Team:Uppsala/Zea-Strain
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.
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.
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.
