<div style="padding-bottom:3%"> The zeaxanthin producing strain is important starting point of our project. However, there was a previous concern about the production not being stable and optimal. Therefore, we decided to test production of protein and plasmid DNA under different conditions. For the testing we chose three different temperatures (30 °C, 37 °C and 42 °C) and four different media (LB, TB, M9 and SOB). As LB is usually the standard media for experiments. We decided to use LB with the basic E. coli strain of DH5α to give a baseline for our measurements. We have also tested two super-rich media (TB and SOB) in attempt to produce higher yield of bacteria in shorter time. To test the possible growth in minimal media we chose M9. </div>
<div style="padding-bottom:3%"> The zeaxanthin producing strain is important starting point of our project. However, there was a previous concern about the production not being stable and optimal. Therefore, we decided to test production of protein and plasmid DNA under different conditions. For the testing we chose three different temperatures (30 °C, 37 °C and 42 °C) and four different media (LB, TB, M9 and SOB). As LB is usually the standard media for experiments. We decided to use LB with the basic E. coli strain of DH5α to give a baseline for our measurements. We have also tested two super-rich media (TB and SOB) in attempt to produce higher yield of bacteria in shorter time. To test the possible growth in minimal media we chose M9. </div>
<div class="miniheader"> How we did it </div>
<div class="miniheader"> How we did it </div>
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<div style="padding-bottom:3%"> Zeaxanthin has previously been expressed in E. coli by iGEM teams using a plasmid. We have transformed the plasmid into DH5alpha cells and tested by colony PCR. The positive colonies were sent to sequencing which came out positive. Then we used the positive colonies to for Growth assay testing. </div>
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<div style="padding-bottom:3%"> Zeaxanthin has previously been expressed in E. coli by iGEM teams using a plasmid. We have transformed the plasmid into DH5alpha cells and tested by colony PCR. The positive colonies were sent to sequencing which came out positive. Then we used the positive colonies to for Growth assay testing.
<figcaption class="figure-caption figtext" style="padding-bottom: 2%; text-align:center;"> figure 1.Colony PCR after transformation of the zeaxanthin strain. Marker used is 1 Kb.</figcaption>
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</figure>
<div class="miniheader"> Growth Assay </div>
<div class="miniheader"> Growth Assay </div>
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The transform strain was inoculated into overnight culture of 5 ml in LB, TB, M9 and SOB with chloramphenicol. DH5-alpha strain was grown overnight in LB. All the cultures were grown at 37 °C on a shaker. Then they were inoculated into fresh media to a total volume of 100 ml and OD600 of 0.05. M9 culture did not results in any growth on two occasion and was discarded as the strain probably is not capable of growing on minimal media. Cultures were placed on a shaker in three temperatures (30°C, 37°C and 40°C) and induced with IPTG to a final concentration of 1mM. After inoculating the OD600 was measured every 30 minutes for 350 minutes. </div>
The transform strain was inoculated into overnight culture of 5 ml in LB, TB, M9 and SOB with chloramphenicol. DH5-alpha strain was grown overnight in LB. All the cultures were grown at 37 °C on a shaker. Then they were inoculated into fresh media to a total volume of 100 ml and OD600 of 0.05. M9 culture did not results in any growth on two occasion and was discarded as the strain probably is not capable of growing on minimal media. Cultures were placed on a shaker in three temperatures (30°C, 37°C and 40°C) and induced with IPTG to a final concentration of 1mM. After inoculating the OD600 was measured every 30 minutes for 350 minutes. </div>
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<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 <a href="http://parts.igem.org/Part:BBa_K864150">BBa_K864150</a>) carrying chloramphenicol resistance gene and Bacillus subtilis levansucrase sacB gene that is lethal for gram-negative bacteria when expressed in presence of sucrose. </div>
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<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: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>.
