Difference between revisions of "Team:Dundee/labjournal manu"

Line 1,831: Line 1,831:
  
 
                   <p><b>Results:</b>
 
                   <p><b>Results:</b>
−
                     <a data-toggle="modal" data-target="#dundee15_chr_igem040" class="journal-protocol">Confirmation of insert size.</a>
+
                     <p><a data-toggle="modal" data-target="#dundee15_chr_igem040" class="journal-protocol">Confirmation of insert size.</a></p>
−
                     <a data-toggle="modal" data-target="#dundee15_chr_150729_chrb-combined" class="journal-protocol">Confirmation of sequence.</a>
+
                     <p><a data-toggle="modal" data-target="#dundee15_chr_150729_chrb-combined" class="journal-protocol">Confirmation of sequence.</a></p>
 
                   </p>
 
                   </p>
  
Line 1,847: Line 1,847:
 
       <div class="border-day">
 
       <div class="border-day">
 
         <div class="box">
 
         <div class="box">
−
             <span class="box-title">Day 4</span> <!--Title of collapsible box-->
+
             <span class="box-title">Day 4 - 30/07</span> <!--Title of collapsible box-->
−
               <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew1-4"></span>
+
               <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew6-4"></span>
−
                 <div id="collapsiblew1-4" class="collapse box-content">
+
                 <div id="collapsiblew6-4" class="collapse box-content">
  
  
−
                   <p><b>Aim of experiment:</b> To prepare <a href="http://parts.igem.org/Part:BBa_K1058008">BBa_K1058008</a> for sequence confirmation, storage, and further processing.</p>
+
                   <p><b>Aim of experiment:</b>Digest of pSB1C3-pChr with SpeI/PstI for insertion of GFP. Repeat of unsuccessful transformation from 28/07.</p>
  
 
                   <p><b>Protocols Used:</b>
 
                   <p><b>Protocols Used:</b>
−
                     <p><a data-toggle="modal" data-target="#plating-modal" class="journal-protocol"> Click here to see our plating protocol.</a></p>
+
                     <p><a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol">Restriction digest</a> of pSB1C3-pChr with SpeI/PstI for insertion of GFP.</p>
−
                     <p><a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol"> Click here to see our overnight culture protocol.</a></p></p>
+
                     <p><a data-toggle="modal" data-target="#ligation-modal" class="journal-protocol">Ligation</a> of GFPmut2, previously digested with XbaI/PstI.</p>
 +
                    <p><a data-toggle="modal" data-target="#transformation-modal" class="journal-protocol">Transformation</a> of pChrGFP into JM110.</p>
 +
                  </p>
  
−
                   <p><b>Results:</b> N/A</p>
+
                   <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_150731_pChrGFP-ligations" class="journal-protocol"> Figure 22 </a></p>
  
−
                  <!-- For images in the results use the following line of code:
 
  
−
                  <p><b>Results:</b><a data-toggle="modal" data-target="#lbpa-figure1-modal" class="journal-protocol"> Figure 1 </a></p>
+
                   <p><b>Next Steps:</b> Purification of the Plasmids for confirmation of insert size and sequence.</p>
−
 
+
−
                  under data-target refer to a modal specifically made for each image under image modals section-->
+
−
 
+
−
 
+
−
                   <p><b>Next Steps:</b> Purification of the Plasmid, containing <a href="http://parts.igem.org/Part:BBa_K1058008">BBa_K1058008</a> and confirmation of size and sequence.</p>
+
 
                    
 
                    
 
                 </div>
 
                 </div>
Line 1,878: Line 1,874:
 
       <div class="border-day">
 
       <div class="border-day">
 
         <div class="box">
 
         <div class="box">
−
             <span class="box-title">Day 5</span> <!--Title of collapsible box-->
+
             <span class="box-title">Day 5 - 31/07</span> <!--Title of collapsible box-->
−
               <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew1-5"></span>
+
               <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew6-5"></span>
−
                 <div id="collapsiblew1-5" class="collapse box-content">
+
                 <div id="collapsiblew6-5" class="collapse box-content">
  
