eco <i>∆IS150::CP25-crtI /pSIM5-tet</i> + <i>∆bglGFB::cat-sacB</i> (P1 lysate) →  Transduction (<i>camR</i>) →  Eco <i>∆IS150::CP25-crtI ∆bgl::cat-sacB /pSIM5-tet</i></div>


      <div class="miniheader"> Step 3 </div>
      <div style="padding-bottom:3%"> Lambda red was performed to replace this newly inserted <i>cat-sacB-T0</i> cassette with a construct containing geranylgeranyl diphosphate synthase gene <i>crtE</i> and phytoene synthase gene <i>crtB</i>. The clones with the correct sequence changed color to light red. Our lycopene producing strain was constructed!</div>
      <div style="padding-bottom:3%"> Eco <i>∆IS150::CP25-crtI ∆bgl::cat-sacB /pSIM5-tet</i> + <i>pFAB46-crtEB</i> (PCR product) → Lambda Red (<i>sucR</i>) →  Eco <i>∆IS150::CP25-crtI ∆bgl::pFAB46-crtEB</i> →  Lycopene!</div>
      <div style="padding-bottom:3%"> Another transduction with P1 bacteriophage was done with the strain containing <i>crtZY</i> as a donor strain and the strain containing <i>crtEBI</i> as a recipient strain. The clones with the correct sequence turned bright yellow. 
      <div style="padding-bottom:3%"> Eco <i>∆IS150::CP25-crtI ∆bgl::pFAB46-crtEB</i> + <i>∆gsp::pFAB70-crtZY gspC::dhfr</i> (P1 lysate) →  Transduction (<i>tmpR</i>) →  Eco <i>IS150::CP25-crtI ∆bgl::pFAB46-crtEB ∆gsp::pFAB70-crtZY gspC::dhfr</i> →  Zeaxanthin! </div>

      <div style="padding-bottom:3%"> <b>We got our zeaxanthin producing strain with the whole pathway from FPP to zeaxanthin integrated into the chromosome!</b> All of the steps were confirmed with PCR, gel electrophoresis and sequencing. We were able to extract and purify the expensive yellow zeaxanthin compound from our strain, see figure 3.</div>

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        <img src="https://static.igem.org/mediawiki/2017/0/0d/Uppsala-ZeaBottle.png" class="figure-img img-fluid picturerow">
        <img src="https://static.igem.org/mediawiki/2017/b/bf/Uppsala-ZeaTube.png" class="figure-img img-fluid picturerow">
        <figcaption class="figure-caption figtext" style="padding-bottom: 3%; padding-left:0%"> Figure 3. Left: Large scale expression of zeaxanthin from the zeaxanthin producing <i>E. coli</i> strain. Right: Extracted and purified zeaxanthin.</figcaption>
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      <div style="padding-bottom:3%"> Besides observing the extracted zeaxanthin by eye we performed an absorbance measurement in the UV-Vis spectra (figure 4). Here we compared the absorbance spectra after extraction from the zeaxanthin strain, from wildtype <i>E. coli</i> and a zeaxanthin standard. The zeaxanthin strain had two peaks at 460 and 482 nm which were not present in wildtype <i>E. coli</i>. These peaks were also present in the standard, therefore we can conclude that our produced strain produces zeaxanthin. For the measurements the extracted compounds were dissolved in toluene.</div>

      <figure class="figure">
      <img src="https://static.igem.org/mediawiki/2017/c/c2/Extracted_Zeaxanthin.png" style="display: block; margin: auto; width:60%; height: auto;">
        <figcaption class="figure-caption figtext" style="padding-bottom: 2%; text-align:center;"> Figure 4. Absorbance spectra for the extraction of zeaxanthin. Mg1665 constitutes the negative control (the same extraction protocol on wildtype <i>E. coli</i>).</figcaption>


      <div class="miniheader">Combining the Zeaxanthin Producing Strain and the Crocin Pathway Enzymes</div>
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After creating the zeaxanthin producing strain, we combined it with the plasmid containing the extended crocin pathway which gave us an <i>E. coli</i> strain including the entire production pathway from FPP to crocin. The three enzyme BioBricks in the zeaxanthin-crocin pathway were assembled to one plasmid using 3A assembly(link 3A assembly protocol) and was inserted into the zeaxanthin producing E.coli strain using electroporation(link Electroporation protocol). The resulting plate can be seen in figure X. </div>
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      <img src="https://static.igem.org/mediawiki/2017/1/18/Uppsala-ZeaPlate.png" style="display: block; margin: auto; width:60%; height: auto;">
        <figcaption class="figure-caption figtext" style="padding-bottom: 2%; text-align:center;"> Figure 4. Plate with Zeaxanthin expressing E. coli strain transformed with plasmid containing all three crocin pathway enzymes CaCCD2, CsADH2946 and UGTCs2.</figcaption>
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The color of the colonies changes slightly at each addition of another enzyme construct (another step in the crocin pathway). This is an indication that something is indeed happening with the bacterial production when we introduce our pathway steps, see figure X. 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>
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<div style="padding-bottom:3%"> We wanted to analyze the compounds found after extraction of zeaxinthin strains with and without plasmids from the crocin pathway. Thin layer chromatography (TLC) was used to do this on a zeaxanthin strain, a zeaxanthin strain containing <a href="https://2017.igem.org/Team:Uppsala/Parts">BBa_K2423005</a>, a zeaxanthin containing a plasmid with both CaCCD2 and CsADH2946, standards for crocetin and crocetin dialdehyde and a wild-type <i>E. coli</i> strain.  
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      <img src="https://static.igem.org/mediawiki/2017/d/d0/TLC_Uppsala.png" style="display:block; margin: auto; width:60%; height: auto;">
      <figcaption class="figure-caption figtext" style="padding-bottom: 2%; text-align:center;"> Figure 5. Thin layer chromatography for zeaxanthin producing strains with and without BioBricks in the crocin pathway, standards for crocetin, crocetin dialdehyde and negative control strain (MG1665). Samples from left to right: zeaxanthin producing strain, zeaxanthin producing strain with <a href="https://2017.igem.org/Team:Uppsala/Parts">BBa_K2423005</a>, zeaxanthin producing strain with a combined CaCCD2 and CsADH2946 plasmid, crocetin, crocetin dialdehyde and MG1665.</figcaption>


      <div style="padding-bottom:3%"> Our team would like to express a special appreciation and thanks to Erik Wistrand-Yuen who provided us with the starting strains and the protocols for the lambda red method and who spent hours guiding and instructing us and providing practical help. Without him this success would not have been possible. </div>

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