<div style="padding-bottom:3%"> Phytoene dehydrogenase gene crtI was amplified with PCR from zeaxanthin BioBrick and the homologies were added with the primers. Lambda red was performed to replace cat-sacB-T0 selection cassette with crtI, and the cells were plated on sucrose agar plates to select for transformants that had successfully integrated the crtI gene.</div>
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<div style="padding-bottom:3%"> To determine which media and temperature is optimal for the best DNA yield cultures in LB, SOB and TB in 37 °C and 42 °C. This experiment was not done for 30 °C as the growth there was generally very slow. The plasmids were prepared according to the protocol for PureLink Quick Plasmid Miniprep kit from Invitrogen. </div>
<div style="padding-bottom:3%"> A construct with beta-carotene hydroxylase gene crtZ and lycopene cyclase gene crtY was inserted with lambda red into another E. coli strain. </div>
<div style="padding-bottom:3%"> The trimethoprim resistance gene dhfr (dihydrofolate reductase) was inserted with lambda red into the strain that now contained crtZY genes. The genes we placed very close to each other to enable the following transduction with P1 bacteriophage.</div>
<div style="padding-bottom:3%"> The trimethoprim resistance gene dhfr (dihydrofolate reductase) was inserted with lambda red into the strain that now contained crtZY genes. The genes we placed very close to each other to enable the following transduction with P1 bacteriophage.</div>
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.
Testing the Slovenia Zeaxanthine strain
In this experiment we tested a strain of E.Coli created by Slovenia iGEM team in 2010 that produces Zeaxanthin. The strain contains a biobrick BBa_K323122. This biobrick includes five genes that convert farnesyl pyrophospate to zeaxanthin. The biobrick is under a lac operon and therefore expression of the enzymes can be induced by IPTG.
The zeaxanthin producing strain is important starting point of our project. However, there was a previous concern about the production not being stable and optimal. Therefore, we decided to test production of protein and plasmid DNA under different conditions. For the testing we chose three different temperatures (30 °C, 37 °C and 42 °C) and four different media (LB, TB, M9 and SOB). As LB is usually the standard media for experiments. We decided to use LB with the basic E. coli strain of DH5α to give a baseline for our measurements. We have also tested two super-rich media (TB and SOB) in attempt to produce higher yield of bacteria in shorter time. To test the possible growth in minimal media we chose M9.
How we did it
Zeaxanthin has previously been expressed in E. coli by iGEM teams using a plasmid. We have transformed the plasmid into DH5alpha cells and tested by colony PCR. The positive colonies were sent to sequencing which came out positive. Then we used the positive colonies to for Growth assay testing.
figure 1.Colony PCR after transformation of the zeaxanthin strain. Marker used is 1 Kb.
Growth Assay
The transform strain was inoculated into overnight culture of 5 ml in LB, TB, M9 and SOB with chloramphenicol. DH5-alpha strain was grown overnight in LB. All the cultures were grown at 37 °C on a shaker. Then they were inoculated into fresh media to a total volume of 100 ml and OD600 of 0.05. M9 culture did not results in any growth on two occasion and was discarded as the strain probably is not capable of growing on minimal media. Cultures were placed on a shaker in three temperatures (30°C, 37°C and 40°C) and induced with IPTG to a final concentration of 1mM. After inoculating the OD600 was measured every 30 minutes for 350 minutes.
Plasmid Preparation
To determine which media and temperature is optimal for the best DNA yield cultures in LB, SOB and TB in 37 °C and 42 °C. This experiment was not done for 30 °C as the growth there was generally very slow. The plasmids were prepared according to the protocol for PureLink Quick Plasmid Miniprep kit from Invitrogen.
Step 2
The trimethoprim resistance gene dhfr (dihydrofolate reductase) was inserted with lambda red into the strain that now contained crtZY genes. The genes we placed very close to each other to enable the following transduction with P1 bacteriophage.
Lambda red was performed to replace this newly inserted cat-sacB-T0 cassette with a construct containing geranylgeranyl diphosphate synthase gene crtE and phytoene synthase gene crtB. The clones with the correct sequence changed color to light red. Our lycopene producing strain was constructed!
Another transduction with P1 bacteriophage was done with the strain containing crtZY as a donor strain and the strain containing crtEBI as a recipient strain. The clones with the correct sequence turned bright yellow. We got our zeaxanthin producing strain 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, see figure 3.
Figure 3. Left: Large scale expression of zeaxanthin from the zeaxanthin producing E. coli strain. Right: Extracted and purified zeaxanthin.
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 and from wildtype E. coli. The zeaxanthin strain had two peaks at 460 and 482 nm which were not present in wildtype E. coli. For the measurements the extracted compounds were dissolved in toluene.
Figure 4. Absorbance spectra for the extraction of zeaxanthin. Mg1665 constitutes the negative control (the same extraction protocol on wildtype E. coli).
Our team would like to express a special appreciation and thanks to Erik Wistrand-Yuen who provided us with the strains and the protocols for the method and who spent hours guiding and instructing us and providing practical help. Without him this success would not have been possible.
