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Team:Bielefeld-CeBiTec/Notebook/Journal/CO2-fixation/Sep
From 2014.igem.org
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<li>We tried to finish our carboxysome with and without <i>csoS1D</i> but we could not find a correct clone until the begining of october. We suggested that our colony PCR did not work but also with a restriction digestion we got no result for this construct.</li> | <li>We tried to finish our carboxysome with and without <i>csoS1D</i> but we could not find a correct clone until the begining of october. We suggested that our colony PCR did not work but also with a restriction digestion we got no result for this construct.</li> | ||
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| + | <li><b><i>Purification of the carboxysome</b></i></li> | ||
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| + | <li>The results of our experiments suggest that higher IPTG concentrations are needed for a efffective protein expression of the carboxysome. For this reason, we induced the protein expression with a final IPTG concentration of 0,5 mM. The purification was carried out using the protocol as described in our <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Journal/CO2-fixation/Aug#Week2" target="_blank"> labjournal </a>, but slightly modificated. The culture volumen was upscaled to 1 litre. For a more effective cell lysis, the sonification protocol was modified using eight cylces a one min with one min cooling intervals for sonification. </li> | ||
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| + | → The purification was not succesful, as you could not recognize a visible band in the gradient. | ||
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Revision as of 15:35, 16 October 2014
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September |
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- glpX
- We tried to find and isolate pSB1K3_glpX.
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~1300 bp)
- Plasmid isolation of pSB1K3_glpX
- csoS1-4
- We tried to amplify csoS1-4 (shell proteins) for a fusion with GFP.
- PCR amplification (fw-GFP-csoS1B, rv-rv-GFP-csoS1A)
- Annealing temperature: 54 °C
- Bands as expected (~4000 bp (~1800 bp csoS1-4, ~2200 bp pSB1C3))
- PCR products were purified
- csoS1-4 and GFP (BBa_K1465222)
- We tried to assemble the shell proteins of the carboxysome and GFP.
- Gibson Assembly with pSB1C3_csoS1-4 and GFP
- Transformation with electrocompotetent cells
- Colony PCR (fw-csoS1A-GFP, rv-csoS1B-GF)
- Annealing temperature: 68 °C
- Bands as expected (~1000 bp)
- Plasmid isolation of pSB1C3_csoS1-4_GFP
- Restriction digestion with NotI
- Bands as expected (~2000 bp and ~2400 bp)
Agarose gel from restriction digestion. As a Ladder we used GeneRuler™ 1 kb DNA Ladder from Thermo Scientific.
- csoS1-4_GFP and T7
- We tried to assemble our pSB1C3_csoS1-4_GFP construct with the T7 promotor.
- BioBrick Assembly (Suffix)
- Transformation with electrocompotetent cells
- Colony PCR (VF-Primer, rv_csoS4A_PstI)
- Annealing temperature: 55 °C
- Bands as expected (~320 bp)
- can and csoS1-4 (BBa_K1465208)
- This week we tried to find positive clones of our transformation.
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~3600 bp)
- Plasmid isolation of pSB1C3_can_csoS1-4
- Restriction digestion with EcoRI and PstI
- Bands as expected (~2100 bp and ~3300 bp)
Agarose gel from restriction digestion. As a Ladder we used GeneRuler™ 1 kb DNA Ladder from Thermo Scientific.
- can_csoS1-4 and csoS1D (BBa_K1465209)
- We tried to assemble our pSB1C3_can_csoS1-4 construct with csoS1D.
- BioBrick Assembly (Suffix)
- Transformation with electrocompotetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~4300 bp)
- Plasmid isolation of pSB1C3_can_csoS1-4_csoS1D
- Restriction digestion with NotI
- Bands as expected (~2000 bp and ~4000 bp)
Agarose gel from restriction digestion. As a Ladder we used GeneRuler™ 1 kb DNA Ladder from Thermo Scientific.
- Hneap and T7
- This week we tried to assemble the T7 promotor with the Hneap.
- BioBrick Assembly (Suffix)
- Transformation with chemocompetent cells
- glpX and ptac (BBa_K1465229)
- This week we tried to bring glpX in the pSB1C3 backbone under the control of the ptac promotor.
- BioBrick Assembly (Suffix)
- Transformation with electrocompotetent cells
- Colony PCR (fw_pSB1_SBPase, rv_pSB1_SBPase)
- Annealing temperature: 54 °C
- Bands as expected (~2100 bp)
- Plasmid isolation of pSB1C3_ptac_glpX
- Restriction digestion with NotI
- Bands as expected (~2000 bp and ~2200 bp)
- Purification of the carboxysome
- Protein expression is induced with a final IPTG concentration of 0,5 mM. The purification was carried out using the protocol as described in our labjournal , but slightly modificated. The culture volumen was upscaled to 1 litre. For a more effective cell lysis, the sonification protocol was modified using longer intervalls for sonication and for cooling. → The purification was not succesful, as you could not recognize a visible band in the gradient.
- Hneap and T7
- We tried to find positiv clones of pSB3A2_T7_Hneap.
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~2000 bp)
- Plasmid isolation of pSB1A2_T7_Hneap
- Restriction digestion with EcoRI and PstI
- Bands as expected (~2000 bp and ~1800 bp)
Agarose gel from restriction digestion. As a Ladder we used GeneRuler™ 1 kb DNA Ladder from Thermo Scientific.
- sap (BBa_K1465204)
- We tried to assemble both parts of sap.
