Week 3 (13/7~19/7)

- Miniprep: extracted plasmids containing Δ9, Δ12 and Δ15 genes

- Restriction cut at XbaI and PstI sites of each type of extracted plasmids

Week 5 (27/7~31/7)

- Restriction cut at XbaI and SpeI for promoter R0010 from plasmid pSB1C3
->no band with matching size of promoter showed after running gel

- Restriction cut at XbaI and PstI forΔ9, Δ12 andΔ15 genes

- Restriction cut at XbaI and PstI on plasmid pSB1C3

- Ligation of each gene into each pSB1C3 plasmid

- Transformation of suspected-to-be modified plasmids into E.coli (DH5αK12 strain)

- Check clone by colony PCR and gel electrophoresis
-> Three times of gel electrophoreses were carried
-> The results were not desired, either no band in gel photo or incorrect sizes of band

- Primer design for colony PCR & sequencing
-> The first designed primers were based on the original iGEM primers, i.e. VF2 and VR, but with extended annealing sequences. They were used for sequencing
-> The second designed primers included more annealing sequences and also RBS sequence. They would be used as the other approach if the processing transformed plasmids did not really have inserts

- Extraction of plasmids containing RBS B0030 and Δ9, Δ12 andΔ15 genes with RBS sequences

- Repeated restriction cut at XbaI and PstI sites of gene carrying plasmids

- ligation of genes to PSB1C3 with XbaI and PstI sites cut and transformation with newly extracted genes

- Colonies picked for further clone check with three types of primers, including the original iGEM primers and two newly designed primers
-> The transformed bacteria seemed to have target inserts after running gel

- Sequencing of the transformed bacteria
-> all sequences matched as expected

- Miniprep of the extracted plasmids with Δ9, Δ12 andΔ15 genes in pSB1C3 plasmids

- Restriction digestion at SpeI and PstI sites ofΔ9 gene containing plasmids, XbaI and PstU sites ofΔ12, Δ15 gene containing plasmids

- Gel electrophoresis in order to collectΔ9 gene containing plasmids, Δ12 and Δ15 genes

- Gel purification of the collected products

- Ligation of Δ9 gene containing plasmids andΔ12 gene insert

- Transformation of suspected-to-be modified plasmids into E.coli (DH5αK12 strain)

- Clone check by PCR and gel electrophoresis to see if grown bacteria contained Δ9 and Δ12 genes or not
-> Result matched, transformation was sucessful