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Team:Bielefeld-CeBiTec/Notebook/Journal/CO2-fixation/Aug
From 2014.igem.org
(Difference between revisions)
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<li>pSB1A2_T7</li> | <li>pSB1A2_T7</li> | ||
<li>pSB1C3_p<sub>tac</sub></li> | <li>pSB1C3_p<sub>tac</sub></li> | ||
| - | <li> | + | <li>pSB1K3_p<sub>Tet</sub></li> |
</ul> | </ul> | ||
<li>Inserts (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Xba</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>) | <li>Inserts (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Xba</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>) | ||
| Line 89: | Line 89: | ||
<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">transform</a> the promotor from another distribution plate (2013) | <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">transform</a> the promotor from another distribution plate (2013) | ||
</ul> | </ul> | ||
| - | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> of | + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> of pSB1K3_p<sub>Tet</sub>_prkA, pSB1K3_<sub>Tet</sub>_Hneap, |
| - | + | pSB1K3_<sub>Tet</sub>_SELAN and pSB1K3_<sub>Tet</sub>_sRNA:pfkA (<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> | </li> | ||
<ul> | <ul> | ||
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<br> | <br> | ||
| - | <li><b><i>prkA</i></b></li> | + | <li><b><i>prkA</i> and pHnCBscoS1D</b></li> |
<ul> <!-- prkA --> | <ul> <!-- prkA --> | ||
<li>We tried to amplify <i>prkA</i> again and to assemble it with the plasmid pHnCBcsoS1D.</li> | <li>We tried to amplify <i>prkA</i> again and to assemble it with the plasmid pHnCBcsoS1D.</li> | ||
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<li><b><i>csoS1-4</i></b></li> | <li><b><i>csoS1-4</i></b></li> | ||
<ul> | <ul> | ||
| - | <li>Not successful | + | <li>Not successful.</li> |
→ Because of the wrong sequence the constructs <i>pSB1C3_can_csoS1-4</i> and <i>pSB1C3_can_csoS1-4_csoS1D</i> are also incorrect and have to be made again. | → Because of the wrong sequence the constructs <i>pSB1C3_can_csoS1-4</i> and <i>pSB1C3_can_csoS1-4_csoS1D</i> are also incorrect and have to be made again. | ||
</ul> | </ul> | ||
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<li><b><i>can</i></b></li> | <li><b><i>can</i></b></li> | ||
<ul> | <ul> | ||
| - | <li>Four to five mutations | + | <li>Four to five mutations at the same positions as before. Maybe we got the wrong accession number, so we will further work with our <i>can</i> construct.</li> |
</ul> | </ul> | ||
<li><b><i>csoS1-4</i></b></li> | <li><b><i>csoS1-4</i></b></li> | ||
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<li><b><i>Selan</i></b></li> | <li><b><i>Selan</i></b></li> | ||
<ul> | <ul> | ||
| - | <li>Not successful | + | <li>Not successful.</li> |
</ul> | </ul> | ||
<li><b><i>sRNA:pfkA</i></b></li> | <li><b><i>sRNA:pfkA</i></b></li> | ||
<ul> | <ul> | ||
| - | <li>Not successful | + | <li>Not successful.</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> | + | <li><b>pHnCBcsoS1D_prkA</b></li> |
<ul> | <ul> | ||
| - | <li>This week we tried to | + | <li>This week we tried to transform the pHnCBcsoS1D_prkA construct.</li> |
<ul> | <ul> | ||
| - | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols# | + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells</li> |
| + | <font color="red"><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#Primer1" target="_blank">Primer1</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Primer2" target="_blank">Primer2</a>) | ||
| + | </li> | ||
<ul> | <ul> | ||
| - | + | <li>Annealing temperature: ...</li> | |
| - | + | <li>Bands (not) as expected (~... bp)</li> | |
| - | </ul> | + | </ul></font> |
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</ul> | </ul> | ||
</ul> | </ul> | ||
| - | + | <font color="red"><li><b><i>csoS1-4</i></b></li></font> | |
<ul> | <ul> | ||
| - | <li>This week we tried to | + | <li>This week we tried again to amplify and transform the shell proteins.</li> |
<ul> | <ul> | ||
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</ul> | </ul> | ||
</ul> | </ul> | ||
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| - | <li>< | + | <br> |
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| + | <li><b><i>prkA</i> and <i>p<sub>Tet</i></b></li> | ||
<ul> | <ul> | ||
| - | + | <li>This week we tried to assemble the <i>p<sub>Tet</sub></i> promotor with the <i>prkA</i>.</li> | |
| - | + | <ul> | |
| - | + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix)</li> | |
| - | + | <ul> | |
| + | <li>Backbone (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>Spe</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li> | ||
| + | <ul> | ||
| + | <li>pSB1K3_p<sub>Tet</li> | ||
| + | </ul> | ||
| + | <li>Insert (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>Xba</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li> | ||
| + | <ul> | ||
| + | <li><i>prkA</i></li> | ||
| + | </ul> | ||
| + | </ul> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells</li> | ||
| + | → Only a few colonies grow. Maybe the TetR (repressor) is not strong enough so the <i>prkA</i> is too toxic. | ||
| + | </ul> | ||
</ul> | </ul> | ||
| - | < | + | |
| - | + | <br> | |
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| + | <li><b>GFP</b></li> | ||
<ul> | <ul> | ||
| - | + | <li>This week we tried isolate GFP from the parts distribution</li> | |
| - | + | <ul> | |
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of [Construct]</li> | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of [Construct]</li> | ||
</ul> | </ul> | ||
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</ul> | </ul> | ||
<li>[...] was succesful</li> | <li>[...] was succesful</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>Annealing temperature: 55 °C</li> |
| - | <li>Bands | + | <li>Bands as expected ( bp)</li> |
</ul> | </ul> | ||
</ul> | </ul> | ||
Revision as of 19:50, 20 September 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.
