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Team:Bielefeld-CeBiTec/Notebook/Journal/CO2-fixation/Aug
From 2014.igem.org
(Difference between revisions)
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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> <!-- gesamte Liste --> | <ul> <!-- gesamte Liste --> | ||
| - | <li><b>Promotors | + | <li><b>Promotors T7, p<sub>tac</sub> and p<sub>Tet</sub></b></li> |
<ul> <!-- Promotors --> | <ul> <!-- Promotors --> | ||
<li>We tried to assemble some inserts with three different promotors to test which one is the best choice.</li> | <li>We tried to assemble some inserts with three different promotors to test which one is the best choice.</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 p<sub>tac</sub>, p<sub>tac</sub>, T7, <i>prkA, Hneap, SELAN, sRNA:pfkA</i> and <i> can </i></li> |
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix)</li> | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix)</li> | ||
<ul> | <ul> | ||
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<li>Bands as expected (~300 bp)</li> | <li>Bands as expected (~300 bp)</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>_prkA, pSB1C3_p<sub>tac</sub>_Hneap, |
| - | + | pSB1C3_p<sub>tac</sub>_SELAN, pSB1C3_p<sub>tac</sub>_sRNA_pfkA and pSB1A2_T7</li> | |
</ul> | </ul> | ||
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<li><b>Purification vector</b></li> | <li><b>Purification vector</b></li> | ||
<ul> | <ul> | ||
| - | <li>This week we tried to amplify the | + | <li>This week we tried to amplify the T7_RBS promotor for the purification vector.</li> |
<ul> | <ul> | ||
<font color="red"><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#Primer1" target="_blank">Primer1</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Primer2" target="_blank">Primer2</a>)</li> | <font color="red"><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#Primer1" target="_blank">Primer1</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Primer2" target="_blank">Primer2</a>)</li> | ||
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<br> | <br> | ||
| - | <li><b><i>sRNA:pfkA</i> and | + | <li><b><i>sRNA:pfkA</i> and T7</b></li> |
<ul> | <ul> | ||
<li>We tried to assemble the <i>sRNA:pfkA</i> construct and pSB1A2_T7</li> | <li>We tried to assemble the <i>sRNA:pfkA</i> construct and pSB1A2_T7</li> | ||
Revision as of 13:10, 15 October 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 pSB1C3_ptac_prkA, pSB1C3_ptac_Hneap, pSB1C3_ptac_SELAN, pSB1C3_ptac_sRNA_pfkA and pSB1A2_T7
- csoS1-4 (shell proteins csoS4AB and csoS1CAB)
- 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.
Agarose gel from the restriction digestion with SpeI and XbaI. As a Ladder we used GeneRuler™ 1 kb DNA Ladder from Thermo Scientific.
- 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 to amplify and transform the shell proteins again.
- 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)
- All PCR products were purified
- Restriction digestion with DpnI
- Gibson Assembly with csoS1-4 and pSB1C3
- Transformation with electrocompotetent cells
- 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 did grow. Maybe the TetR (repressor) is not strong enough so the prkA is too toxic.
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~2200 bp)
- Hneap and pTet
- This week we tried to assemble the pTet promotor with the Hneap.
- BioBrick Assembly (Suffix)
- Transformation with electrocompotetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~3000 bp)
- sap_2
- This week we tried to bring the synthesized sap_2 construct in the pJet vector with blunt end cloning. We also amplified the backbonefor an assembly.
- Blunt-End cloning of sap_2 in the pJet vector
- Colony PCR (Primer1, Primer2)
- Annealing temperature: ... °C
- Bands as expected (~1500 bp)
- PCR amplification of the backbone (pSB1C3) (pSB1C3_pre_sap_1, pSB1C3_suf_sap2)
- Annealing temperature: 55 °C
- Bands as expected (~2100 bp)
- GFP BioBrick (BBa_E0040)
- This week we tried isolate the green fluorescent protein GFP (pSB1A2_GFP) of the parts distribution 2014.
- Plasmid isolation of pSB1A2_GFP
- Purification vector
- This week we tried to amplify the T7_RBS promotor for the purification vector.
- PCR amplification (Primer1, Primer2)
- Annealing temperature: ...
- Bands (not) as expected (... bp)
- Sequencing
- glpX
- Not successful. It was a sequence of RFP. We will start again, this time with pSB1K3_RFP with an ampicillin resistance for the backbone.
- csoS1-4
- Not successful.
- Selan
- Not successful. Too many insertion mutations.
- sRNA:pfkA
- Successful. We got the right sequence.
- can
- ???
- sap_2
- We tried to isolate pJet_sap_2 for further experiments.
- Plasmid isolation of pJet_sap_2
- glpX
- We tried to amplify the pSB1K3 backbone for an assembly with glpX.
- 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 pSB1K3
- Transformation with electrocompotetent cells
- csoS1-4
- We tried to find correct colonies of the psB1C3_csoS1-4 construct so another part of the carboxysome is complete.
- Colony PCR (fw_pSB1C3_csoS4A, rv_csoS1_pSB1C3)
- Annealing temperature: 65 °C
- Bands (not) as expected (~1600 bp)
- Plasmid isolation of pSB1C3_csoS1-4
- Restriction digestion with EcoRI and PstI
- Bands as expected (~2000 bp backbone, ~1800 bp insert)
- sRNA:pfkA and ptac
- We tried to assemble the sRNA:pfkA construct and pSB1C3_ptac
- BioBrick Assembly (Suffix)
- Backbone (digested with SpeI, PstI)
- pSB1C3_ptac
- Insert (digested with XbaI, PstI)
- sRNA:pfkA
- Transformation with electrocompotetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~1700 bp)
- sRNA:pfkA and T7
- We tried to assemble the sRNA:pfkA construct and pSB1A2_T7
- BioBrick Assembly (Suffix)
- Backbone (digested with SpeI, PstI)
- pSB1A2_T7
- Insert (digested with XbaI, PstI)
- sRNA:pfkA
- Transformation with electrocompotetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~900 bp)
- Plasmid isolation of pSB1A2_T7_sRNA:pfkA
- GFP
- We tried to amplify GFP for a fusion with the shell proteins.
- PCR amplification (fw-csoS1A-GFP, rv-csoS1B-GFP)
- Annealing temperature: 54 °C
- Bands as expected (~750 bp)
- PCR products were purified out of the gel
- can and csoS1-4
- We tried to assemble can and csoS1-4 for the carboxysome.
- BioBrick Assembly (Prefix)
- Transformation with electrocompotetent cells
