Team:CityU HK/notebook/lablog

- 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)

Week 3 (13/7~19/7)

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

Week 4 (20/7~26/7)

- PCR of ‘tesA and ‘tesA BB

- PCR purification of ‘tesA and ‘tesA BB

- Prepare LB agar plates(‘tesA BB)

Week 5 (27/7~31/7)

- Digestion of ‘tesA BB and pSB1C3 plasmid with EcoRI & PstI restriction enzymes

- PCR purification for digested ‘tesA BB

- Sub-cloning of ‘tesA BB into pSB1C3 plasmid

- Transformation of ‘tesA BB into W3110 E.coli

- Pick colonies of the transformed E. coli with ‘tesA BB

Week 1 (1/8~2/8)

- Cell lysis of picked E. coli with ‘tesA BB

- Colonies PCR of ‘tesA BB by using VF2 & VR primer

- Send the ‘tesA BB plasmid for sequencing

Week 2 (3/8~9/8)

- 2nd PCR of ‘tesA
∵ The first PCR product of ‘tesA was degraded over a long period of storage time

- PCR purification of ‘tesA PCR product

- 1st Digestion of ‘tesA by using XbaI & PstI restriction enzyme
-> After running gel, no fragment showed in pilot ( other plasmid with the Xbal & Pstl respectively) , could not determine whether the digestion was success or not

- 2nd digestion of ‘tesA by using XbaI & PstI restriction enzyme with adding one more control pilot that is only plasmid with no enzyme
-> Still no fragment presented in pilot, we wonder whether the pilot plasmid or the restriction enzyme had problem

- 3rd digestion of lac promoter in pSB1C3 plasmid & pbad promoter in pSB1C3 plasmid by using EcoRI, XbaI, SpeI & PstI restriction enzyme
-> All of the restriction enzyme worked normally, but the pbad promoter in pSB1C3 plasmid was degraded

- 4th digestion of ‘tesA by using XbaI & PstI restriction enzyme

- Sub-cloning of‘tesA into rbs in pSB1C3 plasmid (Part: BBa_B0030)

Week 3 (10/8~16/8)

- 1st Transformation of ‘tesA with rbs (BBa_B0030) into W3110 E.coli

- Pick colonies of the transformed E. coli with ‘tesA & rbs (BBa_B0030)

- Send the‘tesA with rbs (BBa_B0030) plasmid for sequencing
∵ The sequencing result showed the coding error of the rbs
-> Used BBa_B0034 rbs in pSB1C3 plasmid to replace BBa_B0030 rbs for 2nd cloning

Week 4 (17/8~23/8)

- 3rd PCR of tesA’
∵ The 2nd PCR product of ‘tesA was used up

- PCR purification of ‘tesA PCR product

- 5th digestion of ‘tesA by using XbaI and PstI restriction enzyme

- Sub-cloning of ‘tesA into rbs in pSB1A2 plasmid (Part: pSB1A2- BBa_B0034)

- Miniprep of pbad promoter (Part: pSB1C3-BBa_I13453)

- 2nd tramsformation of ‘tesA with rbs (BBa_B0034) into W3110 E.coli

- 1st digestion of pbad promoter (BBa_I13453) by using SpeI & PstI restriction enzyme

Week 5 (24/8~30/8)

- Pick colonies of the transformed E. coli with ‘tesA & rbs (BBa_B0034)

- Cell lysis of the picked E. coli with ‘tesA & rbs (BBa_B0034)

- Colonies PCR of ‘tesA & rbs (BBa_B0034) by using VF2 & VR primer

- Send the‘tesA with rbs (BBA-B0034) plasmid for sequencing

- Miniprep of ‘tesA with rbs (BBa_B0034)

- 1st digestion of ‘tesA with rbs (BBa_B0034) by using XbaI & PstI restriction enzyme

- Gel purification of digested ‘tesA with rbs (BBa_B0034)

- PCR purification of pbad promoter (BBa_I13453) digested in week 4