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Team:Bielefeld-CeBiTec/Notebook/Journal/C02-fixation/Jul
From 2014.igem.org
(Difference between revisions)
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<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with glpX_1, glpX_2 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>DpnI</i></a>) | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with glpX_1, glpX_2 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>DpnI</i></a>) | ||
</li> | </li> | ||
| - | < | + | <ul> → The colonies became red, what means that RFP from the backbone was expressed. We tried to take another backbone, pSB1K3_RFP, instead of pSB1C3_RFP. |
| + | </ul> | ||
| + | |||
</ul> | </ul> | ||
</ul> | </ul> | ||
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<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of csoS1D | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of csoS1D | ||
</li> | </li> | ||
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</ul> | </ul> | ||
</ul> | </ul> | ||
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<li><b>Phosphoribulokinase (prkA)</b></li> | <li><b>Phosphoribulokinase (prkA)</b></li> | ||
<ul> | <ul> | ||
| - | <li>This week we tried to transform the | + | <li>This week we tried to transform the pSB1C3_prkA construct</li> |
<ul> | <ul> | ||
<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#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> | ||
| + | <li>Annealing temperature: 55 °C</li> | ||
| + | <li>Bands as expected (~1300 bp)</li> | ||
| + | <ul>→ Because of a double band, we made a <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">plasmid isolation</a> and another colony PCR but with gene specific primer. | ||
| + | </ul> | ||
| + | </ul> | ||
<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#prkA_pHn_fwd" target="_blank">prkA_pHn_fwd</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#prkA_pHn_rev" target="_blank">prkA_pHn_rev</a>) | <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#prkA_pHn_fwd" target="_blank">prkA_pHn_fwd</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#prkA_pHn_rev" target="_blank">prkA_pHn_rev</a>) | ||
<ul> | <ul> | ||
| - | <li>Annealing temperature: | + | <li>Annealing temperature: 55 °C</li> |
<li>Bands as expected (~1060 bp)</li> | <li>Bands as expected (~1060 bp)</li> | ||
<div class="element" style="width:150px"> | <div class="element" style="width:150px"> | ||
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</ul> | </ul> | ||
</li> | </li> | ||
| - | |||
</ul> | </ul> | ||
</ul> | </ul> | ||
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<li><b>sRNA:pfkA</b></li> | <li><b>sRNA:pfkA</b></li> | ||
<ul> | <ul> | ||
| - | <li>This week we tried to transform the | + | <li>This week we tried to transform the pSB1C3_sRNA:pfkA construct</li> |
<ul> | <ul> | ||
<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#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> | ||
| + | <li>Annealing temperature: 55 °C</li> | ||
| + | <li>Bands as expected (~600 bp)</li> | ||
| + | </ul> | ||
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of sRNA:pfkA</li> | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of sRNA:pfkA</li> | ||
</ul> | </ul> | ||
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<li><b>RuBisCO of <i>H. neapolitanus</i></b></li> | <li><b>RuBisCO of <i>H. neapolitanus</i></b></li> | ||
<ul> | <ul> | ||
| - | <li>This week we tried to transform the | + | <li>This week we tried to transform the pSB1C3_Hneap construct</li> |
<ul> | <ul> | ||
<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>) |
<ul> | <ul> | ||
| - | <li>Annealing temperature: | + | <li>Annealing temperature: 55 °C</li> |
<li>Bands as expected (~2100 bp)</li> | <li>Bands as expected (~2100 bp)</li> | ||
<div class="element" style="width:150px"> | <div class="element" style="width:150px"> | ||
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<li>We plated sample number 1 for a plasmid isolation.</li> | <li>We plated sample number 1 for a plasmid isolation.</li> | ||
</ul> | </ul> | ||
| - | |||
</li> | </li> | ||
| - | |||
| - | |||
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of Hneap</li> | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of Hneap</li> | ||
</ul> | </ul> | ||
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<br> | <br> | ||
| - | <li><b> | + | <li><b>BioBricks <a href="http://parts.igem.org/Part:BBa_I719005">(BBa_I719005)</a></b></li> |
| - | + | ||
| - | + | ||
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| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
<ul> | <ul> | ||
| - | <li>This week we tried | + | <li>This week we tried isolate the T7 promotor (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 | + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1A2_T7</li> |
</ul> | </ul> | ||
</ul> | </ul> | ||
Revision as of 12:46, 9 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: 55 °C
- 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 (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.
- 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 (part 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
- Pore protein (csoS1D) of the carboxysom
- 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 with (fw_pSB1_SBPase, rv_SBPase_XbaI and fw_SBPase_XbaI, rv_pSB1_SBPase)
- Annealing temperature: 55 °C
- Bands as expected (~400 bp, ~600 bp)
- PCR products were purified
- PCR amplification of the backbone of glpX (fw_SBPase_pSB1C3, rv_SBPase_pSB1)
- 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)
- Carbnonic anhydrase (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
- This week we tried to transform the construct of can 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)
- This week we tried to transform the pSB1C3_prkA construct
- 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
- This week we tried to transform the pSB1C3_sRNA:pfkA construct
- 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
- This week we tried to transform the pSB1C3_Hneap construct
- 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 Hneap
- BioBricks (BBa_I719005)
- This week we tried isolate the T7 promotor (pSB1A2_T7) of the parts distribution 2013.
- Plasmid isolation of pSB1A2_T7
- → The colonies became red, what means that RFP from the backbone was expressed. We tried to take another backbone, pSB1K3_RFP, instead of pSB1C3_RFP.
- → Because of a double band, we made a plasmid isolation and another colony PCR but with gene specific primer.