  
−
                   <p><b>Aim of experiment:</b> To prepare <a href="http://parts.igem.org/Part:BBa_K1058008">BBa_K1058008</a> for sequence confirmation, storage, and further processing.</p>
+
                   <p><b>Aim of experiment:</b>Overnight cultures for subsequent plasmid purification of pSB1C3-pChr-GFP.</p>
  
 
                   <p><b>Protocols Used:</b>
 
                   <p><b>Protocols Used:</b>
−
                     <p><a data-toggle="modal" data-target="#plating-modal" class="journal-protocol"> Click here to see our plating protocol.</a></p>
+
                     <p><a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol">Overnight culture</a>of pSB1C3-pChr-GFP.</p>
−
                    <p><a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol"> Click here to see our overnight culture protocol.</a></p></p>
+
                  </p>
  
 
                   <p><b>Results:</b> N/A</p>
 
                   <p><b>Results:</b> N/A</p>
Line 1,898: Line 1,894:
  
  
−
                   <p><b>Next Steps:</b> Purification of the Plasmid, containing <a href="http://parts.igem.org/Part:BBa_K1058008">BBa_K1058008</a> and confirmation of size and sequence.</p>
+
                   <p><b>Next Steps:</b> Purification of the Plasmids for confirmation of insert size and sequence.</p>
 
                    
 
                    
 
                 </div>
 
                 </div>
Line 1,909: Line 1,905:
 
       <div class="border-day">
 
       <div class="border-day">
 
         <div class="box">
 
         <div class="box">
−
             <span class="box-title">Day 6</span> <!--Title of collapsible box-->
+
             <span class="box-title">Day 6 - 01/08</span> <!--Title of collapsible box-->
−
               <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew1-6"></span>
+
               <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew6-6"></span>
−
                 <div id="collapsiblew1-6" class="collapse box-content">
+
                 <div id="collapsiblew6-6" class="collapse box-content">
  
  
−
                   <p><b>Aim of experiment:</b> To prepare <a href="http://parts.igem.org/Part:BBa_K1058008">BBa_K1058008</a> for sequence confirmation, storage, and further processing.</p>
+
                   <p><b>Aim of experiment:</b> Purification of pSB1C3-pChr-GFP for confirmation of insert size and sequence.</p>
  
 
                   <p><b>Protocols Used:</b>
 
                   <p><b>Protocols Used:</b>
−
                     <p><a data-toggle="modal" data-target="#plating-modal" class="journal-protocol"> Click here to see our plating protocol.</a></p>
+
                     <p><a data-toggle="modal" data-target="#miniprep-modal" class="journal-protocol">Miniprep</a> for purification of pSB1C3-pChr-GFP.</p>
−
                     <p><a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol"> Click here to see our overnight culture protocol.</a></p></p>
+
                     <p><a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol">Restriction digest</a> of purified plasmid with EcoRI/PstI for confirmation of insert size.</p>
 +
                  </p>
  
−
                   <p><b>Results:</b> N/A</p>
+
                   <p><b>Results:</b>
 +
                    <p><a data-toggle="modal" data-target="#dundee15_chr_igem043" class="journal-protocol"> Figure 24 </a> Gel for size confirmation.</p>
 +
                    <p><a data-toggle="modal" data-target="#dundee15_chr_150801_pchrgfp" class="journal-protocol"> Figure 24 </a> Sequence confirmation.</p>
 +
                  </p>
  
−
                  <!-- For images in the results use the following line of code:
+
                   <p><b>Next Steps:</b>Overnight cultures in preparation for western blotting for characterisation of expression of both, GFP, and ChrB.</p>
−
 
+
−
                  <p><b>Results:</b><a data-toggle="modal" data-target="#lbpa-figure1-modal" class="journal-protocol"> Figure 1 </a></p>
+
−
 
+
−
                  under data-target refer to a modal specifically made for each image under image modals section-->
+
−
 
+
−
 
+
−
                   <p><b>Next Steps:</b> Purification of the Plasmid, containing <a href="http://parts.igem.org/Part:BBa_K1058008">BBa_K1058008</a> and confirmation of size and sequence.</p>
+
 
