Team:Uppsala/Zea-Strain

PROJECT DESCRIPTION

ZEA-STRAIN

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 with the genes necessary for zeaxanthin production (Fig.1) [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.

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?]*

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?]*

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.