Team:Bielefeld-CeBiTec/Notebook/Journal/CO2-fixation/Aug

From 2014.igem.org

(Difference between revisions)
Line 291: Line 291:
             </div>
             </div>
             <div class="content">
             <div class="content">
 +
              <ul>
 +
        <li><b>SBPase (glpX)</b></li>
 +
        <ul>
 +
    <li>This week we tried to amplify glpX</li>
 +
                    <ul>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Primer1" target="_blank">Primer1</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Primer2" target="_blank">Primer2</a>) of pSB1C3-RFP backbone</li>
 +
              <ul>
 +
          <li>Annealing temperature: ...</li>
 +
          <li>Bands (not) as expected (... bp)</li>
 +
              </ul>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Primer1" target="_blank">Primer1</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Primer2" target="_blank">Primer2</a>) of glpX</li>
 +
              <ul>
 +
          <li>Annealing temperature: ...</li>
 +
          <li>Bands (not) as expected (... bp)</li>
 +
              </ul>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>DpnI</i></a></li>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with glpX_1 and  glpX_2 on pSB1C3</li>
 +
              <ul>
 +
          <li>Bands (not) as expected (... bp)</li>
 +
              </ul>
 +
                      <li>Another amplification and purification was tried.</li>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Primer1" target="_blank">Primer1</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Primer2" target="_blank">Primer2</a>)</li>
 +
              <ul>
 +
          <li>Annealing temperature: ...</li>
 +
          <li>Bands as expected (~1000 bp) but also additional bands</li>
 +
              </ul>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of glpX</li>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>PstI</i></a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>EcoRI</i></a></li>
 +
              <ul>
 +
          <li>Bands not as expected</li>
 +
              </ul>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Primer1" target="_blank">Primer1</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Primer2" target="_blank">Primer2</a>)</li>
 +
              <ul>
 +
          <li>Annealing temperature: ...</li>
 +
          <li>Bands (not) as expected (... bp)</li>
 +
              </ul>
 +
                    </ul>
 +
        </ul>
 +
        <li><b>pHnCBcsoS1D, prkA</b></li>
 +
        <ul>
 +
    <li>This week we tried to combine the addgene plasmid with the prkA</li>
 +
                    <ul>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells</li>
 +
                    </ul>
 +
        </ul>
 +
                <li><b>csoS1-4</b></li>
 +
        <ul>
 +
    <li>This week we tried again to amplify the shell proteins</li>
 +
                    <ul>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Primer1" target="_blank">Primer1</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Primer2" target="_blank">Primer2</a>)</li>
 +
              <ul>
 +
          <li>Annealing temperature: ...</li>
 +
          <li>Bands (not) as expected (... bp)</li>
 +
              </ul>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>DpnI</i></a></li>
 +
              <ul>
 +
          <li>Bands (not) as expected (... bp)</li>
 +
              </ul>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with csoS1-4 on pSB1C3</li>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Primer1" target="_blank">Primer1</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Primer2" target="_blank">Primer2</a>)</li>
 +
              <ul>
 +
          <li>Annealing temperature: ...</li>
 +
          <li>Bands (not) as expected (... bp)</li>
 +
              </ul>
 +
                    </ul>
 +
        </ul>
 +
                <li><b>prkA and RuBisCO</b></li>
 +
        <ul>
 +
    <li>This week we tried the pTet for prkA and RuBisCO</li>
 +
                    <ul>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrickSuffix" target="_blank">BioBrick Assembly Suffix</a>:</li>
 +
              <ul>
 +
          <li>Backbones (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank">[Enzyme]</a>)</li>
 +
          <ul>
 +
    <li>pTet</li>
 +
          </ul>
 +
        <li>Inserts (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank">[Enzyme]</a>)</li>
 +
        <ul>
 +
    <li>prkA and Hneap</li>
 +
        </ul>
 +
              </ul>
 +
              <li>[...] was succesful</li>
 +
                    </ul>
 +
        </ul>
 +
                <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Primer1" target="_blank">Primer1</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Primer2" target="_blank">Primer2</a>)</li>
 +
        <ul>
 +
    <li>Annealing temperature: ...</li>
 +
    <li>Bands (not) as expected (... bp)</li>
 +
                    <li>Only some colonies, TetR (repressor) not strong enough in the pTet_prkA construct</li>
 +
        </ul>
 +
        <li><b>GFP</b></li>
 +
        <ul>
 +
    <li>This week we tried isolate GFP from the parts distribution</li>
 +