                    
 
                    
 
                 </div>
 
                 </div>
Line 1,940: Line 1,933:
 
       <div class="border-day">
 
       <div class="border-day">
 
         <div class="box">
 
         <div class="box">
−
             <span class="box-title">Day 7</span> <!--Title of collapsible box-->
+
             <span class="box-title">Day 7 - 02/08</span> <!--Title of collapsible box-->
−
               <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew1-7"></span>
+
               <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew6-7"></span>
−
                 <div id="collapsiblew1-7" class="collapse box-content">
+
                 <div id="collapsiblew6-7" class="collapse box-content">
  
  
−
                   <p><b>Aim of experiment:</b> To prepare <a href="http://parts.igem.org/Part:BBa_K1058008">BBa_K1058008</a> for sequence confirmation, storage, and further processing.</p>
+
                   <p><b>Aim of experiment:</b>Overnight cultures of sequence confirmed ChrB, ChrBopt, pChrGFP1 (from 15/07), pChrGFP3 and pChrGFP4 (31/07) for subsequent western blotting.</p>
  
 
                   <p><b>Protocols Used:</b>
 
                   <p><b>Protocols Used:</b>
−
                    <p><a data-toggle="modal" data-target="#plating-modal" class="journal-protocol"> Click here to see our plating protocol.</a></p>
+
                     <p><a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol"> Overnight culture</a> of named constituents.</p>
−
                     <p><a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol"> Click here to see our overnight culture protocol.</a></p></p>
+
                  </p>
  
 
                   <p><b>Results:</b> N/A</p>
 
                   <p><b>Results:</b> N/A</p>
Line 1,960: Line 1,953:
  
  
−
                   <p><b>Next Steps:</b> Purification of the Plasmid, containing <a href="http://parts.igem.org/Part:BBa_K1058008">BBa_K1058008</a> and confirmation of size and sequence.</p>
+
                   <p><b>Next Steps:</b> Western blotting for characterisation of expression of both, GFP, and ChrB.</p>
 
                    
 
                    
 
                 </div>
 
                 </div>
Line 4,361: Line 4,354:
 
       <div class="modal-body">
 
       <div class="modal-body">
 
       <img src="https://static.igem.org/mediawiki/2015/a/a4/Dundee15_chr_150729_ChrB-combined.png" style="width:400px;height:auto">'  
 
       <img src="https://static.igem.org/mediawiki/2015/a/a4/Dundee15_chr_150729_ChrB-combined.png" style="width:400px;height:auto">'  
 +
 +
      <!--insert link of uploaded files here-->
 +
         
 +
      </div>
 +
      <div class="modal-footer">
 +
        <button type="button" class="btn btn-primary" data-dismiss="modal">Close</button>
 +
      </div>
 +
    </div>
 +
  </div>
 +
</div>
 +
 +
 +
<div class="modal fade" id="dundee15_chr_150731_pChrGFP-ligations" tabindex="-1" role="dialog" aria-labelledby="myModalLabel">
 +
  <div class="modal-dialog" role="document">
 +
    <div class="modal-content">
 +
      <div class="modal-header">
 +
        <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">&times;</span></button>
 +
        <div class="modal-title" id="myModalLabel">Figure 23: Plates after ligation of GFP into pSB1C3-pChr. Left: Ratio Insert:Vector 2:1; Right: Ration Insert:Vector 3:1.
 +
        </div>
 +
      </div>
 +
      <div class="modal-body">
 +
      <img src="https://static.igem.org/mediawiki/2015/5/54/Dundee15_chr_150731_pChrGFP-ligations.png" style="width:400px;height:auto">'
 +
 +
      <!--insert link of uploaded files here-->
 +
         
