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Team:Bielefeld-CeBiTec/Notebook/Journal/CO2-fixation/Sep
From 2014.igem.org
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<li><b><i>Purification of the carboxysome</b></i></li> | <li><b><i>Purification of the carboxysome</b></i></li> | ||
<ul> | <ul> | ||
| - | <li>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 <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 L. For a more effective cell lysis, the sonification protocol was modified using longer intervalls for sonication and for cooling. </li> | + | <li>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 <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 L. For a more effective cell lysis, the sonification protocol was modified using longer intervalls for sonication and for cooling. Furthermore, the ultracentrifugation was carried out for a longer time (45 min), to obtain a better concentration and seperation. </li> |
→ The purification was not succesful, as you could not recognize a visible band in the gradient. | → The purification was not succesful, as you could not recognize a visible band in the gradient. | ||
Revision as of 15:58, 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 L. For a more effective cell lysis, the sonification protocol was modified using longer intervalls for sonication and for cooling. Furthermore, the ultracentrifugation was carried out for a longer time (45 min), to obtain a better concentration and seperation. → 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
- Protein expression is still 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. A larger culture volume of 1,6 L was used. The resultion pellet was resuspended in 20 mL TEMB buffer. The cell lysis was carried out this time using a French pressure cell (Aminco SLM Instrument) with a pressure of 5000 psig (around 345 bar). The cell suspension was homogenisated two times in the French pressure cell. Before the step of ultracentrifugation, the resulting pellet was resuspended in only 1,5 mL TEMB buffer. → 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
- Purification of the carboxysome
- Protein expression is still 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. A larger culture volume of 1,6 L was used. The resultion pellet was resuspended in 20 mL TEMB buffer. The cell lysis was carried out this time using a French pressure cell (Aminco SLM Instrument) with a pressure of 5000 psig (around 345 bar). The cell suspension was homogenisated two times in the French pressure cell. Before the step of ultracentrifugation, the resulting pellet was resuspended in only 1,5 mL TEMB buffer. → The purification was not succesful, as you could not recognize a visible band in the gradient.
- 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.