                    <ul>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of [Construct]</li>
 +
                    </ul>
 +
        </ul>
 +
        <li><b>Purification vector</b></li>
 +
        <ul>
 +
    <li>This week we tried amplify the T7 promotor and RBS for the purification vector.</li>
 +
                    <ul>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Primer1" target="_blank">Primer1</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Primer2" target="_blank">Primer2</a>)</li>
 +
              <ul>
 +
          <li>Annealing temperature: ...</li>
 +
          <li>Bands (not) as expected (... bp)</li>
 +
              </ul>
 +
                    </ul>
 +
        </ul>           
 +
        <li><b>fbaA</b></li>
 +
        <ul>
 +
    <li>This week we tried amplify first part of fbaA for purification.</li>
 +
                    <ul>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Primer1" target="_blank">Primer1</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Primer2" target="_blank">Primer2</a>)</li>
 +
              <ul>
 +
          <li>Annealing temperature: ...</li>
 +
          <li>Bands (not) as expected (... bp)</li>
 +
              </ul>
 +
                    </ul>
 +
        </ul>
 +
        <li><b>tktA</b></li>
 +
        <ul>
 +
    <li>This week we tried amplify both parts of tktA for purification.</li>
 +
                    <ul>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Primer1" target="_blank">Primer1</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Primer2" target="_blank">Primer2</a>)</li>
 +
              <ul>
 +
          <li>Annealing temperature: ...</li>
 +
          <li>Bands (not) as expected (... bp)</li>
 +
              </ul>
 +
                    </ul>
 +
        </ul>
 +
        <li><b>gapA</b></li>
 +
        <ul>
 +
    <li>This week we tried amplify both parts of gapA for purification.</li>
 +
                    <ul>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Primer1" target="_blank">Primer1</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Primer2" target="_blank">Primer2</a>)</li>
 +
              <ul>
 +
          <li>Annealing temperature: ...</li>
 +
          <li>Bands (not) as expected (... bp)</li>
 +
              </ul>
 +
                    </ul>
 +
        </ul>
 +
        <li><b>IntCBD</b></li>
 +
        <ul>
 +
    <li>This week we tried amplify the intein tag for the purification vector.</li>
 +
                    <ul>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Primer1" target="_blank">Primer1</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Primer2" target="_blank">Primer2</a>)</li>
 +
              <ul>
 +
          <li>Annealing temperature: ...</li>
 +
          <li>Bands (not) as expected (... bp)</li>
 +
              </ul>
 +
                    <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with T7 RBS and  IntCBD on pSB1C3</li>
 +
                    </ul>
 +
        </ul>
 +
        <li><b>T7 Promotor</b></li>
 +
        <ul>
 +
    <li>This week we tried the T7 promotor for prkA and Hneap</li>
 +
                    <ul>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrickSuffix" target="_blank">BioBrick Assembly Suffix</a>:</li>
 +
              <ul>
 +
          <li>Backbones (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank">[Enzyme]</a>)</li>
 +
          <ul>
 +
    <li>T7</li>
 +
          </ul>
 +
          <li>Inserts (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank">[Enzyme]</a>)</li>
 +
          <ul>
 +
    <li>prkA and Hneap</li>
 +
          </ul>
 +
                </ul>
 +
                <li>[...] was succesful</li>
 +
                        <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Primer1" target="_blank">Primer1</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Primer2" target="_blank">Primer2</a>)</li>
 +
                <ul>
 +
          <li>Annealing temperature: ...</li>
 +
          <li>Bands (not) as expected (... bp)</li>
 +
                </ul>
 +
                    </ul>
 +
        </ul>
 +
 +
 +
 +
 +
        <li><b>Sequencing</b></li>
 +
        <ul>
 +
    <li>pSB1C3_glpX</li>
 +
                    <ul>
 +
                      <li>not successful. It was a sequence of a fluorescence protein (RFP backbone used)</li>
 +
                    </ul>
 +
                    <li>pSB1C3_csoS4ABcsoS1CAB</li>
 +
                    <ul>
 +
                      <li>not successful. It was another sequence. We think of a contamination.</li>
 +
                    </ul>
 +
                    <li>Selan</li>
 +
                    <ul>
 +
                      <li>too many insertion mutations</li>
 +
                    </ul>
 +
                    <li>sRNA:pfkA</li>
 +
                    <ul>
 +
                      <li>successful</li>
 +
                    </ul>
 +
                </ul>
 +
</ul>
 +
 +
</ul>
         </div>
         </div>
       </div>
       </div>
Line 307: Line 509:
             </div>
             </div>
             <div class="content">
             <div class="content">
 +
 +
         </div>
         </div>
       </div>
       </div>