 +
      </div>
 +
      <div class="modal-footer">
 +
        <button type="button" class="btn btn-primary" data-dismiss="modal">Close</button>
 +
      </div>
 +
    </div>
 +
  </div>
 +
</div>
 +
 +
 +
<div class="modal fade" id="dundee15_chr_igem043" tabindex="-1" role="dialog" aria-labelledby="myModalLabel">
 +
  <div class="modal-dialog" role="document">
 +
    <div class="modal-content">
 +
      <div class="modal-header">
 +
        <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">&times;</span></button>
 +
        <div class="modal-title" id="myModalLabel">Figure 24: Gel for size confirmation of pSB1C3-pChr-GFP (Top). The bottom lanes are unrelated to this project. Samples 3 and 4 were submitted for sequencing.
 +
        </div>
 +
      </div>
 +
      <div class="modal-body">
 +
      <img src="https://static.igem.org/mediawiki/2015/7/7e/Dundee15_chr_igem043.png" style="width:400px;height:auto">'
 +
 +
      <!--insert link of uploaded files here-->
 +
         
 +
      </div>
 +
      <div class="modal-footer">
 +
        <button type="button" class="btn btn-primary" data-dismiss="modal">Close</button>
 +
      </div>
 +
    </div>
 +
  </div>
 +
</div>
 +
 +
 +
<div class="modal fade" id="dundee15_chr_150801_pchrgfp" tabindex="-1" role="dialog" aria-labelledby="myModalLabel">
 +
  <div class="modal-dialog" role="document">
 +
    <div class="modal-content">
 +
      <div class="modal-header">
 +
        <button type="button" class="close" data-dismiss="modal" aria-label="close" aria-hidden="true" role="dialog" tabindex="-1"><span aria-hidden="true">&times;</span></button>
 +
        <div class="modal-title" id="myModalLabel">Figure 25: Gel for size confirmation of pSB1C3-pChr-GFP (Top). The bottom lanes are unrelated to this project. Samples 3 and 4 were submitted for sequencing.
 +
        </div>
 +
      </div>
 +
      <div class="modal-body">
 +
      <img src="https://static.igem.org/mediawiki/2015/2/2c/Dundee15_chr_150801_pSB1C3-pChr-GFP.png" style="width:400px;height:auto">'
  
 
       <!--insert link of uploaded files here-->
 
       <!--insert link of uploaded files here-->

Revision as of 04:43, 13 August 2015

LABJOURNAL

BioSpray

Our forensic toolkit aims to use synthetic biology approaches to improve on the current methods used by crime scene investigators.

Chromium Detector

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Curabitur a tincidunt elit. Aliquam porta nibh at enim luctus, auctor consequat dolor vehicula.

Fingerprint Aging

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Curabitur a tincidunt elit. Aliquam porta nibh at enim luctus, auctor consequat dolor vehicula.

22/06 - 28/06

Summary

This week was all about getting started with the newly arrived biobrick BBa_K1058008

Day 1

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 BBa_K1058008 and confirmation of size and sequence.

Day 2

Aim of experiment: To purify BBa_K1058008 for sequence confirmation.

Protocols Used:

Click here to see our miniprep protocol.

Click here to see our sequencing protocol.

Results Miniprep:

Sample Name

Concentration

Unit

Blank

-

-

BBa_K1058008

190.93

ng/µl

Results Sequencing: Figure 1

Next Steps: PCR of parts of BBa_K1058008.

29/06 - 05/07

Summary

During this week, BBa_K1058008 was disassembled into its constituent parts, pChr, and ChrB.

Day 1

Aim of experiment: To break down BBa_K1058008 into the promoter region pChr, and the repressor gene, ChrB, while adding standard prefix and suffix to each. Furthermore optimisation of first 15 codons of ChrB in order to increase the overall rate of transcription. Restriction digest of the respective ends and subsequent ligation of pChr and ChrB into pSB1C3 for submission to registry.

Protocols Used:

Click here to see our PCR protocol.

Click here to see our gel extraction protocol.

Click here to see our restriction digest protocol.

Results:

Figure 2: Agarose gel after PCR of pChr and ChrB.

As can be seen on the image of the gel, PCR of ChrB was not successful. pChr was further processed by restriction digest with EcoRI and PstI

Next Steps: Repeat of PCR of ChrB. Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

06/07 - 12/07

Summary

During this week ligations of pChr and ChrB into pSB1C3 were finalised and the constituent parts of our newly designed chromate sensing system were prepared for ligation and transformation.

