Difference between revisions of "Team:NUS Singapore/Notebook/Group3"

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         <h3>
 
         <h3>
 
         <span>1 June, 2016</span>
 
         <span>1 June, 2016</span>
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             <a href="http://tympanus.net/Tutorials/TypographyEffects/">Milestone 3: : Overall cloning design</a>
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             <a href="http://tympanus.net/Tutorials/TypographyEffects/">Milestone 1 : Overall cloning design</a>
 
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         <h3>
 
         <h3>
 
         <span>4 July, 2016</span>
 
         <span>4 July, 2016</span>
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             <a href="http://tympanus.net/Tutorials/TypographyEffects/">Milestone 3: : Construction of pLuxR-LuxI-HA </a>
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             <a>Milestone 3: : Construction of pLuxR-LuxI-HA </a>
 
         </h3>
 
         </h3>
 
     </div>
 
     </div>
 
</div>
 
</div>
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<div class="ss-row ss-large">
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  <div class="ss-left">
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    <h3>
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      <span>11 July, 2016</span>
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      <a>Milestone 4: Screening of pLuxR-invasin constructs </a>
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    </h3>
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<p><img src="https://static.igem.org/mediawiki/2016/c/c6/T--NUS_Singapore--log3_16.png" style="width: 100%;"></p>
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    </div>
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  </div>
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<p><img src="https://static.igem.org/mediawiki/2016/0/08/T--NUS_Singapore--log3_17.png" style="width: 100%;"></p>
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        </div>
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    <div class="ss-right">
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        <h3>
 +
        <span>1 Aug, 2016</span>
 +
            <a>Milestone 5: Screening of pLuxR-LLO construct</a>
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        </h3>
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    </div>
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</div>
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     <h3>
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       <span>1 Aug, 2016</span>
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       <span>8 Aug, 2016</span>
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       <a>Milestone 2: Construct HasA expression plasmid</a>
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       <a>Milestone 6: Construction of pLuxR-GFP plasmid</a>
 
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<p><img src="https://static.igem.org/mediawiki/2016/2/2b/T--NUS_Singapore--log3_18.png" style="width: 100%;"></p>
  
 
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     </div>
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            <p>
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<p><img src="https://static.igem.org/mediawiki/2016/a/a3/T--NUS_Singapore--log3_19.png" style="width: 100%;"></p>
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                28 July: RIOT responder circuit custom synthesised in three plasmids (Seq1, Seq 2, Seq3). PCR of fragments successfully carried out</br>
+
        </div>
−
                29 July: Seq3 fragment inserted into Biobrick plasmid pSCB13 via RE digest and ligation</br>
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    </div>
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                30 July: Seq3 in pSCB13 transformed into E.coli</br>
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    <div class="ss-right">
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                2 Aug: Colony screening for Seq3 yielded one positive colony</br>
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−
                3 Aug: Seq3 pSCB13 extracted and inserted with Seq2 via RE digest and ligation</br>
+
−
                4 Aug: Seq2+Seq3 in pSCB13 transformed into E.coli</br>
+
−
                5 Aug: Colony screening for Seq2+Seq3 yielded several positive colonies</br>
+
−
                9 Aug: Seq2+Seq3 pSCB13 extracted and inserted with Seq1 via RE digest and ligation</br>
+
−
                10 Aug: Seq1+Seq2+Seq3 in pSCB13 transformed into E.coli</br>
+
−
                11 Aug: Colonies were picked for overnight culture and Miniprepped</br>
+
−
                12 Aug: RE digest of extracted Seq1+Seq2+Seq3 pSCB13 yielded several positive colonies</br>
+
−
 
+
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            </p>
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            <p style="text-align: center;"><img src="https://static.igem.org/mediawiki/2016/a/a3/T--NUS_Singapore--log2_5.png" style="width: 60%;"></p>
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         <h3>
 
         <h3>
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            <span>12 Aug, 2016</span>
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        <span>1 Aug, 2016</span>
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             <a >Milestone 3: Construct full RIOT Responder operon circuit </a>
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             <a>Milestone 7: Testing the assembled pConst-LuxR-pLuxR-LuxI construct</a>
 
         </h3>
 
         </h3>
 
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     </div>
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    <h3>
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    <h3>
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        <span>22 September, 2016</span>
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      <span>19 Sep, 2016</span>
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        <a >Milestone 4: Induction and Western blot of HasA</a>
+
      <a>Milestone 8: Testing the assembled pConst-LuxR-pLuxR-LLO construct</a>
 
     </h3>
 
     </h3>
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     <div class="log-content ss-circle-deco">
 
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        <p>
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<p><img src="https://static.igem.org/mediawiki/2016/b/bf/T--NUS_Singapore--log3_20.png" style="width: 100%;"></p>
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            14 Sep: Mass liquid culture of HasA bacteria and induction of expression by IPTG</br>
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−
            15 Sep: Lysis of HasA culture</br>
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−
            17 Sep: Purification of HasA by nickel column</br>
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            22 Sep: Completed Western blot of HasA</br>
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        </p>
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        <p style="text-align: center;"><img src="https://static.igem.org/mediawiki/2016/b/bc/T--NUS_Singapore--log2_6.png" style="width: 100%;"></p>
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    <div class="ss-left">
 
