Team:CityU HK/notebook/lablog

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FadD & FadL module's lablog

  • July - Week 4 (20/7~26/7)Open or Close

    - 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

  • July - Week 5 (27/7~2/8)Open or Close

    - 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)




    • TesA module's lablog

    • JulyOpen or Close

      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

    • Early AugustOpen or Close

      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)

    • Late AugustOpen or Close

      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




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