Day 1

Aim of experiment: To repeat PCR of ChrB for subsequent ligation into pSB1C3. Ligation of pChr and ChrB into pSB1C3. Transformation of each into a non-pathogenic lab strain of E. coli.

Overnight culture of strain, containing GFPmut2, as a fluorescent reporter for our Chromate sensing system

Protocols Used:

Click here to see our PCR protocol.

The annealing temperature was lowered in order to increase the chances of accomodating a primer that is not 100% complementary. Furthermore different concentrations of DMSO were tested.

Click here to see our gel extraction protocol.

Click here to see our restriction digest protocol.

Click here to see our ligation protocol. Ligations were made in 2:1 and 3:1 ratio.

Click here to see our Overnight culture protocol.

Results:

Figure 3: Agarose gel after PCR of ChrB.

2 bands were extracted from the gel for further characterisation, the strongest band, hence called ChrBs, and the band directly above, hence called ChrBw.

pChr, ChrBs, and ChrBw were ligated into pSB1C3.

pChr-pSB1C3, ChrBs-pSB1C3, and ChrBw-pSB1C3 were transformed into E. coli MC1061.

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 2

Aim of experiment: Purification of GFPmut2 from overnight cultures. Overnight cultures of recombinant MC1061, containing pChr-pSB1C3, ChrBs-pSB1C3, and ChrBw-pSB1C3 respectively. Overnight culture of pUniprom for subsequent ligation of ChrB

Protocols Used:

Miniprep for plasmid purification of overnight culture of GFPmut2.

PCR for amplification of GFPmut2 and ChrB.

Gel extraction of PCR product pf GFPmut2.

Overnight culture of MC1061 containing pChr-pSB1C3, ChrBs-pSB1C3, and ChrBw-pSB1C3 respectively. Furthermore of a strain containing the vector pUniprom.

Results: Figure 5 Since amplification of ChrB was unsuccessful, it will be repeated.

Next Steps: Sequence and size confirmation of pChr-pSB1C3, ChrBs-pSB1C3, and ChrBw-pSB1C3.

Day 3

Aim of experiment: To purify overnight cultures for size and sequence confirmation. Repeat of PCR amplification of pChr and ChrB. Restriction digestes of all parts in order to produce compatible sticky ends.

Protocols Used:

Miniprep of pChr-pSB1C3, ChrBs-pSB1C3, ChrBw-pSB1C3 and pUniprom.

Results Miniprep:

Sample Name

Concentration

Unit

Blank

-

-

pUniprom1

55.65

ng/µl

pUniprom2

64.20

ng/µl

pChr1

116.58

ng/µl

pChr2

110.13

ng/µl

pChr3

114.65

ng/µl

pChr4

105.63

ng/µl

ChrBs1

151.18

ng/µl

ChrBs2

108.37

ng/µl

ChrBs3

143.13

ng/µl

ChrBs4

183.27

ng/µl

ChrBw1

116.26

ng/µl

ChrBw2

236.99

ng/µl

ChrBw3

219.21

ng/µl

ChrBw4

208.27

ng/µl

Protocols Used:

PCR for amplification of pChr and ChrB.

Restriction digest for confirmation of size of pChr and ChrB.

Results: Figure 6 Agarose gel for size confirmation.

Protocols Used:

Sequencing of pChr and ChrB that showed expectes sizes.

Results: Figure 7 Confirmed sequence of pChr1.

Results: Figure 8 Confirmed sequence of ChrBw4.

Protocols Used:

Restriction digest of gel purified PCR-products of pChr, ChrB, and GFPmut2, using EcoRI/SpeI, BamHI/HindIII, and XbaI/PstI respecitvely.

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 4

Aim of experiment: To prepare pUniprom for insertion of ChrB.

Protocols Used:

Restriction digest of pUniprom with BamHI and HindIII.

Gel extraction of digested pUniprom

Results: Figure 9

Next Steps: Ligation of ChrB into pUniprom.

Aim of experiment: To amplify ChrB version with first 15 codons optimised for insertion into pUniprom.

