Week 3 (13/7~19/7)

- Primer design for the leaderless tesA gene for Fit-coli (‘tesA) & New biobrick (‘tesA BB)

Week 4 (20/7~26/7)

- PCR of the ‘tesA and ‘tesA BB DNA

- PCR purification of the ‘tesA and ‘tesA BB amplicons

- LB agar plates with antibiotics were prepared

Week 5 (27/7~31/7)

- Double digestion on the ‘tesA BB and pSB1C3 plasmids with EcoRI and PstI

- PCR purification on the restriction digested ‘tesA BB DNA fragment

- Sub-cloning of the ‘tesA BB fragment into pSB1C3 plasmid

- Transformation of the ‘tesA BB ligation mixture into E. coli W3110

- E. coli colonies were picked with ‘tesA BB gene

Week 1 (1/8~2/8)

- Lysis of E. coli colonies was done on the ‘tesA BB gene boiling them in hot water

- Colony PCR on the ‘tesA BB gene was performed using VF2 and VR primer pairs

- The ‘tesA BB recombinant plasmids were confirmed by DNA sequencing

Week 2 (3/8~9/8)

- PCR purification was performed on the ‘tesA PCR product

- Double digestion were performed on the ‘tesA PCR product using XbaI and PstI

- Digestion of the lac promoter in pSB1C3 plasmid and the P_BAD promoter in pSB1C3 plasmid using EcoRI, XbaI, SpeI and PstI restriction enzymes

- Sub-cloning of the ‘tesA gene downstream of the RBS in pSB1C3 plasmid (Part: BBa_B0030)

Week 3 (10/8~16/8)

- BBa_B0030 was transformed into the host E. coli W3110

- The identity of transformants of BBa_B0030 was confirmed by DNA sequencing
-> DNA sequencing showed errors in DNA sequence in the RBS so we used the RBS sequence of BBa_B0034 instead of BBa_B0030 RBS

Week 4 (17/8~23/8)

- ‘tesA PCR product was purified with PCR purification kit

- ‘tesA PCR amplicon was doubly digested with XbaI and PstI

- ‘tesA was subcloned downstream of the RBS site in pSB1A2 plasmid (Part: pSB1A2- BBa_B0034)

- P_BAD promoter plasmid (Part: pSB1C3-BBa_I13453) was purified with plasmid DNA purification kit

- ‘tesA with RBS (BBa_B0034) was transformed into E.coli W3110

- P_BAD promoter plasmid (BBa_I13453) was digested with SpeI and PstI

Week 5 (24/8~30/8)

- Colonies of E. coli transformed with ‘tesA & RBS (BBa_B0034) were picked for colony PCR using VF2 & VR primer pairs

- DNA sequence of the transformant plasmids were confirmed by DNA sequencing

- Plasmid DNA of BBa_B0034 was purified

- BBa_B0034 plasmid DNA was digested with XbaI and PstI

- XbaI / PstI digested BBa_B0034 plasmid DNA was purified

- P_BAD promoter (BBa_I13453) plasmid digested with SpeI and PstI was purified