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Team:Bielefeld-CeBiTec/Notebook/Journal/C02-fixation/Jul
From 2014.igem.org
(Difference between revisions)
| Line 74: | Line 74: | ||
<li>Bands as expected (~2070 bp)</li> | <li>Bands as expected (~2070 bp)</li> | ||
</ul> | </ul> | ||
| - | <li>PCR products were <a href= | + | <li>PCR products were <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">purified</a></li> |
</ul> | </ul> | ||
| Line 95: | Line 95: | ||
<li>Bands as expected (~2070 bp)</li> | <li>Bands as expected (~2070 bp)</li> | ||
</ul> | </ul> | ||
| - | <li>PCR products were <a href= | + | <li>PCR products were <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">purified</a></li> |
</ul> | </ul> | ||
</ul> | </ul> | ||
| Line 115: | Line 115: | ||
<li>Bands as expected (~2070 bp)</li> | <li>Bands as expected (~2070 bp)</li> | ||
</ul> | </ul> | ||
| - | <li>PCR products were <a href= | + | <li>PCR products were <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">purified</a></li> |
</ul> | </ul> | ||
</ul> | </ul> | ||
| Line 151: | Line 151: | ||
CFP.</li> | CFP.</li> | ||
<ul> | <ul> | ||
| - | → We will try a <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">DpnI</a> of the template and we will make a <a href= | + | → We will try a <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">DpnI</a> of the template and we will make a <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">purification</a> out of the gel instead of using the PCR product. |
</ul> | </ul> | ||
</ul> | </ul> | ||
| Line 182: | Line 182: | ||
<li>Bands as expected (~13,200 bp)</li> | <li>Bands as expected (~13,200 bp)</li> | ||
</ul> | </ul> | ||
| - | <li>PCR products were <a href= | + | <li>PCR products were <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">purified</a> out of the gel.</li> |
</ul> | </ul> | ||
</ul> | </ul> | ||
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<div class="content" style="margin-right:10%; margin-left:10%"> | <div class="content" style="margin-right:10%; margin-left:10%"> | ||
<ul> | <ul> | ||
| - | <li><b>BioBricks ( | + | <li><b>BioBricks (<a href="http://parts.igem.org/Part:BBa_K731500" target="_blank">BBa_K731500</a> and <a href="http://parts.igem.org/Part:BBa_Q01400" target="_blank">BBa_Q01400</a>)</b></li> |
<ul> | <ul> | ||
| - | <li>This week we tried to | + | <li>This week we tried to isolate promotors of two BioBricks (pSB1C3_P<sub>tac</sub> and pSB1C3_P<sub>Tet</sub>) of the parts distribution.</li> |
<ul> | <ul> | ||
| - | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of | + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1C3_P<sub>tac</sub> and pSB1C3_P<sub>Tet</sub></li> |
</ul> | </ul> | ||
</ul> | </ul> | ||
| - | <li><b>csoS4AB</b></li> | + | |
| + | <br> | ||
| + | |||
| + | <li><b>csoS4AB and csoS1CAB</b></li> | ||
<ul> | <ul> | ||
| - | <li>This week we tried | + | <li>This week we tried to amplify the shell proteins of the carboxysome and the backbone pSB1C3 for 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# | + | <ul><!-- Teil1 --> |
| + | <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#fw_pSB1C3_csoS4A" target="_blank">fw_pSB1C3_csoS4A</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_csoS4A_PstI" target="_blank">rv_csoS4A_PstI</a>)</li> | ||
<ul> | <ul> | ||
| - | <li>Annealing temperature: | + | <li>Annealing temperature: 55 °C</li> |
| - | <li>Bands as expected ( | + | <li>Bands as expected (~180 bp)</li> |
</ul> | </ul> | ||
| - | + | ||
| - | + | <!-- Teil2 --> | |
| - | + | <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#fw_PstI_csoS4A" target="_blank">fw_PstI_csoS4A</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_SpeI_Intergen" target="_blank">rv_SpeI_Intergen</a>)</li> | |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
<ul> | <ul> | ||
| - | <li>Annealing temperature: ...</li> | + | <li>Annealing temperature: 55 °C</li> |
| - | <li>Bands as expected (... bp)</li> | + | <li>Bands as expected (~1200 bp)</li> |
| + | </ul> | ||
| + | |||
| + | <!-- Teil3 --> | ||
| + | <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#fw_SpeI_Intergen" target="_blank">fw_SpeI_Intergen</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_csoS1_pSB1C3" target="_blank">rv_csoS1_pSB1C3</a>)</li> | ||