- Plasmid isolation of ptac, ptac, T7, prkA, Hneap, SELAN, sRNA:pfkA and can
- BioBrick Assembly (Suffix)
- Backbones (digested with SpeI, PstI)
- pSB1A2_T7
- pSB1C3_ptac
- pSB1K3_pTet
- Inserts (digested with XbaI, PstI)
- prkA
- Hneap
- SELAN
- sRNA:pfkA
- Transformation of all constructs with electrocompotetent cells
- Colony PCR of pSB1C3_ptac_prkA, pSB1C3_ptac_Hneap, pSB1C3_ptac_SELAN and pSB1C3_ptac_sRNA:pfkA (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~2500 bp (ptac_prkA), ~3200 bp (ptac_Hneap) ~3200 bp (ptac_SELAN) ~1700 bp (ptac_sRNA:pfkA))
- Colony PCR of pSB1A2_T7_prkA, pSB1A2_T7_Hneap, pSB1A2_T7_SELAN and pSB1A2_T7_sRNA:pfkA (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands not as expected → Showed in all cases a band 400 bp over the expected value. We tried to extract and transform the promotor from another distribution plate (2013)
- Colony PCR of pSB1K3_pTet_prkA, pSB1K3_Tet_Hneap, pSB1K3_Tet_SELAN and pSB1K3_Tet_sRNA:pfkA (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands not as expected → We tried it again.
- Colony PCR of pSB1A2_T7 from the 2013 distribution plate (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~300 bp)
- Plasmid isolation of ptac_prkA, ptac_Hneap, ptac_SELAN, ptac_sRNA_pfkA and pSB1A2_T7
- csoS1-4
- We used the amplified products to assemble and transform them to get the shell protein construct.
- Restriction digestion with DpnI
- Gibson Assembly with csoS1-4 and pSB1C3
- Transformation with electrocompotetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~2100 bp)
- Plasmid isolation
- Restriction digestion with SpeI and XbaI
- Bands as expected (~1800 bp and ~2200 bp)
- can and csoS1-4
- We tried to assemble the shell proteins and the carbonic anhydrase for the carboxysome.
- BioBrick Assembly (Suffix)
- Transformation with electrocompotetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~3600 bp)
- Plasmid isolation of pSB1C3_can_csoS1-4
- glpX
- We tried to assemble and transform the glpX parts again.
- Restriction digestion with DpnI
- Gibson Assembly with glpX_1, glpX_2 and pSB1K3
- Transformation with electrocompotetent cells
- Colony PCR on one part of the fragment (fw-pSB1C3-BBa_B0034-SBPase, rv-SBPase-Schnittstelle)
- Annealing temperature: 55 °C
- Bands as expected (~500 bp)
- Plasmid isolation of glpX
- prkA and pHnCBscoS1D
- We tried to amplify prkA again and to assemble it with the plasmid pHnCBcsoS1D.
- Plasmid isolation of DH5α prkA
- PCR amplification on the isolated prkA (prkA_pHn_fwd, prkA_pHn_rev)
- Annealing temperature: 55 °C
- Bands as expected (~1000 bp)
- PCR products were purified out of the gel
- Gibson Assembly with prkA and pHnCBcsoS1D
- RuBisCO
- We tried to assemble both RuBisCO with pSB1C3_ptac_prkA
- BioBrick Assembly (Suffix)
- Backbone (digested with SpeI, PstI)
- ptac_prkA
- Inserts (digested with XbaI, PstI)
- Hneap
- SELAN
- We assembled pSB1C3_ptac_prkA with Hneap respectively SELAN
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- pSB1C3_ptac_prkA_Hneap
- Bands not as expected (too short). → We will try to ligate pSB1C3_ptac_prkA and Hneap again.
