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Team:Bielefeld-CeBiTec/Notebook/Journal/CO2-fixation/Aug
From 2014.igem.org
(Difference between revisions)
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<li>We tried to assemble some inserts with three different promotors to test which one is the best choice.</li> | <li>We tried to assemble some inserts with three different promotors to test which one is the best choice.</li> | ||
<ul> | <ul> | ||
| - | <li>BioBrick Assembly (Suffix)</li> | + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix)</li> |
<ul> | <ul> | ||
<li>Backbones (digested with SpeI, PstI)</li> | <li>Backbones (digested with SpeI, PstI)</li> | ||
| Line 71: | Line 71: | ||
<li>Only the assembly with the p<sub>Tac</sub> promotor was successful.</li> | <li>Only the assembly with the p<sub>Tac</sub> promotor was successful.</li> | ||
</ul> | </ul> | ||
| - | <li>Colony PCR and plasmid isolation of p<sub>Tac</sub>_prkA, p<sub>Tac</sub>_Hneap, | + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> and plasmid isolation of p<sub>Tac</sub>_prkA, p<sub>Tac</sub>_Hneap, |
p<sub>Tac</sub>_SELAN and p<sub>Tac</sub>_sRNA_pfkA</li> | p<sub>Tac</sub>_SELAN and p<sub>Tac</sub>_sRNA_pfkA</li> | ||
| - | <li>Colony PCR of T7 promotor</li> | + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> of T7 promotor</li> |
<ul> | <ul> | ||
<li>Showed in all cases an band 400 bp over the expected value. We tried to extract and | <li>Showed in all cases an band 400 bp over the expected value. We tried to extract and | ||
tranform the promotor from another distribution plate (2013)</li> | tranform the promotor from another distribution plate (2013)</li> | ||
</ul> | </ul> | ||
| - | <li>Colony PCR of T7 promotor from the 2013 distribution plate</li> | + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> of T7 promotor from the 2013 distribution plate</li> |
<ul> | <ul> | ||
<li>Bands as expected (~300 bp)</li> | <li>Bands as expected (~300 bp)</li> | ||
| Line 88: | Line 88: | ||
<li>We tried to [...]</li> | <li>We tried to [...]</li> | ||
<ul> | <ul> | ||
| - | <li>Colony PCR (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#VF-Primer" target="_blank">VF-Primer</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#VR-Primer" target="_blank">VR-Primer</a>)</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#VF-Primer" target="_blank">VF-Primer</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#VR-Primer" target="_blank">VR-Primer</a>)</li> |
| - | <li>Gibson Assembly</li> | + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a></li> |
<li>Plasmid isolation</li> | <li>Plasmid isolation</li> | ||
<li>Restriction digestion with SpeI and XbaI</li> | <li>Restriction digestion with SpeI and XbaI</li> | ||
| Line 95: | Line 95: | ||
<li>Bands as expected ()</li> | <li>Bands as expected ()</li> | ||
</ul> | </ul> | ||
| - | <li>BioBrick Assembly (Suffix)</li> | + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix)</li> |
<ul> | <ul> | ||
<li>Backbone (digested with SpeI, PstI)</li> | <li>Backbone (digested with SpeI, PstI)</li> | ||
| Line 106: | Line 106: | ||
</ul> | </ul> | ||
</ul> | </ul> | ||
| - | <li>Colony PCR of can_csoS1-4</li> | + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> of can_csoS1-4</li> |
<ul> | <ul> | ||
<li>Bands as expected ()</li> | <li>Bands as expected ()</li> | ||
| Line 116: | Line 116: | ||
<li>We tried to [...]</li> | <li>We tried to [...]</li> | ||
<ul> | <ul> | ||
| - | <li>Gibson Assembly after DpnI digestion</li> | + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> after DpnI digestion</li> |
| - | <li>Colony PCR</li> | + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a></li> |
</ul> | </ul> | ||
</ul> <!-- /glpX --> | </ul> <!-- /glpX --> | ||
| Line 128: | Line 128: | ||
<li>Bands as expected ()</li> | <li>Bands as expected ()</li> | ||
</ul> | </ul> | ||
| - | <li>Gibson Assembly with pHnCBcsoS1D</li> | + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with pHnCBcsoS1D</li> |
</ul> | </ul> | ||
</ul> <!-- /prkA --> | </ul> <!-- /prkA --> | ||
| Line 135: | Line 135: | ||
<li>We tried to...</li> | <li>We tried to...</li> | ||
<ul> | <ul> | ||
| - | <li>BioBrick Assembly (Suffix)</li> | + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix)</li> |
<ul> | <ul> | ||
<li>Backbone (digested with SpeI, PstI)</li> | <li>Backbone (digested with SpeI, PstI)</li> | ||
| Line 148: | Line 148: | ||
<li>We assembled p<sub>Tac</sub>_prkA with <i>Hneap</i> respectively <i>SELAN</i></li> | <li>We assembled p<sub>Tac</sub>_prkA with <i>Hneap</i> respectively <i>SELAN</i></li> | ||
</ul> | </ul> | ||
| - | <li>Colony PCR</li> | + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a></li> |
<ul> | <ul> | ||
<li>p<sub>Tac</sub>_prkA_Hneap</li> | <li>p<sub>Tac</sub>_prkA_Hneap</li> | ||
Revision as of 14:56, 26 August 2014
 |
August |
 |
- 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 an band 400 bp over the expected value. We tried to extract and tranform the promotor from another distribution plate (2013)
- Colony PCR of T7 promotor from the 2013 distribution plate
- Bands as expected (~300 bp)
- csoS1-4
- We tried to [...]
- Colony PCR (VF-Primer, VR-Primer)
- Gibson Assembly
- Plasmid isolation
- Restriction digestion with SpeI and XbaI
- Bands as expected ()
- BioBrick Assembly (Suffix)
- Backbone (digested with SpeI, PstI)
- pSB1C3_can
- Insert (digested with XbaI, PstI)
- csoS1-4
- Colony PCR of can_csoS1-4
- Bands as expected ()
- glpX
- We tried to [...]
- Gibson Assembly after DpnI digestion
- Colony PCR
- prkA
- We tried to [...]
- PCR amplification and purification for insertion in pHnCBcsoS1D
- Bands as expected ()
- Gibson Assembly with pHnCBcsoS1D
- 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 with 100%. 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).
