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Team:Bielefeld-CeBiTec/Notebook/Journal/C02-fixation/Jul
From 2014.igem.org
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| - | <li><b>csoS1D of the carboxysome</b></li> | + | <li><b><i>csoS1D</i> of the carboxysome</b></li> |
<ul> | <ul> | ||
| - | <li>This week we tried to amplify the backbone pSB1C3 for csoS1D and csoS1D itself.</li> | + | <li>This week we tried to amplify the backbone pSB1C3 for <i>csoS1D</i> and <i>csoS1D</i> itself.</li> |
<ul> | <ul> | ||
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> of csoS1D (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#csoS1D_fwd" target="_blank">csoS1D_fwd</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#csoS1D_rev" target="_blank">csoS1D_rev</a>)</li> | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> of csoS1D (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#csoS1D_fwd" target="_blank">csoS1D_fwd</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#csoS1D_rev" target="_blank">csoS1D_rev</a>)</li> | ||
Revision as of 16:33, 15 September 2014
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July |
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- pHnCBcsoS1D
- This week we tried to isolate the plasmid of the strain ordered from addgene
- Plasmid isolation of pHnCBcsoS1D
- csoS1D of the carboxysome
- This week we tried to amplify the backbone pSB1C3 for csoS1D and csoS1D itself.
- PCR amplification of csoS1D (csoS1D_fwd, csoS1D_rev)
- Annealing temperature: 55 °C
- Bands as expected (~703 bp)
- PCR amplification of pSB1C3 (pSB1C3_suf_csoS1D, pSB1C3_pre_csoS1D)
- Annealing temperature: 55 °C
- Bands as expected (~2070 bp)
- PCR products were purified
- Carbonic anhydrase (can) of the carboxysome
- This week we tried to amplify the backbone pSB1C3 for can and can itself.
- PCR amplification of can (csoS3_can_fwd, csoS3_can_rev)
- Annealing temperature: 55 °C
- Bands as expected (~1605 bp)
- PCR amplification of pSB1C3 (pSB1C3_pre_can, pSB1C3_suf_can)
- Annealing temperature: 55 °C
- Bands as expected (~2070 bp)
- PCR products were purified
- Shell associated protein of the carboxysome (first part of the protein)
- This week we tried to amplify the backbone pSB1C3 for sap and the first part of sap: sap_1.
- PCR amplification of sap_1 (csoS2_sap_1_fwd, csoS2_sap_1_rev)
- Annealing temperature: 55 °C
- Bands as expected (~1235 bp)
- PCR amplification of pSB1C3 (pSB1C3_pre_sap_1, pSB1C3_suf_sap2)
- Annealing temperature: 55 °C
- Bands as expected (~2070 bp)
- PCR products were purified
- csoS1D
- This week we tried to assemble csoS1D with the backbone pSB1C3 and to transform the construct.
- Gibson Assembly with csoS1D and pSB1C3
- Transformation with electrocompotetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands not as expected (~1250 bp, expected size: ~1017 bp). Size looked like template insert CFP.
- Carbonic anhydrase (can)
- This week we tried to assemble can with the backbone pSB1C3 and to transform the construct.
- Gibson Assembly with can and pSB1C3
- Transformation with electrocompotetent cells
- Transformation was not successful (no colonies on the plates).
- pHnCBcsoS1D backbone
- This week we tried to amplify the backbone of the plasmid.
- PCR amplification (pHnCBcsoS1D_fwd, pHnCBcsoS1D_rev)
- Annealing temperature: 55 °C
- Bands as expected (~13,200 bp)
- PCR products were purified out of the gel.
-
→ We will try a restriction digestion with DpnI of the template and we will make a purification out of the gel instead of using the PCR product.
- BioBricks (BBa_K731500 and BBa_Q01400)
- This week we tried to isolate promotors of two BioBricks (pSB1C3_Ptac and pSB1C3_PTet) of the parts distribution.
- Plasmid isolation of pSB1C3_Ptac and pSB1C3_PTet
- csoS4AB and csoS1CAB
- This week we tried to amplify the shell proteins of the carboxysome and the backbone pSB1C3 for the shell proteins.
- PCR amplification (fw_pSB1C3_csoS4A, rv_csoS4A_PstI)
- Annealing temperature: 55 °C
- Bands as expected (~180 bp)
- PCR amplification (fw_PstI_csoS4A, rv_SpeI_Intergen)
- Annealing temperature: 55 °C
- Bands as expected (~1200 bp)
- PCR amplification (fw_SpeI_Intergen, rv_csoS1_pSB1C3)
- Annealing temperature: 55 °C
- Bands as expected (~350 bp)
- PCR amplification of the backbone (fw_csoS1_pSB1C3, rv_pSB1C3_csoS4A)
- Annealing temperature: 55 °C
- Bands as expected (~2070 bp)
- Shell associated protein (part 1)
- This week we tried to amplify and purify sap_1 and the backbone pSB1C3.