Protocols Used:

PCR of optimised ChrB from the sequence confirmed ChrBw4-pSB1C3, adding restriction sites compatible to pUniprom.

Gel extraction of optimised ChrBafter PCR.

Results: Figure 10

Next Steps: Ligation of ChrB and codon optimised ChrB into pUniprom.

Day 5

Aim of experiment: Ligation of ChrB and optimised ChrB into pUniprom. Ligation of pChr and GFPmut2 into pSB1C3

Protocols Used:

Ligation of ChrB and optimised ChrB into pUniprom via BamHI and HindIII restriction sites. Ligation of pChr to GFPmut2 via Spe/Xba link and ligation of pChrGFP construct into pSB1C3 EcoRI/PstI restriction sites.

Results: N/A

Next Steps:Transformation of abovementioned ligations.

13/07 - 19/07

Summary

During this week successful transformations were identified and propagated.

Day 1

Aim of experiment: Transform recombinant vectors into E. coli chassis.

Protocols Used:

Transformation of ChrB-pUniprom, optimised ChrB-pUniprom and pChr-GFPmut2-pSB1C3 into JM110.

Results: Figure 11

Aim of experiment: Backup of digested pSB1C3 for the case that a ligation with 2 inserts does not work.

Protocols Used:

Restriction digest of pSB1C3 with EcoRI/SpeI for subsequent insertion of pChr.

Results:N/A

Next Steps: Store digested plasmid at -20oC.

Day 2

Aim of experiment: Purification of recombinant vectors for sequence and size confirmation.

Protocols Used:

Overnight culture of JM110 with ChrB-pUniprom, optimised ChrB-pUniprom and pChr-GFPmut2-pSB1C3.

Patch plating of the same colonies.

Results: N/A

Next Steps: Purification of the recombinant plasmids for confirmation of size and sequence.

Day 3

Aim of experiment: Purification of recombinant plasmids for confirmation of size and sequence.

Protocols Used:

Miniprep of ChrB-pUniprom, optimised ChrB-pUniprom and pChr-GFPmut2-pSB1C3.

Results:

Sample Name

Concentration

Unit

Blank

-

-

pSB1C3-pChr-GFPmut2_1

382.32

ng/µl

pSB1C3-pChr-GFPmut2_2

355.31

ng/µl

pSB1C3-pChr-GFPmut2_3

209.85

ng/µl

pSB1C3-pChr-GFPmut2_4

217.57

ng/µl

pUniprom-ChrB-opt_1

150.51

ng/µl

pUniprom-ChrB-opt_2

214.17

ng/µl

pUniprom-ChrB-opt_3

140.85

ng/µl

pUniprom-ChrB-opt_4

115.85

ng/µl

pUniprom-ChrB_1

134.72

ng/µl

pUniprom-ChrB_2

111.41

ng/µl

pUniprom-ChrB_3

111.31

ng/µl

pUniprom-ChrB_4

138.9

ng/µl

Protocols Used:

Restriction digest of ChrB-pUniprom and optimised ChrB-pUniprom with BamHI and HindIII. Restriction digest of pChr-GFPmut2-pSB1C3 with EcoRI/PstI.

Results: Figure 1

Protocols Used:

Colony PCR of pUniprom-ChrB-opt for elucidation of insert size or presence of insert.

Results: Figure 13 Gel after colony PCR. Colonies 2, 4, 9, and 12 were selected for sequencing.

Protocols Used:

Sequencing of pSB1C3-pChr-GFP.

Results: Figure 14 Sequencing result of pSB1C3-pChr-GFPmut2_1. The Result for pSB1C3-pChr-GFPmut2_2 was not as expected, albeit in the correct orientation.

Next Steps: Confirmation of insert size and sequence of ChrB-opt.

Day 4

Aim of experiment: To prepare ChrB-opt colonies 2, 4, 9, and 12 for sequencing.

Protocols Used:

Click here to see our overnight culture protocol.

Results:N/A

Next Steps: Purification of recombinant plasmids.

Day 5

Aim of experiment: To purify recombinant plasmids for sequence confirmation.

Protocols Used:

Miniprep of ChrB-opt 2, 4, 9, 12.

