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Team:Bielefeld-CeBiTec/Notebook/Journal/C02-fixation/Jul
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<td style="padding:30px; width:400px"><h1> July </h1></td> | <td style="padding:30px; width:400px"><h1> July </h1></td> | ||
<td width="100px" align="right"><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Journal/CO2-fixation/Aug"><img src="https://static.igem.org/mediawiki/2014/9/9b/Bielefeld-CeBiTec_2014-08-14_Arrow-right.png" width="50px"> </a> </td> | <td width="100px" align="right"><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Journal/CO2-fixation/Aug"><img src="https://static.igem.org/mediawiki/2014/9/9b/Bielefeld-CeBiTec_2014-08-14_Arrow-right.png" width="50px"> </a> </td> | ||
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<li><b>pHnCBcsoS1D</b></li> | <li><b>pHnCBcsoS1D</b></li> | ||
<ul> | <ul> | ||
| - | <li>This week we tried to isolate the plasmid of the strain ordered from addgene</li> | + | <li>This week we tried to isolate the plasmid of the strain ordered from addgene. We used it as template for the party of our carboxysome.</li> |
<ul> | <ul> | ||
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pHnCBcsoS1D</li> | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pHnCBcsoS1D</li> | ||
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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 (<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> | + | <li><b>p<sub>tac</sub> and p<sub>Tet</sub> 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 isolate | + | <li>This week we tried to isolate promoters 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 pSB1C3_P<sub>tac</sub> and pSB1C3_P<sub>Tet</sub></li> | <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> | ||
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<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with <i>glpX_1, glpX_2</i> and pSB1C3 (with and without <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>Dpn</i>I</a>) | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with <i>glpX_1, glpX_2</i> and pSB1C3 (with and without <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>Dpn</i>I</a>) | ||
</li> | </li> | ||
| - | <ul> → The transformation was not successful. We | + | <ul> → The transformation was not successful. We had to make the assemble and transformation again. |
</ul> | </ul> | ||
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</li> | </li> | ||
</ul> | </ul> | ||
| + | → Sequencing of the construct was not successful. We got five mutations so it will be done again. | ||
</ul> | </ul> | ||
<br> | <br> | ||
| - | <li><b><i>csoS1D</i></ | + | <li><b><i>csoS1D</i> <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1465207" target="_blank">(BBa_K1465207)</a></li></b> |
<ul> | <ul> | ||
| - | <li>This week we tried to transform the construct of <i> | + | <li>This week we tried to transform the construct of <i>csoS1D</i> with the backbone pSB1C3.</li> |
<ul> | <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>Dpn</i>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>Dpn</i>I</a></li> | ||
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<br> | <br> | ||
| - | <li><b>Phosphoribulokinase (<i>prkA</i>)</b></li> | + | <li><b>Phosphoribulokinase (<i>prkA</i>) <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1465201" target="_blank">(BBa_K1465201)</a></b></li> |
<ul> | <ul> | ||
<li>This week we tried to assemble <i>prkA</i> of the gene synthesis and the backbone pSB1C3. We aimed to transform the pSB1C3_prkA construct construct.</li> | <li>This week we tried to assemble <i>prkA</i> of the gene synthesis and the backbone pSB1C3. We aimed to transform the pSB1C3_prkA construct construct.</li> | ||
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<br> | <br> | ||
| - | <li><b><i>sRNA:pfkA</i></b></li> | + | <li><b><i>sRNA:pfkA</i> <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1465225" target="_blank">(BBa_K1465225)</a></b></li> |
<ul> | <ul> | ||
<li>This week we tried to assemble <i>sRNA:pfkA</i> of the gene synthesis and the backbone pSB1C3. We aimed to transform the pSB1C3_sRNA:pfkA construct.</li> | <li>This week we tried to assemble <i>sRNA:pfkA</i> of the gene synthesis and the backbone pSB1C3. We aimed to transform the pSB1C3_sRNA:pfkA construct.</li> | ||
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<br> | <br> | ||
| - | <li><b>RuBisCO of <i>H. neapolitanus</i></b></li> | + | <li><b>RuBisCO of <i>H. neapolitanus</i> <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1465202" target="_blank">(BBa_K1465202)</a></b></li> |
<ul> | <ul> | ||
<li>This week we tried to assemble <i>Hneap</i> of the gene synthesis and the backbone pSB1C3. We aimed to transform the pSB1C3_Hneap construct.</li> | <li>This week we tried to assemble <i>Hneap</i> of the gene synthesis and the backbone pSB1C3. We aimed to transform the pSB1C3_Hneap construct.</li> | ||
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</li> | </li> | ||
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of <i>Selan</i></li> | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of <i>Selan</i></li> | ||
| + | |||
</ul> | </ul> | ||
| + | →Sequencing of the construct was not successful. We got a few mutations so it will be done again. | ||
</ul> | </ul> | ||
<br> | <br> | ||
| - | <li><b> | + | <li><b>T7 BioBrick <a href="http://parts.igem.org/Part:BBa_I719005" target="_blank">(BBa_I719005)</a></b></li> |
<ul> | <ul> | ||
| - | <li>This week we tried isolate the T7 | + | <li>This week we tried isolate the T7 promoter (pSB1A2_T7) of the parts distribution 2013.</li> |
<ul> | <ul> | ||
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1A2_T7</li> | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1A2_T7</li> | ||
Latest revision as of 09:44, 17 October 2014
July |
 |
- pHnCBcsoS1D
- This week we tried to isolate the plasmid of the strain ordered from addgene. We used it as template for the party of our carboxysome.
