Team:CityU HK/notebook/lablog 2

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     <a href="http://www6.cityu.edu.hk/bhdbapp/deptweb/index.html" target="_blank"><img src="https://static.igem.org/mediawiki/2014/9/98/CityU_HK_bchlogo.png" width="80"></a>
     <a href="http://www6.cityu.edu.hk/bhdbapp/deptweb/index.html" target="_blank"><img src="https://static.igem.org/mediawiki/2014/9/98/CityU_HK_bchlogo.png" width="80"></a>
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       <h3>Stay connected!</h3>
       <h3>Stay connected!</h3>

Revision as of 04:35, 16 October 2014

Bootstrap 101 Template

TesA Module's Lablog

  • JulyOpen or Close

    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

  • Early AugustOpen or Close

    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 PBAD 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)

  • Late AugustOpen or Close

    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)

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

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

    - PBAD 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

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