Results:

Sample Name

Concentration

Unit

Blank

-

-

pUniprom-ChrB-opt_2

255.35

ng/µl

pUniprom-ChrB-opt_4

146.77

ng/µl

pUniprom-ChrB-opt_9

211.91

ng/µl

pUniprom-ChrB-opt_12

181.08

ng/µl

Protocols Used:

Sequencing of ChrB-opt 2, 4, 9, 12.

Results: ChrB showed HindIII restriction sites and was hence cleaved into partial inserts.

Next Steps:Use alternative cloning protocol for ChrB and ChrBopt.

20/07 - 26/07

Summary

During this week an alternative cloning strategy for ChrB and ChrB-opt was employed. Furthermore, the pChr-GFP construct was cloned again in a stepwise fashion in order to clone it in the correct orientation.

Day 1 - 20/07

Aim of experiment:To compare colonies on the patch plate of pSB1C3-pChr-GFP by differentially visualising colonies that do or do not express GFP under UV light.

Results:Figure 15 pSB1C3-pChr-GFP in JM110.

Next Steps:Size confirmation and sequencing of newly selected colonies.

Day 2 - 21/07

Aim of experiment:Purification of newly selected colonies containing pSB1C3-pChr-GFP for subsequent sequence confirmation.

Protocols Used:

Miniprep for purification of pSB1C3-pChr-GFP.

Results:

Sample Name

Concentration

Unit

Blank

-

-

pSB1C3-pChr-GFP_1

313.63

ng/µl

pSB1C3-pChr-GFP_5

673.70

ng/µl

pSB1C3-pChr-GFP_6

678.02

ng/µl

pSB1C3-pChr-GFP_7

537.87

ng/µl

Next Steps:Sequencing of pSB1C3-pChr-GFP samples 1, 5, 6, and 7.

Protocols Used:

Sequencing of pSB1C3-pChr-GFP samples 1, 5, 6, and 7.

Results: Figure 16 Alignment of sequencing results.

Next Steps:Stepwise ligation of pChr-GFP into pSB1C3.

Protocols Used:

Restriction digest of pUniprom with BamHI/PstI for subsequent insertion of ChrB and ChrB-opt.

p>Results: Figure 17 Gel image

Next Steps:Insertion of ChrB and ChrB-opt respectively via BamHI/PstI.

Day 3 - 22/07

Aim of experiment:Ligation of pChr into pSB1C3 via Eco/Spe link.

Protocols Used:

Ligation of pChr(08/07/15) into pSB1C3(13/07/15)

Results: N/A

Aim of experiment:Transformation

Protocols Used:

Transformation of pSB1C3-pChr into JM110.

Results: Figure 18 Plates after transformation of pChr into pSB1C3.

Next Steps:Sequence confirmation of pChr in pSB1C3.

Day 4 - 23/07

Aim of experiment: To prepare pSB1C3-pChr for sequence confirmation

Protocols Used:

Overnight culture of pSB1C3-pChr.

Patch-plating of the same colonies of pSB1C3-pChr.

Results: N/A

Next Steps: Purification of the recombinant vector for sequence confirmation.

Day 5 - 24/07

Aim of experiment:Purification of pSB1C3-pChr for sequence confirmation.

Protocols Used:

Miniprep of pSB1C3-pChr.

Results: Insert table here

Aim of experiment:Sequence confirmation of pSB1C3-pChr.

Protocols Used:

Sequencing of samples 1 and 2 of pSB1C3-pChr.

Results: Figure 19 Alignment of sequenced pSB1C3-pChr vectors with pChr sequence.

Next Steps: Digest of pSB1C3-pChr with SpeI/PstI and ligation of GFPmut2.

Day 6 - 25/07

Aim of experiment: Amplification of ChrB and optimised ChrB

Protocols Used:

PCR-mplification of ChrB and ChrBopt.

Results: N/A

Next Steps:Ligation and Transformation of ChrB and optimised ChrB.

Day 7 - 26/07

Aim of experiment: Insertion of ChrB and optimised ChrB into pUniprom vector.

