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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.
- BioBrick Assembly (Suffix)
- Backbones (digested with SpeI, PstI)
- T7
- pTac
- pTet
- Inserts (digested with XbaI, PstI)
- prkA
- Hneap
- SELAN
- sRNA:pfkA
- Only the assembly with the pTac promotor was successful.
- Colony PCR and plasmid isolation of pTac_prkA, pTac_Hneap, pTac_SELAN and pTac_sRNA_pfkA
- Colony PCR of T7 promotor
- Showed in all cases an band 400 bp over the expected value. We tried to extract and tranform the promotor from another distribution plate (2013)
- Colony PCR of T7 promotor from the 2013 distribution plate
- Bands as expected (~300 bp)
- csoS1-4
- We tried to [...]
- glpX
- We tried to [...]
- Gibson Assembly after DpnI digestion
- Colony PCR
- prkA
- We tried to [...]
- PCR amplification and purification for insertion in pHnCBcsoS1D
- Bands as expected ()
- Gibson Assembly with pHnCBcsoS1D
- RuBisCO
- We tried to...
- BioBrick Assembly (Suffix)
- Backbone (digested with SpeI, PstI)
- pTac_prkA
- Inserts (digested with XbaI, PstI)
- Hneap
- SELAN
- We assembled pTac_prkA with Hneap respectively SELAN
- Colony PCR
- pTac_prkA_Hneap
- Bands es expected ()
- pTac_prkA_SELAN
- Bands as expected ()
- Plasmid isolation of pTac_prkA_Hneap and pTac_prkA_SELAN
- Sequencing
- csoS1D
- Successful. We got the right sequence with 100%. 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).