- Plasmid isolation of pHnCBcsoS1D
- Pore protein (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 (sap) 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: 55 °C
- Bands not as expected (~1250 bp, expected size: ~1017 bp). Size looked like template insert CFP.
- 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 (pHnCBcsoS1D_fwd, pHnCBcsoS1D_rev)
- Annealing temperature: 55 °C
- 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.
- ptac and pTet BioBricks (BBa_K731500 and BBa_Q01400)
- This week we tried to isolate promoters of two BioBricks (pSB1C3_Ptac and pSB1C3_PTet) of the parts distribution.
- Plasmid isolation of pSB1C3_Ptac and pSB1C3_PTet
- Shell proteins (csoS4AB and csoS1CAB) of the carboxysome
- 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)
- sap_1
- This week we tried to amplify and purify sap_1 and the backbone pSB1C3.
- PCR amplification of sap_1 (csoS2_sap_1_fwd, csoS2_sap_1_rev)
- Annealing temperature: 55 °C
- Bands as expected (~1235 bp)
- PCR products were purified out of the gel
- PCR amplification of the backbone of sap (pSB1C3_pre_sap_1, pSB1C3_suf_sap2)
- Annealing temperature: 55 °C
- Bands as expected (~2070 bp)
- PCR products were purified out of the gel
- csoS1D
- This week we tried to amplify and purify csoS1D and the backbone pSB1C3.
- PCR amplification of csoS1D (csoS1D_fwd, csoS1D_rev)
- Annealing temperature: 55 °C
- Bands as expected (~700 bp)
- PCR products were purified out of the gel
- PCR amplification of the backbone of sap (pSB1C3_pre_csoS1D, pSB1C3_suf_csoS1D)
- Annealing temperature: 55 °C
- Bands as expected (~2070 bp)
- PCR products were purified out of the gel
- SBPase (glpX)
- This week we tried to amplify and assemble both parts of glpX and the backbone pSB1C3.
- PCR amplification of glpX (fw_pSB1_SBPase, rv_SBPase_XbaI and fw_SBPase_XbaI, rv_pSB1_SBPase)
- Annealing temperature: 55 °C
- Bands as expected (~400 bp, ~600 bp)
- Annealing temperature: 55 °C
- Bands as expected (~2070 bp)
- PCR products were purified
- Gibson Assembly with glpX_1, glpX_2 and pSB1C3 (with and without restriction digestion with DpnI)
- can
- This week we tried to transform the construct of can with the backbone pSB1C3.
- Restriction digestion with DpnI
- Gibson Assembly with can and pSB1C3
- Transformation with electrocompotetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~1900 bp)
- Plasmid isolation of can
- csoS1D (BBa_K1465207)
- This week we tried to transform the construct of csoS1D with the backbone pSB1C3.
- Restriction digestion with DpnI
- Gibson Assembly with csoS1D and pSB1C3
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~1000 bp)
- Plasmid isolation of csoS1D
- Phosphoribulokinase (prkA) (BBa_K1465201)
- This week we tried to assemble prkA of the gene synthesis and the backbone pSB1C3. We aimed to transform the pSB1C3_prkA construct construct.
- Restriction digestion with DpnI
- Gibson Assembly with prkA and pSB1C3
- Transformation with electrocompotetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~1300 bp)
- Colony PCR (prkA_pHn_fwd, prkA_pHn_rev)
- Annealing temperature: 55 °C
- Bands as expected (~1060 bp)
- sRNA:pfkA (BBa_K1465225)
- This week we tried to assemble sRNA:pfkA of the gene synthesis and the backbone pSB1C3. We aimed to transform the pSB1C3_sRNA:pfkA construct.
- Restriction digestion with DpnI
- Gibson Assembly with sRNA:pfkA and pSB1C3
- Transformation with electrocompotetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~600 bp)
- Plasmid isolation of sRNA:pfkA
- RuBisCO of H. neapolitanus (BBa_K1465202)
- This week we tried to assemble Hneap of the gene synthesis and the backbone pSB1C3. We aimed to transform the pSB1C3_Hneap construct.
- Restriction digestion with DpnI
- Gibson Assembly with Hneap and pSB1C3
- Transformation with electrocompotetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~2100 bp)
- Plasmid isolation of Hneap
- RuBisCO of S. elongatus
- This week we tried to assemble Selan of the gene synthesis and the backbone pSB1C3. We aimed to transform the pSB1C3_Selan construct.
- Restriction digestion with DpnI
- Gibson Assembly with Selan and pSB1C3
- Transformation with electrocompotetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~2100 bp)
- We plated sample number 1 for a plasmid isolation.
- Plasmid isolation of Selan
- T7 BioBrick (BBa_I719005)
- This week we tried isolate the T7 promoter (pSB1A2_T7) of the parts distribution 2013.
- Plasmid isolation of pSB1A2_T7
- → The transformation was not successful. We had to make the assemble and transformation again.
- → Because of a double band, we made a plasmid isolation and another colony PCR but with gene specific primer.
