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Team:Bielefeld-CeBiTec/Notebook/Journal/CO2-fixation/Sep
From 2014.igem.org
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| + | <li><b><i>csoS1-4_GFP</i> and T7_sap</b></li> | ||
| + | <ul> | ||
| + | <li>We tried to assemble our GFP construct with the shell associated protein <i>sap</i> and the T7 promotor.</li> | ||
| + | <ul> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Prefix)</li> | ||
| + | <ul> | ||
| + | <li>Backbone (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>EcoR</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Xba</i>I</a>)</li> | ||
| + | <ul> | ||
| + | <li>pSB1C3</li> | ||
| + | </ul> | ||
| + | <li>Insert (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>EcoR</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Spe</i>I</a>)</li> | ||
| + | <ul> | ||
| + | <li><i>[Construct]</i></li> | ||
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Revision as of 20:32, 14 October 2014
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September |
 |
- 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
- 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
- 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
- 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
- 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)
- 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
- 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.
- 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)
- tkt
- This week we want to purify the enzymes 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 want to purify the enzymes 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
- This week we want to purify the enzymes for the SBPase assay.
- 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 ...