| + | <ul> | ||
| + | <li>Annealing temperature: 55 °C</li> | ||
| + | <li>Bands as expected (~350 bp)</li> | ||
| + | </ul> | ||
| + | |||
| + | <!-- Teil4 (Backbone) --> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> of the backbone (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_csoS1_pSB1C3" target="_blank">fw_csoS1_pSB1C3</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_pSB1C3_csoS4A" target="_blank">rv_pSB1C3_csoS4A</a>)</li> | ||
| + | <ul> | ||
| + | <li>Annealing temperature: 55 °C</li> | ||
| + | <li>Bands as expected (~2070 bp)</li> | ||
</ul> | </ul> | ||
</ul> | </ul> | ||
</ul> | </ul> | ||
| - | <li><b> | + | |
| + | <br> | ||
| + | |||
| + | <font color="red"><li><b>Shell associated protein</b></li> | ||
<ul> | <ul> | ||
| - | <li>This week we tried to purify the | + | <li>This week we tried to amplify and purify the sap_1 and the backbone pSB1C3.</li> |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
<ul> | <ul> | ||
<li>Purification of can with gel extraction</li> | <li>Purification of can with gel extraction</li> | ||
| Line 252: | Line 259: | ||
</ul> | </ul> | ||
</ul> | </ul> | ||
| + | |||
| + | <br> | ||
| + | |||
<li><b>Pore protein (csoS1D) of the carboxysom</b></li> | <li><b>Pore protein (csoS1D) of the carboxysom</b></li> | ||
<ul> | <ul> | ||
| - | <li>This week we tried to purify the | + | <li>This week we tried to amplify and purify csoS1D and the backbone.</li> |
<ul> | <ul> | ||
<li>Purification of can with gel extraction</li> | <li>Purification of can with gel extraction</li> | ||
| Line 260: | Line 270: | ||
<li>Bands not as expected (~703 bp)</li> | <li>Bands not as expected (~703 bp)</li> | ||
</ul> | </ul> | ||
| - | </ul> | + | </ul></font> |
</ul> | </ul> | ||
| Line 292: | Line 302: | ||
</ul> | </ul> | ||
</ul> | </ul> | ||
| + | |||
| + | <br> | ||
<li><b>Carbnonic anhydrase (can)</b></li> | <li><b>Carbnonic anhydrase (can)</b></li> | ||
<ul> | <ul> | ||
| Line 309: | Line 321: | ||
</ul> | </ul> | ||
</ul> | </ul> | ||
| + | |||
| + | <br> | ||
| + | |||
<li><b>csoS1D</b></li> | <li><b>csoS1D</b></li> | ||
<ul> | <ul> | ||
| Line 330: | Line 345: | ||
</ul> | </ul> | ||
</ul> | </ul> | ||
| + | |||
| + | <br> | ||
| + | |||
<li><b>Phosphoribulokinase (prkA)</b></li> | <li><b>Phosphoribulokinase (prkA)</b></li> | ||
<ul> | <ul> | ||
| Line 347: | Line 365: | ||
</ul> | </ul> | ||
</ul> | </ul> | ||
| + | |||
| + | <br> | ||
| + | |||
<li><b>sRNA:pfkA</b></li> | <li><b>sRNA:pfkA</b></li> | ||
<ul> | <ul> | ||
| Line 355: | Line 376: | ||
</ul> | </ul> | ||
</ul> | </ul> | ||
| + | |||
| + | <br> | ||
| + | |||
<li><b>RuBisCO of <i>H. neapolitanus</i></b></li> | <li><b>RuBisCO of <i>H. neapolitanus</i></b></li> | ||
<ul> | <ul> | ||
| Line 376: | Line 400: | ||
</ul> | </ul> | ||
</ul> | </ul> | ||
| + | |||
| + | <br> | ||
| + | |||
<li><b>RuBisCO of <i>S. elongatus</i></b></li> | <li><b>RuBisCO of <i>S. elongatus</i></b></li> | ||
<ul> | <ul> | ||
| Line 383: | Line 410: | ||
</ul> | </ul> | ||
</ul> | </ul> | ||
| + | |||
| + | <br> | ||
| + | |||
<li><b>BioBricks (BBa_I719005)</b></li> | <li><b>BioBricks (BBa_I719005)</b></li> | ||
<ul> | <ul> | ||
Revision as of 17:46, 6 September 2014
 |
July |
 |
- pHnCBcsoS1D
- This week we tried to isolate the plasmid of the strain ordered from addgene
- Plasmid isolation of pHnCBcsoS1D
- csoS1D of the carboxysome
- This week we tried to amplify the backbone pSB1C3 for csoS1D and csoS1D itself.
- PCR amplification of csoS1D (csoS1D_fwd, csoS1D_rev)
- Annealing temperature: 55 °C
- Bands as expected (~703 bp)
- PCR amplification of pSB1C3 (pSB1C3_suf_csoS1D, pSB1C3_pre_csoS1D)
- Annealing temperature: 55 °C
- Bands as expected (~2070 bp)
- PCR products were purified
- Carbonic anhydrase (can) of the carboxysome
- This week we tried to amplify the backbone pSB1C3 for can and can itself.
- PCR amplification of can (csoS3_can_fwd, csoS3_can_rev)
- Annealing temperature: 55 °C
- Bands as expected (~1605 bp)
- PCR amplification of pSB1C3 (pSB1C3_pre_can, pSB1C3_suf_can)
- Annealing temperature: 55 °C
- Bands as expected (~2070 bp)
- PCR products were purified
- Shell associated protein of the carboxysome (first part of the protein)
- This week we tried to amplify the backbone pSB1C3 for sap and the first part of sap: sap_1.
