- 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 by miniprep

- Restriction digestion of fadL, fadD inserts by XbaI and PstI & BBa_R0040, BBa_B0030 (rbs) by SpeI and PstI
-> No band is observed for BBa_B0030 after digestion
-> Gel photo showing extracted BBa_B0030 is contaminated with RNA
-> Lead to overestimation of BBa_B0030 concentration for digestion

- Purification of digested fadL, fadD inserts, BBa_R0040 products

- Ligation of digested fadD insert into BBa_B0030 vector

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

- Repeat extraction of BBa_B0030 by miniprep, product is treated with RNase A

- Restriction digestion of Bba_B0030 by SpeI and PstI

- Purification of digested Bba_B0030

- Ligation of fadL insert into BBa_R0040 vector

- LB plate preparation with Chloramphenicol (34ug/ml)

Week 1 (3/8~9/8)

- DH5α competent cell is transformed with pSB1C3_ B0030-fadD by heat shock

- Screening for pSB1C3_ R0040-fadL clones by colony PCR
-> non-specific bands observed

- Screening for pSB1C3_ B0030-fadD clones by colony PCR
-> non-specific bands observed

- Repeat screening for pSB1C3_ R0040-fadL clones by colony PCR
-> Bands with expected size are observed in 2 clones and sent for sequencing

- Repeat screening for pSB1C3_ B0030-fadD clones by colony PCR
-> Bands with expected size are observed in 6 clones and sent for sequencing

Week 2 (10/8~16/8)

- Sequencing results show negative result for the sent clones:
-> Missing one to two base pair in fadL gene in pSB1C3_ R0040-fadL clones
Missing ribosomal binding site in pSB1C3_ B0030-fadD clones

- Repeat screening for pSB1C3_ R0040-fadL clones by colony PCR
-> Bands with expected size are observed in 4 clones and sent for sequencing

- PCR amplification of fadD, fadL inserts with 1°C higher annealing temperature

- Purification of fadD, fadL PCR products

- Restriction digestion of fadL, fadD inserts by XbaI and PstI

- Extraction of BBa_B0034 (biobricks with ribosomal binding site) by miniprep

- Extraction of BBa_R0040 by miniprep

- Restriction digestion of BBa_R0040 by SpeI and PstI

- Purification of digested BBa_R0040 product

Week 3 (17/8~23/8)

- Sequencing results show negative result for the sent clones last week:
-> Missing one to two base pair in fadL gene in pSB1C3_ R0040-fadL clones

- Ligation of fadL insert into BBa_R0040 vector

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

- Restriction digestion of BBa_B0034 by SpeI and PstI

- Purification of digested BBa_B0034 product

- Ligation of fadD insert into BBa_B0034 vector

- LB plate preparation with ampicilin (100ug/ml) for selection of pSB1A2 clones

- DH5α competent cell is transformed with pSB1A2_ B0034-fadD by heat shock

- Screening for pSB1C3_ R0040-fadL clones by colony PCR
-> Bands with expected size are observed in 1 clones and sent for sequencing

Week 4 (24/8~30/8)

- Screening for pSB1A2_ B0034-fadD clones by colony PCR
-> Bands with expected size are observed in 4 clones and sent for sequencing

- PCR amplification of fadD, fadL inserts with ?°C higher annealing temperature

- LB plate preparation with ampicilin (100ug/ml)