- pSB1C3_ptac_prkA_SELAN
- Bands as expected (~4200 bp)
- Plasmid isolation of ptac_prkA_Hneap and ptac_prkA_SELAN
- can_csoS1-4 and csoS1D
- We tried to assemble pSB1C3_can_csoS1-4 and csoS1D and transform the construct.
- BioBrick Assembly Suffix:
- Transformation with electrocompotetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~4300 bp)
- Sequencing
- csoS1D
- Successful. We got the right sequence. The first part of the carboxysome is complete.
- can
- Five mutations. Another sequencing will follow.
- csoS1-4
- Not successful. → Because of the wrong sequence the constructs pSB1C3_can_csoS1-4 and pSB1C3_can_csoS1-4_csoS1D are also incorrect and have to be made again.
- glpX
- Not successful. All samples showed the sequence of CFP from the backbone. We will start again, this time with pSB1C3_RFP for the backbone.
- glpX
- This week we tried to amplify the backbone pSB1C3 for glpX again. We took the pSB1C3_RFP as a template.
- PCR amplification of the pSB1C3_RFP backbone (fw_SBPase_pSB1C3, rv_SBPase_pSB1)
- Annealing temperature: 54 °C
- Bands as expected (~2100 bp)
- PCR products were purified
- Restriction digestion with DpnI
- Gibson Assembly with glpX_1, glpX_2 and pSB1C3
- Transformation with electrocompotetent cells → Not successful. We will take another plasmid, pSB1K3.
- csoS1-4
- We tried to isolate the right plasmid again.
- Plasmid isolation of pSB1C3_csoS1-4
- Restriction digestion with PstI and EcoRI as a control
- Bands as expected (~2100 bp (backbone) and ~1700 bp (insert))
- Sequencing
- can
- Four to five mutations at the same positions as before. Maybe we got the wrong accession number, so we will further work with our can construct.
- csoS1-4
- Not successful.
- Selan
- Not successful.
- sRNA:pfkA
- Not successful.
- pHnCBcsoS1D_prkA
- This week we tried to transform the pHnCBcsoS1D_prkA construct.
- Transformation with electrocompotetent cells
- Colony PCR (Primer1, Primer2)
- Annealing temperature: ...
- Bands (not) as expected (~... bp)
- csoS1-4
- This week we tried again to amplify and transform the shell proteins.
- prkA and pTet
- This week we tried to assemble the pTet promotor with the prkA.
- BioBrick Assembly (Suffix)
- Transformation with electrocompotetent cells → Only a few colonies grow. Maybe the TetR (repressor) is not strong enough so the prkA is too toxic.
- GFP
- This week we tried isolate GFP from the parts distribution
- Plasmid isolation of [Construct]
- Purification vector
- This week we tried amplify the T7 promotor and RBS for the purification vector.
- PCR amplification (Primer1, Primer2)
- Annealing temperature: ...
- Bands (not) as expected (... bp)
- fbaA
- This week we tried amplify second part of fbaA for purification.
- PCR amplification (fbaA_purify_rev, fbaA_PstI_fw)
- Annealing temperature: ...
- Bands (not) as expected (... bp)
- tktA
- This week we tried amplify both parts of tktA for purification.
- PCR amplification (tktA_purify_fw, tktA_PstI_rev and tktA_purify_rev, tktA_PstI_fw)
- Annealing temperature: ...
- Bands (not) as expected (... bp)
- gapA
- This week we tried amplify both parts of gapA for purification.
- PCR amplification (gapA_purify_fw, gapA_NotI_rev and gapA_purify_rev, gapA_NotI_fw)
- Annealing temperature: ...
- Bands (not) as expected (... bp)
- IntCBD
- This week we tried amplify the intein tag for the purification vector.
- PCR amplification (Primer1, Primer2)
- Annealing temperature: ...
- Bands (not) as expected (... bp)
- Gibson Assembly with T7 RBS and IntCBD on pSB1C3
- T7 Promotor
- This week we tried the T7 promotor for prkA and Hneap
- BioBrick Assembly Suffix:
- [...] was succesful
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected ( bp)
- Sequencing
- pSB1C3_glpX
- not successful. It was a sequence of a fluorescence protein (RFP backbone used)
- pSB1C3_csoS4ABcsoS1CAB
- not successful. It was another sequence. We think of a contamination.
- Selan
- too many insertion mutations
- sRNA:pfkA
- successful
