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Team:Bielefeld-CeBiTec/Notebook/Journal/CO2-fixation/Aug
From 2014.igem.org
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August |
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- 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
- pSB1C3_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 pSB1C2_pTet_prkA, pSB1C2_Tet_Hneap, pSB1C2_Tet_SELAN and pSB1C2_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 and ptac_sRNA_pfkA
- 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)
- 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
- 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 as expected (~4200 bp)
- pSB1C3_ptac_prkA_SELAN
- Bands as expected (~4200 bp)
- Plasmid isolation of ptac_prkA_Hneap and ptac_prkA_SELAN
- can_csoS1-4 and csoS1D
- csoS1D
- Successful. We got the right sequence. The first part of the carboxysome is complete.
- can
- Five mutations. Another sequencing will follow.
- glpX
- Not successful. We got the CFP sequence instead of the glpX sequence, so we will try another backbone (pSB1C3_RFP).
- SBPase (glpX)
- This week we tried to amplify glpX
- PCR amplification (Primer1, Primer2) of pSB1C3-RFP backbone
- Annealing temperature: ...
- Bands (not) as expected (... bp)
- PCR amplification (Primer1, Primer2) of glpX
- Annealing temperature: ...
- Bands (not) as expected (... bp)
- Restriction digestion with DpnI
- Gibson Assembly with glpX_1 and glpX_2 on pSB1C3
- Bands (not) as expected (... bp)
- Another amplification and purification was tried.
- csoS1D
- This week we tried to do a BioBrick Assembly.
- BioBrick Assembly Suffix:
- Backbones (digested with EcoRI and XbaI)
- csoS1D
- Inserts (digested with digested with EcoRI and SpeI
- can_csoS1-4
- [...] was succesful
- csoS1-4
- This week we tried to isolate the plasmid.
- Plasmid isolation of csoS1-4
- Restriction digestion with PstI and EcoRI
- Bands (not) as expected (... bp)
- Sequencing
- Plasmid isolation of can, Selan, Hneap, can_csoS1-4
- can
- 4 to 5 mutations on the same positions. Maybe we got the wrong accession number
- csoS1D
- correct sequence
- csoS1-4
- not successful
- Selan
- not successful
- sRNA:pfkA
- not successful
- SBPase (glpX)
- This week we tried to amplify glpX
- PCR amplification (Primer1, Primer2) of pSB1C3-RFP backbone
- Annealing temperature: ...
- Bands (not) as expected (... bp)
- PCR amplification (Primer1, Primer2) of glpX
- Annealing temperature: ...
- Bands (not) as expected (... bp)
- Restriction digestion with DpnI
- Gibson Assembly with glpX_1 and glpX_2 on pSB1C3
- Bands (not) as expected (... bp)
- Another amplification and purification was tried.
- Colony PCR (Primer1, Primer2)
- Annealing temperature: ...
- Bands as expected (~1000 bp) but also additional bands
- Plasmid isolation of glpX
- Restriction digestion with PstI and EcoRI
- Bands not as expected
- Colony PCR (Primer1, Primer2)
- Annealing temperature: ...
- Bands (not) as expected (... bp)
- pHnCBcsoS1D, prkA
- This week we tried to combine the addgene plasmid with the prkA
- Transformation with electrocompotetent cells
- csoS1-4
- This week we tried again to amplify the shell proteins
- PCR amplification (Primer1, Primer2)
- Annealing temperature: ...
- Bands (not) as expected (... bp)
- Restriction digestion with DpnI
- Bands (not) as expected (... bp)
- Gibson Assembly with csoS1-4 on pSB1C3
- Colony PCR (Primer1, Primer2)
- Annealing temperature: ...
- Bands (not) as expected (... bp)
- prkA and RuBisCO
- This week we tried the pTet for prkA and RuBisCO
- BioBrick Assembly Suffix:
- Backbones (digested with [Enzyme])
- pTet
- Inserts (digested with [Enzyme])
- prkA and Hneap
- [...] was succesful
- Colony PCR (Primer1, Primer2)
- Annealing temperature: ...
- Bands (not) as expected (... bp)
- Only some colonies, TetR (repressor) not strong enough in the pTet_prkA construct
- 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 (Primer1, Primer2)
- Annealing temperature: ...
- Bands (not) 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