- PCR amplification of sap_1 (csoS2_sap_1_fwd, csoS2_sap_1_rev)
- Annealing temperature: 55 °C
- Bands as expected (~1235 bp)
- PCR products were purified out of the gel
- PCR amplification of the backbone of sap (pSB1C3_pre_sap_1, pSB1C3_suf_sap2)
- Annealing temperature: 55 °C
- Bands as expected (~2070 bp)
- PCR products were purified out of the gel
- Pore protein (csoS1D) of the carboxysom
- This week we tried to amplify and purify csoS1D and the backbone pSB1C3.
- PCR amplification of csoS1D (csoS1D_fwd, csoS1D_rev)
- Annealing temperature: 55 °C
- Bands as expected (~700 bp)
- PCR products were purified out of the gel
- PCR amplification of the backbone of sap (pSB1C3_pre_csoS1D, pSB1C3_suf_csoS1D)
- Annealing temperature: 55 °C
- Bands as expected (~2070 bp)
- PCR products were purified out of the gel
- SBPase (glpX)
- This week we tried to amplify and assemble both parts of glpX and the backbone pSB1C3.
- PCR amplification with (fw_pSB1_SBPase, rv_SBPase_XbaI and fw_SBPase_XbaI, rv_pSB1_SBPase)
- Annealing temperature: 55 °C
- Bands as expected (~400 bp, ~600 bp)
- PCR products were purified
- PCR amplification of the backbone of glpX (fw_SBPase_pSB1C3, rv_SBPase_pSB1)
- Annealing temperature: 55 °C
- Bands as expected (~2070 bp)
- PCR products were purified
- Gibson Assembly with glpX_1, glpX_2 and pSB1C3 (with and without restriction digestion with DpnI)
- Carbnonic anhydrase (can)
- This week we tried to transform the construct of can with the backbone pSB1C3.
- Restriction digestion with DpnI
- Gibson Assembly with can and pSB1C3
- Transformation with electrocompotetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~1900 bp)
- Plasmid isolation of can
- csoS1D
- This week we tried to transform the construct of can with the backbone pSB1C3.
- Restriction digestion with DpnI
- Gibson Assembly with csoS1D and pSB1C3
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~1000 bp)
- Plasmid isolation of csoS1D
- Phosphoribulokinase (prkA)
- This week we tried to assemble prkA of the gene synthesis and the backbone pSB1C3. We aimed to transform the pSB1C3_prkA construct construct.
- Restriction digestion with DpnI
- Gibson Assembly with prkA and pSB1C3
- Transformation with electrocompotetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~1300 bp)
- Colony PCR (prkA_pHn_fwd, prkA_pHn_rev)
- Annealing temperature: 55 °C
- Bands as expected (~1060 bp)
- sRNA:pfkA
- This week we tried to assemble sRNA:pfkA of the gene synthesis and the backbone pSB1C3. We aimed to transform the pSB1C3_sRNA:pfkA construct.
- Restriction digestion with DpnI
- Gibson Assembly with sRNA_pfkA and pSB1C3
- Transformation with electrocompotetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~600 bp)
- Plasmid isolation of sRNA:pfkA
- RuBisCO of H. neapolitanus
- This week we tried to assemble Hneap of the gene synthesis and the backbone pSB1C3. We aimed to transform the pSB1C3_Hneap construct.
- Restriction digestion with DpnI
- Gibson Assembly with Hneap and pSB1C3
- Transformation with electrocompotetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~2100 bp)
- We plated sample number 1 for a plasmid isolation.
- Plasmid isolation of Hneap
- RuBisCO of S. elongatus
- This week we tried to assemble Selan of the gene synthesis and the backbone pSB1C3. We aimed to transform the pSB1C3_Selan construct.
- Restriction digestion with DpnI
- Gibson Assembly with Selan and pSB1C3
- Transformation with electrocompotetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~2100 bp)
- We plated sample number 1 for a plasmid isolation.
- Plasmid isolation of Selan
- BioBricks (BBa_I719005)
- This week we tried isolate the T7 promotor (pSB1A2_T7) of the parts distribution 2013.
- Plasmid isolation of pSB1A2_T7
- → The colonies became red, what means that RFP from the backbone was expressed. We tried to take another backbone, pSB1K3_RFP, instead of pSB1C3_RFP.
- → Because of a double band, we made a plasmid isolation and another colony PCR but with gene specific primer.
