- PCR amplification of fadD, fadL inserts

- Purification of fadD, fadL PCR products

- LB plate preparation with chloramphenicol (34ug/ml) for selection of pSB1C3 clones

- DH5α competent cells are transformed with BBa_B0030 and BBa_R0040 separately by heat shock

- 1st Extraction of BBa_B0030, BBa_R0040 by miniprep
-> low product yield of BBa_R0040

- Extraction of BBa_R0040, by miniprep
-> low product yield

- New batch of BBa_R0040 transformants are prepared in LB broth

- Extraction of BBa_R0040 were performed with plasmid miniprep kit

- Restriction digestion were performed on:
fadL, fadD inserts with XbaI and PstI
BBa_R0040, BBa_B0030 with SpeI and PstI
Results: No band was observed for BBa_B0030 after digestion
-> Gel photo showed the extracted BBa_B0030 DNA was contaminated with RNA leading to overestimation of BBa_B0030 concentration for restriction digestion

- Purification was performed on the digested fadL and fadD inserts and BBa_R0040 products

- Ligation of digested fadD insert into BBa_B0030 vector was performed

- DH5α competent cell was transformed with pSB1C3_ R0040-fadL by heat shock protocol

- BBa_B0030 was performed again with plasmid DNA miniprep kit, the product was treated with additional RNase A

- Restriction digestion of BBa_B0030 was performed with SpeI and PstI

- Digested BBa_B0030 was purified with PCR purification kit

- fadL insert was ligated into BBa_R0040 vector

- LB plate with chloramphenicol (34 µg/mL) was prepared

Week 1 (3/8~9/8)

- DH5α competent cells were transformed with pSB1C3_ B0030-fadD by heat shock protocol

- pSB1C3_ R0040-fadL clones was screened with colony PCR method
-> non-specific bands were observed

- pSB1C3_ B0030-fadD clones were screened by colony PCR method
-> non-specific bands were observed

- Repeated colony PCR analysis on the pSB1C3_ R0040-fadL clones
-> Bands with expected size were observed in two clones and sent for DNA sequencing

- pSB1C3_ B0030-fadD clones were screening again with colony PCR method
-> DNA Bands with expected size were observed in 6 clones and sent for DNA sequencing

Week 2 (10/8~16/8)

- Sequencing results show that the transformants contained errors in DNA sequences
-> Missing one to two base pairs in the fadL gene in the pSB1C3_ R0040-fadL clones
-> Missing the ribosomal binding site in the pSB1C3_ B0030-fadD clones

- Repeat screening for pSB1C3_ R0040-fadL clones were performed with the colony PCR method
-> Bands with expected size were observed in 4 clones and sent for DNA sequencing

- PCR amplification of fadD, fadL genes with higher annealing temperature

- Purification of fadD, fadL PCR products

- Double restriction digestion on fadL, fadD PCR products with XbaI and PstI

- Extraction of BBa_B0034 (biobricks with the ribosomal binding site) plasmid by plasmid DNA miniprep kit

- Extraction of BBa_R0040 plasmid with plasmid DNA miniprep kit

- Double restriction digestion on BBa_R0040 plasmid with SpeI and PstI

- Purification of the digested BBa_R0040 plasmid

Week 3 (17/8~23/8)

- Sequencing results showed errors in DNA sequences in the clones prepared last week
-> Missing one to two base pairs in the fadL gene in the pSB1C3_ R0040-fadL plasmid

- Ligation was performed on the fadL insert and the BBa_R0040 vector

- DH5α competent cell was transformed with the pSB1C3_ R0040-fadL plasmid by the heat shock protocol

- Double restriction digestion on BBa_B0034 by SpeI and PstI

- Purification of the digested BBa_B0034 plasmid

- Ligation were performed on the fadD insert and the BBa_B0034 vector

- LB plate with ampicillin (100 µg/mL) were prepared for the selection of pSB1A2 clones

- DH5α competent cells were transformed with pSB1A2_ B0034-fadD by the heat shock protocol

- Screening for pSB1C3_ R0040-fadL clones were done by the colony PCR method
-> DNA bands with expected size were observed in only one clone and sent for DNA sequencing

Week 4 (24/8~30/8)

- Screening on the pSB1A2_ B0034-fadD clones were done by the colony PCR method
-> DNA bands with expected size were observed in 4 clones and sent for DNA sequencing

- PCR amplification of the fadD, fadL inserts were performed at higher annealing temperature

- LB plates with ampicillin (100 µg/mL) were prepared

- Sequencing results:
The identity of the DNA insert in pSB1C3_ R0040-fadL clone was confirmed by DNA sequencing.
Changes in DNA sequence were found in the fadD gene in pSB1A2_ B0034-fadD clones