"
Team:Bielefeld-CeBiTec/Notebook/Journal/CO2-fixation/Aug
From 2014.igem.org
(Difference between revisions)
| Line 50: | Line 50: | ||
<div class="content" > | <div class="content" > | ||
<ul> <!-- gesamte Liste --> | <ul> <!-- gesamte Liste --> | ||
| - | + | <li><b>Promotors T7, p<sub>Tac</sub> and p<sub>Tet</sub></b></li> | |
<ul> <!-- Promotors --> | <ul> <!-- Promotors --> | ||
| - | <li>We tried to | + | <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>BioBrick Assembly (Suffix)</li> | ||
| Line 84: | Line 84: | ||
</ul> | </ul> | ||
</ul> <!-- /Promotors --> | </ul> <!-- /Promotors --> | ||
| - | + | <li><b><i>csoS1-4</i></b></li> | |
<ul> <!-- csoS1-4 --> | <ul> <!-- csoS1-4 --> | ||
<li>We tried to [...]</li> | <li>We tried to [...]</li> | ||
| Line 112: | Line 112: | ||
</ul> | </ul> | ||
</ul> <!-- /csoS1-4 --> | </ul> <!-- /csoS1-4 --> | ||
| - | + | <li><b><i>glpX</i></b></li> | |
<ul> <!-- glpX --> | <ul> <!-- glpX --> | ||
<li>We tried to [...]</li> | <li>We tried to [...]</li> | ||
| Line 120: | Line 120: | ||
</ul> | </ul> | ||
</ul> <!-- /glpX --> | </ul> <!-- /glpX --> | ||
| - | + | <li><b><i>prkA</i></b></li> | |
<ul> <!-- prkA --> | <ul> <!-- prkA --> | ||
<li>We tried to [...]</li> | <li>We tried to [...]</li> | ||
<ul> | <ul> | ||
| - | <li>PCR amplification and purification for insertion in pHnCBcsoS1D</li> | + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">purification</a> for insertion in pHnCBcsoS1D</li> |
<ul> | <ul> | ||
<li>Bands as expected ()</li> | <li>Bands as expected ()</li> | ||
| Line 165: | Line 165: | ||
<ul> <!-- Sequencing --> | <ul> <!-- Sequencing --> | ||
<li><i>csoS1D</i></li> | <li><i>csoS1D</i></li> | ||
| + | <ul> | ||
<li>Successful. We got the right sequence with 100%. The first part of the carboxysome is | <li>Successful. We got the right sequence with 100%. The first part of the carboxysome is | ||
complete.</li> | complete.</li> | ||
| + | </ul> | ||
<li><i>can</i></li> | <li><i>can</i></li> | ||
| + | <ul> | ||
<li>Five mutations. Another sequencing will follow.</li> | <li>Five mutations. Another sequencing will follow.</li> | ||
| + | </ul> | ||
<li><i>glpX</i></li> | <li><i>glpX</i></li> | ||
| - | <li>Not successful. We got the CFP sequence instead of the <i>glpX</i> sequence, so we will try another backbone (pSB1C3_RFP).</li> | + | <ul> |
| + | <li>Not successful. We got the CFP sequence instead of the <i>glpX</i> sequence, so we will try | ||
| + | another backbone (pSB1C3_RFP).</li> | ||
| + | </ul> | ||
</ul> <!-- /Sequencing --> | </ul> <!-- /Sequencing --> | ||
Revision as of 14:46, 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 [...]
- 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).
