Team:CityU HK/notebook/lablog 2

From 2014.igem.org

(Difference between revisions)
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                             <div class="st-content">
                             <div class="st-content">
                                 <h2 class="title"><b>Week 3 (13/7~19/7)</b></h2>
                                 <h2 class="title"><b>Week 3 (13/7~19/7)</b></h2>
-
                                 <p><b>-</b> Primer design for leaderless tesA for Fit-coli (‘tesA) & New biobrick (‘tesA BB)</p>
+
                                 <p><b>-</b> Primer design for the leaderless tesA gene for Fit-coli (‘tesA) & New biobrick (‘tesA BB)</p>
                                 <h2 class="title"><b>Week 4 (20/7~26/7)</b></h2>
                                 <h2 class="title"><b>Week 4 (20/7~26/7)</b></h2>
-
                                 <p><b>-</b> PCR of ‘tesA and ‘tesA BB</p>
+
                                 <p><b>-</b> PCR of the ‘tesA and ‘tesA BB DNA</p>
-
                                 <p><b>-</b> PCR purification of ‘tesA and ‘tesA BB</p>
+
                                 <p><b>-</b> PCR purification of the ‘tesA and ‘tesA BB amplicons</p>
-
                                 <p><b>-</b> Prepare LB agar plates(‘tesA BB)</p>
+
                                 <p><b>-</b> LB agar plates with antibiotics were prepared</p>
                                 <h2 class="title"><b>Week 5 (27/7~31/7)</b></h2>
                                 <h2 class="title"><b>Week 5 (27/7~31/7)</b></h2>
-
                                 <p><b>-</b> Digestion of ‘tesA BB and pSB1C3 plasmid with EcoRI & PstI restriction enzymes</p>
+
                                 <p><b>-</b> Double digestion on the ‘tesA BB and pSB1C3 plasmids with EcoRI and PstI </p>
-
                                 <p><b>-</b> PCR purification for digested ‘tesA BB</p>
+
                                 <p><b>-</b> PCR purification on the restriction digested ‘tesA BB DNA fragment</p>
-
                                 <p><b>-</b> Sub-cloning of ‘tesA BB into pSB1C3 plasmid</p>
+
                                 <p><b>-</b> Sub-cloning of the ‘tesA BB fragment into pSB1C3 plasmid</p>
-
                                 <p><b>-</b> Transformation of ‘tesA BB into W3110 E.coli</p>
+
                                 <p><b>-</b> Transformation of the ‘tesA BB ligation mixture into <i>E. coli </i>W3110</p>
-
                                 <p><b>-</b> Pick colonies of the transformed E. coli with ‘tesA BB</p>
+
                                 <p><b>-</b> <i>E. coli</i> colonies were picked with ‘tesA BB gene</p>
                             </div>
                             </div>
                         </li>
                         </li>
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                             <div class="st-content">
                                 <h2 class="title"><b>Week 1 (1/8~2/8)</b></h2>
                                 <h2 class="title"><b>Week 1 (1/8~2/8)</b></h2>
-
                                 <p><b>-</b> Cell lysis of picked E. coli with ‘tesA BB</p>
+
                                 <p><b>-</b> Lysis of E. coli colonies was done on the ‘tesA BB gene boiling them in hot water </p>
-
                                 <p><b>-</b> Colonies PCR of ‘tesA BB by using VF2 & VR primer</p>
+
                                 <p><b>-</b> Colony PCR on the ‘tesA BB gene was performed using VF2 and VR primer pairs</p>
-
                                 <p><b>-</b> Send the ‘tesA BB plasmid for sequencing</p>
+
                                 <p><b>-</b> The ‘tesA BB recombinant plasmids were confirmed by DNA sequencing</p>
                                 <h2 class="title"><b>Week 2 (3/8~9/8)</b></h2>
                                 <h2 class="title"><b>Week 2 (3/8~9/8)</b></h2>
-
                                 <p><b>-</b> 2nd PCR of ‘tesA <br>∵ The first PCR product of ‘tesA was degraded over a long period of storage time</p>
+
                                 <p><b>-</b> PCR purification was performed on the ‘tesA PCR product</p>
-
                                 <p><b>-</b> PCR purification of ‘tesA PCR product</p>
+
                                 <p><b>-</b> Double digestion were performed on the ‘tesA PCR product using <i>XbaI</i> and <i>PstI</i></p>
-
                                <p><b>-</b> 1st Digestion of ‘tesA by using XbaI & PstI restriction enzyme<br>-> 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</p>
+
                                 <p><b>-</b> Digestion of the lac promoter in pSB1C3 plasmid and the P<sub>BAD</sub> promoter in pSB1C3 plasmid using <i>EcoRI</i>, <i>XbaI</i>, <i>SpeI</i> and <i>PstI</i> restriction enzymes</p>
-
                                 <p><b>-</b> 2nd digestion of ‘tesA by using XbaI & PstI restriction enzyme with adding one more control pilot that is only plasmid with no enzyme<br>-> Still no fragment presented in pilot, we wonder whether the pilot plasmid or the restriction enzyme had problem</p>
+
                                 <p><b>-</b> Sub-cloning of the ‘tesA gene downstream of the RBS in pSB1C3 plasmid (Part: BBa_B0030)</p>
-
                                <p><b>-</b> 3rd digestion of lac promoter in pSB1C3 plasmid & pbad promoter in pSB1C3 plasmid by using EcoRI, XbaI, SpeI & PstI restriction enzyme<br>-> All of the restriction enzyme worked normally, but the pbad promoter in pSB1C3 plasmid was degraded</p>
+
-
                                <p><b>-</b> 4th digestion of ‘tesA by using XbaI & PstI restriction enzyme</p>
+
-
                                 <p><b>-</b> Sub-cloning of‘tesA into rbs in pSB1C3 plasmid (Part: BBa_B0030)</p>
+
                                  