Protocols Used:

Ligation of ChrB and optimised ChrB into vector pUniprom.

Results: N/A

Next Steps: Transformation of ligated vectors.

27/07 - 02/08

Summary

Final touches were made on the chromate system. All parts were ligated into their final vectors during this week. Preparations were made for characterisation of each of the parts.

Day 1 - 27/07

Aim of experiment:Transformation of the repressors, ChrB and optimised ChrB, into JM110

Protocols Used:

Transformation of ChrB and optimised ChrB into JM110

Results: Figure 1

Next Steps: Purification of the plasmids for sequence confirmation.

Day 2 - 28/07

Aim of experiment:Processing of pSB1C3-pChr from 24/07 for insertion of GFP.

Protocols Used:

Restriction digest of pSB1C3-pChr with SpeI/PstI for insertion of GFP.

Ligation of GFPmut2, previously digested with XbaI/PstI.

Results: N/A

Next Steps: Transformation of recombinant vector for confirmation of size and sequence.

Aim of experiment:Overnight culture from plates of ChrB and optimised ChrB.

Protocols Used:

Overnight culture of of ChrB and optimised ChrB.

Results: N/A

Next Steps: Purification of plasmids for sequence confirmation.

Day 3 - 29/07

Aim of experiment:Purification of ChrB and optimised ChrB for confirmation of size and sequence.

Protocols Used:

Miniprep of ChrB and ChrBopt.

Restriction Digest of the purified vectors for confirmation of size.

Sequencing of ChrB and ChrBopt.

Results:

Confirmation of insert size.

Confirmation of sequence.

Next Steps: Confirmation of expression of ChrB and ChrBopt by western blotting.

Day 4 - 30/07

Aim of experiment:Digest of pSB1C3-pChr with SpeI/PstI for insertion of GFP. Repeat of unsuccessful transformation from 28/07.

Protocols Used:

Restriction digest of pSB1C3-pChr with SpeI/PstI for insertion of GFP.

Ligation of GFPmut2, previously digested with XbaI/PstI.

Transformation of pChrGFP into JM110.

Results: Figure 22

Next Steps: Purification of the Plasmids for confirmation of insert size and sequence.

Day 5 - 31/07

Aim of experiment:Overnight cultures for subsequent plasmid purification of pSB1C3-pChr-GFP.

Protocols Used:

Overnight cultureof pSB1C3-pChr-GFP.

Results: N/A

Next Steps: Purification of the Plasmids for confirmation of insert size and sequence.

Day 6 - 01/08

Aim of experiment: Purification of pSB1C3-pChr-GFP for confirmation of insert size and sequence.

Protocols Used:

Miniprep for purification of pSB1C3-pChr-GFP.

Restriction digest of purified plasmid with EcoRI/PstI for confirmation of insert size.

Results:

Figure 24 Gel for size confirmation.

Figure 24 Sequence confirmation.

Next Steps:Overnight cultures in preparation for western blotting for characterisation of expression of both, GFP, and ChrB.

Day 7 - 02/08

Aim of experiment:Overnight cultures of sequence confirmed ChrB, ChrBopt, pChrGFP1 (from 15/07), pChrGFP3 and pChrGFP4 (31/07) for subsequent western blotting.

Protocols Used:

Overnight culture of named constituents.

Results: N/A

Next Steps: Western blotting for characterisation of expression of both, GFP, and ChrB.

Week Beginning 22/06

Summary

This week was all about getting started with the newly arrived biobrick BBa_K1058008

Day 1

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 2

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 3

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 4

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 5

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 6

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 7

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Week Beginning 22/06

Summary

This week was all about getting started with the newly arrived biobrick BBa_K1058008

Day 1

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 2

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 3

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 4

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 5

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 6

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 7

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Week Beginning 22/06

Summary

This week was all about getting started with the newly arrived biobrick BBa_K1058008

Day 1

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 2

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 3

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 4

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 5

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 6

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 7

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Week Beginning 22/06

Summary

This week was all about getting started with the newly arrived biobrick BBa_K1058008

Day 1

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 2

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 3

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 4

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 5

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 6

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 7

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.