- PCR amplification of sap_1 (csoS2_sap_1_fwd, csoS2_sap_1_rev)
- Annealing temperature: 55 °C
- Bands as expected (~1235 bp)
- PCR amplification of pSB1C3 (pSB1C3_pre_sap_1, pSB1C3_suf_sap2)
- Annealing temperature: 55 °C
- Bands as expected (~2070 bp)
- PCR products were purified
- csoS1D
- This week we tried to assemble csoS1D with the backbone pSB1C3 and to transform the construct.
- Gibson Assembly with csoS1D and pSB1C3
- Transformation with electrocompotetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: ...
- Bands not as expected (~1250 bp, expected size: ~1017 bp). Size looked like template insert CFP.
- Carbonic anhydrase (can)
- This week we tried to assemble can with the backbone pSB1C3 and to transform the construct.
- Gibson Assembly with can and pSB1C3
- Transformation with electrocompotetent cells
- Transformation was not successful (no colonies on the plates).
- pHnCBcsoS1D backbone
- This week we tried to amplify the backbone of the plasmid.
- PCR amplification (Primer1, Primer2)
- Annealing temperature: ...
- Bands as expected (~13,200 bp)
- PCR products were purified out of the gel.
-
→ We will try a restriction digestion with DpnI of the template and we will make a purification out of the gel instead of using the PCR product.
- BioBricks (BBa_K731500 and BBa_Q01400)
- This week we tried to isolate promotors of two BioBricks (pSB1C3_Ptac and pSB1C3_PTet) of the parts distribution.
- Plasmid isolation of pSB1C3_Ptac and pSB1C3_PTet
- csoS4AB and csoS1CAB
- This week we tried to amplify the shell proteins of the carboxysome and the backbone pSB1C3 for the shell proteins.
- PCR amplification (fw_pSB1C3_csoS4A, rv_csoS4A_PstI)
- Annealing temperature: 55 °C
- Bands as expected (~180 bp)
- PCR amplification (fw_PstI_csoS4A, rv_SpeI_Intergen)
- Annealing temperature: 55 °C
- Bands as expected (~1200 bp)
- PCR amplification (fw_SpeI_Intergen, rv_csoS1_pSB1C3)
- Annealing temperature: 55 °C
- Bands as expected (~350 bp)
- PCR amplification of the backbone (fw_csoS1_pSB1C3, rv_pSB1C3_csoS4A)
- Annealing temperature: 55 °C
- Bands as expected (~2070 bp)
- Shell associated protein
- This week we tried to amplify and purify the sap_1 and the backbone pSB1C3.
- Purification of can with gel extraction
- Bands as expected (~1235 bp)
- Pore protein (csoS1D) of the carboxysom
- This week we tried to amplify and purify csoS1D and the backbone.
- Purification of can with gel extraction
- Bands not as expected (~703 bp)
- SBPase (glpX)
- This week we tried to amplify both parts of glpX.
- PCR amplification (Primer1, Primer2 and Primer3, Primer4)
- Annealing temperature: ...
- Bands (not) as expected (... bp)
- Carbnonic anhydrase (can)
- This week we tried to transform the construct.
- Gibson Assembly with csoS1D and pSB1C3
- Restriction digestion with DpnI
- Bands (not) as expected (... bp)
- Colony PCR (Primer1, Primer2)
- Annealing temperature: ...
- Bands as expected (1900 bp)
- Plasmid isolation of can
- csoS1D
- This week we tried to transform the construct.
- Gibson Assembly with csoS1D and pSB1C3
- Restriction digestion with DpnI
- Bands (not) as expected (... bp)
- Colony PCR (Primer1, Primer2)
- Annealing temperature: ...
- Bands as expected (1000 bp)
- Restriction digestion with EcoRI and XbaI
- Bands (not) as expected (... bp)
- Plasmid isolation of csoS1D
- Phosphoribulokinase (prkA)
- This week we tried to transform the fragments of the gene synthesis
- Transformation with electrocompotetent cells
- Colony PCR (prkA_pHn_fwd, prkA_pHn_rev)
- Annealing temperature: ...
- Bands as expected (~1060 bp)
- Plasmid isolation of prkA
- sRNA:pfkA
- This week we tried to transform the fragments of the gene synthesis
- Transformation with electrocompotetent cells
- Plasmid isolation of sRNA:pfkA
- RuBisCO of H. neapolitanus
- This week we tried to transform the fragments of the gene synthesis
- Transformation with electrocompotetent cells
- Colony PCR (Primer1, Primer2)
- Annealing temperature: ...
- Bands as expected (~2100 bp)
- We plated sample number 1 for a plasmid isolation.
- Plasmid isolation of Hneap
- RuBisCO of S. elongatus
- This week we tried to transform the fragments of the gene synthesis
- Transformation with electrocompotetent cells
- BioBricks (BBa_I719005)
- This week we tried to use the promotor of the BioBrick of the parts distribution.
- Plasmid isolation of pSB1C3_T7 BBa_I719005