Revision as of 12:29, 28 August 2014


August

Week 1     08/04 - 08/10
Week 1     08/04 - 08/10
  • Promotors T7, pTac and pTet
    • We tried to assemble some inserts with three different promotors to test which one is the best choice.
      • BioBrick Assembly (Suffix)
        • Backbones (digested with SpeI, PstI)
          • T7
          • pTac
          • pTet
        • Inserts (digested with XbaI, PstI)
          • prkA
          • Hneap
          • SELAN
          • sRNA:pfkA
        • Only the assembly with the pTac promotor was successful.
      • Colony PCR and plasmid isolation of pTac_prkA, pTac_Hneap, pTac_SELAN and pTac_sRNA_pfkA
      • Colony PCR of T7 promotor
        • Showed in all cases a band 400 bp over the expected value. We tried to extract and transform the promotor from another distribution plate (2013)
      • Colony PCR of T7 promotor from the 2013 distribution plate
        • Bands as expected (~300 bp)
  • csoS1-4
  • glpX
  • prkA
  • RuBisCO
    • We tried to...
      • BioBrick Assembly (Suffix)
        • Backbone (digested with SpeI, PstI)
          • pTac_prkA
        • Inserts (digested with XbaI, PstI)
          • Hneap
          • SELAN
        • We assembled pTac_prkA with Hneap respectively SELAN
      • Colony PCR
        • pTac_prkA_Hneap
          • Bands es expected ()
        • pTac_prkA_SELAN
          • Bands as expected ()
      • Plasmid isolation of pTac_prkA_Hneap and pTac_prkA_SELAN
  • Sequencing
    • csoS1D
      • Successful. We got the right sequence. The first part of the carboxysome is complete.
    • can
      • Five mutations. Another sequencing will follow.
    • glpX
      • Not successful. We got the CFP sequence instead of the glpX sequence, so we will try another backbone (pSB1C3_RFP).
Week 2     08/11 - 08/17
Week 2     08/11 - 08/17
  • SBPase (glpX)
  • csoS1D
  • csoS1-4
  • Sequencing
    • Plasmid isolation of can, Selan, Hneap, can_csoS1-4
    • can
      • 4 to 5 mutations on the same positions. Maybe we got the wrong accession number
    • csoS1D
      • correct sequence
    • csoS1-4
      • not successful
    • Selan
      • not successful
    • sRNA:pfkA
      • not successful
Week 3     08/18 - 08/24
Week 3     08/18 - 08/24
  • SBPase (glpX)
  • pHnCBcsoS1D, prkA
    • This week we tried to combine the addgene plasmid with the prkA
  • csoS1-4
  • prkA and RuBisCO
  • Colony PCR (Primer1, Primer2)
    • Annealing temperature: ...
    • Bands (not) as expected (... bp)
    • Only some colonies, TetR (repressor) not strong enough in the pTet_prkA construct
  • GFP
    • This week we tried isolate GFP from the parts distribution
  • Purification vector
    • This week we tried amplify the T7 promotor and RBS for the purification vector.
  • fbaA
    • This week we tried amplify first part of fbaA for purification.
  • tktA
    • This week we tried amplify both parts of tktA for purification.
  • gapA
    • This week we tried amplify both parts of gapA for purification.
  • IntCBD
  • T7 Promotor
  • Sequencing
    • pSB1C3_glpX
      • not successful. It was a sequence of a fluorescence protein (RFP backbone used)
    • pSB1C3_csoS4ABcsoS1CAB
      • not successful. It was another sequence. We think of a contamination.
    • Selan
      • too many insertion mutations
    • sRNA:pfkA
      • successful

Retrieved from "http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Journal/CO2-fixation/Aug"