- PCR amplification on the pJet plasmid for sap_2 (sap_2_fwd, sap_2_rev)
- Annealing temperature: 55 °C
- Bands as expected (~1500 bp)
- Gibson Assembly with sap_1, sap_2 and pSB1C3
- Transformation with electrocompotetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~3000 bp)
- Restriction digestion with EcoRI and PstI
- Bands as expected (~2000 bp (backbone) and ~2700 bp (insert))
- Restriction digestion with EcoRV
- Bands as expected (~1500 bp and ~3200 bp)
Agarose gel from restriction digestion. As a Ladder we used GeneRuler™ 1 kb DNA Ladder from Thermo Scientific.
- can_csoS1-4 respectively can_csoS1-4_csoS1D and sap
- We tried to finish our carboxysome with and without csoS1D but we could not find a correct clone until the begining of october. We suggested that our colony PCR did not work but also with a restriction digestion we got no result for this construct.
- Purification of the carboxysome
- The results of our experiments suggest that higher IPTG concentrations are needed for a efffective protein expression of the carboxysome. For this reason, we induced the protein expression with a final IPTG concentration of 0,5 mM. The purification was carried out using the protocol as described in our labjournal , but slightly modificated. The culture volumen was upscaled to 1 litre. For a more effective cell lysis, the sonification protocol was modified using eight cylces a one min with one min cooling intervals for sonification. → The purification was not succesful, as you could not recognize a visible band in the gradient.
- T7 and prkA
- This week we wanted to bring the prkA under the control of the T7 promotor.
- BioBrick Assembly (Suffix)
- Transformation with chemocompetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~1400 bp)
- Plasmid isolation of pSB1A2_T7_prkA
- Restriction digestion with EcoRI and PstI
- Bands as expected (~2000 bp and ~1100 bp)
- T7 and sap
- We tried to bring the sap under the control of the T7 promotor.
- BioBrick Assembly (Suffix)
- Transformation with chemocompetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~3000 bp)
- Plasmid isolation of pSB1A2_T7_sap
- Restriction digestion with EcoRI and PstI
- Bands as expected (~2800 bp and ~2000 bp)
- csoS1-4_GFP and T7_sap
- We tried to assemble our GFP construct with the shell associated protein sap and the T7 promotor.
- BioBrick Assembly (Prefix)
- Transformation with electrocompotetent cells
- Transformation with chemocompetent cells
- T7_sap_csoS1-4_GFP (BBa_K1465223)
- We tried find the construct to use it for the microscopy.
- Colony PCR (VF-Primer, Primer2)
- Annealing temperature: 55 °C
- Bands as expected (~3000 bp)
- Plasmid isolation of pSB1C3_T7_sap_csoS1-4_GFP
- Restriction digestion with PstI and EcoRI
- Bands as expected (~2000 bp and ~4600 bp)
- tkt
- This week we wanted to purify the enzyme of tkt for the SBPase assay.
- Cultivation of pet16b_tkt in 250 ml LB medium
- Induction with 1 mM IPTG at OD 0.8. Taking samples:t0, t1, t2, t3, t15, t17
- SDS-Page of cultivation result in correct bands at ~100kD
- His-Tag purification of tkt
- SDS-Page of His-Tag purification result in correct bands at imidazol concentration of ...
- fba
- This week we wanted to purify the enzyme of fba for the SBPase assay.
- Cultivation of pet16b_fba in 250 ml LB medium
- Induction with 1 mM IPTG at OD 0.8. Taking samples:t0, t1, t2, t3, t15, t17
- SDS-Page of cultivation result in correct bands at ~40kD
- His-Tag purification of fba
- SDS-Page of His-Tag purification result in correct bands at imidazol concentration of ...
- glpX (BBa_K1465228 (pSB1C3_glpX))
- This week we wanted to purify the enzyme of
- Cultivation of pet16b_glpX in 250 ml LB medium
- Induction with 1 mM IPTG at OD 0.8. Taking samples:t0, t1, t2, t3, t15, t17
- SDS-Page of cultivation result in correct bands at ~38kD
- His-Tag purification of glpX
- SDS-Page of His-Tag purification result in correct bands at imidazol concentration of ...
- We also tried to bring glpX in the right vector pSB1C3.
- BioBrick Assembly (Suffix)
- Transformation with electrocompotetent cells
- Transformation with chemocompetent cells
- Colony PCR (fw_pSB1_SBPase, rv_pSB1_SBPase)
- Annealing temperature: 54 °C
- Bands as expected (~1300 bp)
- Plasmid isolation of pSB1C3_glpX
- Restriction digestion with PstI and EcoRI
- Bands as expected (~2000 bp and ~1000 bp)
- Additionally we tried to bring glpX under the control of the T7 promotor.
- BioBrick Assembly (Suffix)
- Transformation with chemocompetent cells
- Colony PCR (Primer1, Primer2)
- Annealing temperature: ... °C
- Bands (not) as expected (~... bp)
- prkA and RBS BioBrick (BBa_B0034)
- We recognized that our pSB1C_prkA construct did not have the desired RBS so we tried to take the RBS (pSB1A2_RBS) out of the parts distrubution 2014 and clone it in front of our prkA.
- Plasmid isolation of pSB1A2_RBS
- BioBrick Assembly (Suffix/Prefix)
- Transformation with chemocompetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~1500 bp)
- Plasmid isolation of pSB1A2_prkA
- Restriction digestion with [Enzyme]I and [Enzyme]I
- Bands as expected (~ bp)
- T7_sRNA:pfkA and ptac_sRNA:pfkA
- We made a cultivation with our two constructs to see if there is a difference between induced, not induced and the wild type of E. coli. The cultivations were made in shaking flasks with a volume of 30 ml in 250 ml flasks at 37°C. The induction was with rhamnose for the T7 promotor and IPTG for the ptac. It lasted 13 hours. We made 2 biological and 2 technical replica for each culture. We took two samples (1 ml) of each biological replica for HPLC analysis that followed.
