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Team:Bielefeld-CeBiTec/Notebook/Journal/C02-fixation/Jul
From 2014.igem.org
(Difference between revisions)
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<li><b>csoS1D</b></li> | <li><b>csoS1D</b></li> | ||
<ul> | <ul> | ||
| - | <li>This week we tried to transform the construct.</li> | + | <li>This week we tried to assemble csoS1D with the backbone pSB1C3 and to transform the construct.</li> |
<ul> | <ul> | ||
| - | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with csoS1D | + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with csoS1D and pSB1C3</li> |
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells</li> | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells</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# | + | <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> |
<ul> | <ul> | ||
| - | <li>Annealing temperature: ...</li> | + | <li>Annealing temperature: <font color="red">...</font></li> |
| - | <li>Bands not as expected ( | + | <li>Bands not as expected (~1250 bp, expected size: ~1017 bp). Size looked like template insert |
| - | + | CFP.</li> | |
| - | + | <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=“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> | </ul> | ||
</ul> | </ul> | ||
| + | |||
| + | <br> | ||
| + | |||
<li><b>Carbonic anhydrase (can)</b></li> | <li><b>Carbonic anhydrase (can)</b></li> | ||
| - | + | <ul> | |
| - | + | <li>This week we tried to assemble can with the backbone pSB1C3 and to transform the construct.</li> | |
| - | + | <ul> | |
| - | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with can and | + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with can and pSB1C3</li> |
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells</li> | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells</li> | ||
<ul> | <ul> | ||
| - | <li>Transformation was not | + | <li>Transformation was not successful.</li> |
</ul> | </ul> | ||
</ul> | </ul> | ||
</ul> | </ul> | ||
| + | |||
| + | <br> | ||
| + | |||
<li><b>pHnCBcsoS1D backbone</b></li> | <li><b>pHnCBcsoS1D backbone</b></li> | ||
<ul> | <ul> | ||
| Line 286: | Line 292: | ||
<li>This week we tried to transform the construct.</li> | <li>This week we tried to transform the construct.</li> | ||
<ul> | <ul> | ||
| - | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with csoS1D and | + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with csoS1D and pSB1C3</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>DpnI</i></a></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>DpnI</i></a></li> | ||
<ul> | <ul> | ||
| Line 303: | Line 309: | ||
<li>This week we tried to transform the construct.</li> | <li>This week we tried to transform the construct.</li> | ||
<ul> | <ul> | ||
| - | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with csoS1D and | + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with csoS1D and pSB1C3</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>DpnI</i></a></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>DpnI</i></a></li> | ||
<ul> | <ul> | ||
Revision as of 16:28, 6 September 2014
 |
July |
 |
- pHnCBcsoS1D
- This week we aimed 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)
- 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)
- 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)
- 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.
- 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)
-
→ 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 use promotors of two BioBricks of the parts distribution.
- Plasmid isolation of pSB1C3_pTac and pSB1C3_p_TetR
- csoS4AB
- This week we tried amplifiy shell proteins of the carboxysome.
- PCR amplification (Primer1, Primer2)
- Annealing temperature: ...
- Bands as expected (... bp)
- csoS1CAB
- This week we tried amplifiy shell proteins of the carboxysome.
- PCR amplification (Primer1, Primer2)
- Annealing temperature: ...
- Bands as expected (... bp)
- Carbonic anhydrase
- This week we tried to purify the construct from the gel.
- Purification of can with gel extraction
- Bands not as expected (... bp)
- Shell associated protein
- This week we tried to purify the construct from the gel.
- Purification of can with gel extraction
- Bands as expected (~1235 bp)
- Pore protein (csoS1D) of the carboxysom
- This week we tried to purify the construct from the gel.
- 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
