Week 3 (13/7~19/7)

- Plasmids containing theΔ9, Δ12 and Δ15 desaturase genes were extracted using commercial DNA extraction kits and digested either singly or doubly with the XbaI and PstI restriction enzymes.

Week 5 (27/7~31/7)

- Plasmid pSB1C3 (which carries the R0010 promoter) was doubly digested with the XbaI and SpeI restriction enzymes
-> However, there is no band matching the size of the promoter R0010

- Three plasmids that carry theΔ9, Δ12 andΔ15 desaturase genes were each doubly digested with XbaI and PstI

- Plasmid pSB1C3 was restriction digested with XbaI and PstI

- The Δ9, Δ12 andΔ15 desaturase genes were individually subcloned into the XbaI / PstI site of pSB1C3 and transformed into E.coli DH5α (K12 strain)

- Transformants were checked by colony PCR and gel electrophoresis
-> - Gel electrophoresis analyses were repeated 3 times
-> The results were not promising, either no band was observed in the gel or incorrect size bands were obtained

- Primer design for colony PCR and DNA sequencing
-> The primers were designed based on the original iGEM primers, i.e. VF2 and VR, with a few additional nucleotides. They were also used for DNA sequencing to verify the identity of the insert
-> The primers subsequently designed included additional nucleotide sequences and an RBS sequence.

- Plasmids containing the RBS B0030 and the Δ9, Δ12 andΔ15 genes were purified using commercial DNA extraction kits

- The cloned Δ9, Δ12 andΔ15 genes in pSC1C3 were restriction cut with XbaI and PstI, and sequentially subcloned into the XbaI / PstI site of pSB1C3 and transformed into E. coli

- Transformants were checked by colony PCR with gene-specific primers targeting the Δ9, Δ12 orΔ15 desaturase genes
-> The transformed bacteria seemed to have the target inserts
-> PCR-positive colonies were confirmed by DNA sequencing to carry the desired gene inserts

- Plasmids containing the Δ9, Δ12 andΔ15 genes (in the pSB1C3 backbone) were purified using commercial DNA extraction kits

- The Δ9-containing plasmid was double digested with SpeI / PstI, and the Δ12- andΔ15- containing plasmids were double digested with XbaI / PstI

- Gel electrophoresis was carried out to prepare the Δ9, Δ12 and Δ15 gene fragments for sequential cloning into pSB1C3 to obtain the Δ9-Δ12-Δ15 gene cluster in the pSB1C3 plasmid backbone

- Gel purification of the DNA fragments

- Ligation of Δ9 gene containing plasmids and the Δ12 and Δ15 gene inserts

- Transformation of ligation mixture into E.coli DH5α

- Transformant colonies were checked by colony PCR and gel electrophoresis to determine if bacterial transformants contain the Δ9, Δ12 andΔ15 genes
-> Results confirmed that the transformation was successful