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        <div class="log-content ss-circle-deco">
 
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            <p>19 Sep: RIOT responder bacteria induced with HasA protein at 10-4 M and 10-5 M. Fluorescence microscopy pictures taken at hourly intervals</br>
 
−
                20 Sep: Overnight HasA induced RIOT responder bacteria observed under fluorescent microscope</p>
 
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                <p style="text-align: center;"><img src="https://static.igem.org/mediawiki/2016/4/4d/T--NUS_Singapore--log2_8.png" style="width: 60%;"></p>
 
−
                <p style="text-align: center;">HasA induction of RIOT responder bacteria (overnight) Top: red fluorescence microscopy pictures of (from left to right) negative control, and 10-5 M HasA. Bottom: bright field microscopy pictures of (from left to right) negative control, and 10-5 M HasA.
 
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        <div class="ss-right">
 
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            <h3>
 
−
                <span>20 September, 2016</span>
 
−
                <a>Milestone 6: Induction of RIOT Responder circuit</a>
 
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            <h3>
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             <div class="log-content ss-circle-deco" style="margin-top: 20px;">
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                <span>13 October, 2016</span>
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<p><img src="https://static.igem.org/mediawiki/2016/6/64/T--NUS_Singapore--log3_211.png" style="width: 100%;"></p>
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                <a>Milestone 5: Western blot of Has operon construct</a>
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        <h3>
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        <span>10 Oct, 2016</span>
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                <p>6 Oct: 500mL liquid culture of RIOT Responder bacteria</br>
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            <a>Milestone 9: Testing the assembled pConst-LuxR-pLuxR-GFP construct</a>
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                    7 Oct: Lysis of liquid culture</br>
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                    11 Oct: Nickel column purification of HasR, HasS, and HasI proteins</br>
+
    </div>
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                    12 Oct: SDS PAGE of purification fractions and electrotransfer to PVDF membrane</br>
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</div>
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                    13 Oct: Western Blot and chemiluminescent visualisation of HasR, HasS, and HasI bands</br>
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                    <p style="text-align: center;"><img src="https://static.igem.org/mediawiki/2016/a/ac/T--NUS_Singapore--log2_7.png" style="width: 60%;"></p>
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                     hasPerspective  = false,
 
                     hasPerspective  = false,
 
                      
 
                      
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                     perspective    = hasPerspective && Modernizr.csstransforms3d,
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                     perspective    = hasPerspective,
 
                     // initialize function
 
                     // initialize function
 
                     init            = function() {
 
                     init            = function() {

Revision as of 15:03, 19 October 2016

<!DOCTYPE html> Interactive Points | Codrops

iGEM 2016         NUS SINGAPORE

Default Demo Perspective Demo (Webkit only)

Wet Lab Notebook: RIOT RESPONDER

Jun

2016

Overall Strategies

1 June, 2016 Milestone 1 : Overall cloning design

12 June, 2016 Milestone 2: Construction of pConst-LuxR-HA

Construct 1:

Fig 1. A. LuxR gene amplified from iGEM distribution 2016 with designed primers GEM002 and GEM005. The expected band size of LuxR with overhang is 795 bp. B.LuxR stitiched with terminater (BBa_B0015) with overlapping PCR. C. Constructed full-length RBS-pConstitutive-LuxR-HA-Terminator with expected size of 1034 bp.

July

2016

Construct 2:

Fig 2. A. LuxI gene is cloned from iGEM2016 distribution kit with primers GEM008 and GEM011. The observed band size of LuxI with overhangs is close to the expected band size of 629 bp. B.Terminator BBa_B0015 is amplified using GEM009 and 012 and stitiched with LuxI. C. Promoter-RBS fragment is synthesized and stitiched to LuxI-terminator by PCR reaction. The observed band size for full LuxI construct is close to the expected band size of 936 bp.

4 July, 2016 Milestone 3: : Construction of pLuxR-LuxI-HA

11 July, 2016 Milestone 4: Screening of pLuxR-invasin constructs

Aug

2016

1 Aug, 2016 Milestone 5: Screening of pLuxR-LLO construct

8 Aug, 2016 Milestone 6: Construction of pLuxR-GFP plasmid

1 Aug, 2016 Milestone 7: Testing the assembled pConst-LuxR-pLuxR-LuxI construct

Sep

2016

19 Sep, 2016 Milestone 8: Testing the assembled pConst-LuxR-pLuxR-LLO construct

Oct

2016

10 Oct, 2016 Milestone 9: Testing the assembled pConst-LuxR-pLuxR-GFP construct

  • © NUS_Singapore iGEM 2016
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