                                  
                             </div>
                             </div>
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                             <div class="st-content">
                             <div class="st-content">
                                 <h2 class="title"><b>Week 3 (10/8~16/8)</b></h2>
                                 <h2 class="title"><b>Week 3 (10/8~16/8)</b></h2>
-
                                 <p><b>-</b> 1st Transformation of ‘tesA with rbs (BBa_B0030) into W3110 E.coli</p>
+
                                 <p><b>-</b> BBa_B0030 was transformed into the host <i>E. coli</i> W3110</p>
-
                                <p><b>-</b> Pick colonies of the transformed E. coli with ‘tesA & rbs (BBa_B0030)</p>
+
                                 <p><b>-</b> The identity of transformants of BBa_B0030 was confirmed by DNA sequencing<br> -> DNA sequencing showed errors in DNA sequence in the RBS so we used the RBS sequence of BBa_B0034 instead of BBa_B0030 RBS
-
                                 <p><b>-</b> Send the‘tesA with rbs (BBa_B0030) plasmid for sequencing<br>∵ The sequencing result showed the coding error of the rbs<br> -> Used BBa_B0034 rbs in pSB1C3 plasmid to replace BBa_B0030 rbs for 2nd cloning </p>
+
</p>
                                  
                                  
                                 <h2 class="title"><b>Week 4 (17/8~23/8)</b></h2>
                                 <h2 class="title"><b>Week 4 (17/8~23/8)</b></h2>
-
                                 <p><b>-</b> 3rd PCR of tesA’ <br>∵ The 2nd PCR product of ‘tesA was used up</p>
+
                                 <p><b>-</b> ‘tesA PCR product was purified with PCR purification kit</p>
-
                                 <p><b>-</b> PCR purification of ‘tesA PCR product</p>
+
                                 <p><b>-</b> ‘tesA PCR amplicon was doubly digested with <i>XbaI</i> and <i>PstI</i></p>
-
                                <p><b>-</b> 5th digestion of ‘tesA by using XbaI and PstI restriction enzyme</p>
+
                                 <p><b>-</b> ‘tesA was subcloned downstream of the RBS site in pSB1A2 plasmid (Part: pSB1A2- BBa_B0034)</p>
-
                                 <p><b>-</b> Sub-cloning of ‘tesA into rbs in pSB1A2 plasmid (Part: pSB1A2- BBa_B0034)</p>
+
                                 <p><b>-</b> P<sub>BAD</sub> promoter plasmid (Part: pSB1C3-BBa_I13453) was purified with plasmid DNA purification kit</p>
-
                                 <p><b>-</b> Miniprep of pbad promoter (Part: pSB1C3-BBa_I13453)</p>
+
                                 <p><b>-</b> ‘tesA with RBS (BBa_B0034) was transformed into <i>E.coli</i> W3110</p>
-
                                 <p><b>-</b> 2nd tramsformation of ‘tesA with rbs (BBa_B0034) into W3110 E.coli</p>
+
                                 <p><b>-</b> PBAD promoter plasmid (BBa_I13453) was digested with <i>SpeI</i> and <i>PstI</i></p>
-
                                 <p><b>-</b> 1st digestion of pbad promoter (BBa_I13453) by using SpeI & PstI restriction enzyme</p>
+
                                 <h2 class="title"><b>Week 5 (24/8~30/8)</b></h2>
                                 <h2 class="title"><b>Week 5 (24/8~30/8)</b></h2>
-
                                 <p><b>-</b> Pick colonies of the transformed E. coli with ‘tesA & rbs (BBa_B0034)</p>
+
                                 <p><b>-</b> Colonies of <i>E. coli</i> transformed with ‘tesA & RBS (BBa_B0034) were picked for colony PCR using VF2 & VR primer pairs</p>
-
                                <p><b>-</b> Cell lysis of the picked E. coli with ‘tesA & rbs (BBa_B0034)</p>
+
                                 <p><b>-</b> DNA sequence of the transformant plasmids were confirmed by DNA sequencing</p>
-
                                <p><b>-</b> Colonies PCR of ‘tesA & rbs (BBa_B0034) by using VF2 & VR primer</p>
+
                                 <p><b>-</b> Plasmid DNA of BBa_B0034 was purified</p>
-
                                 <p><b>-</b> Send the‘tesA with rbs (BBA-B0034) plasmid for sequencing</p>
+
                                 <p><b>-</b> BBa_B0034 plasmid DNA was digested with XbaI and PstI</p>
-
                                 <p><b>-</b> Miniprep of ‘tesA with rbs (BBa_B0034)</p>
+
                                 <p><b>-</b> XbaI / PstI digested BBa_B0034 plasmid DNA was purified</p>
-
                                 <p><b>-</b> 1st digestion of ‘tesA with rbs (BBa_B0034) by using XbaI & PstI restriction enzyme</p>
+
                                 <p><b>-</b> P<sub>BAD</sub> promoter (BBa_I13453) plasmid digested with SpeI and PstI was purified</p>
-
                                 <p><b>-</b> Gel purification of digested ‘tesA with rbs (BBa_B0034)</p>
+
-
                                 <p><b>-</b> PCR purification of pbad promoter (BBa_I13453) digested in week 4</p>
+
                             </div>
                             </div>
                         </li>
                         </li>

Revision as of 07:27, 2 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

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