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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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- Promoters T7, ptac and pTet (BBa_K1465213 (pSB1C3_ptac_Hneap), BBa_K1465212 (pSB1C3_ptac))
- We tried to assemble some inserts with three different promoters to test which one is the best choice.
- Plasmid isolation of ptac, ptac, T7, prkA, Hneap, sRNA:pfkA and can
- BioBrick Assembly (Suffix)
- Backbones (digested with SpeI, PstI)
- pSB1A2_T7
- pSB1C3_ptac
- pSB1K3_pTet
- Inserts (digested with XbaI, PstI)
- prkA
- Hneap
- sRNA:pfkA
- Transformation of all constructs with electrocompotetent cells
- Colony PCR of pSB1C3_ptac_prkA, pSB1C3_ptac_Hneap and pSB1C3_ptac_sRNA:pfkA (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~2500 bp (ptac_prkA), ~3200 bp (ptac_Hneap), ~1700 bp (ptac_sRNA:pfkA))
- Colony PCR of pSB1A2_T7_prkA, pSB1A2_T7_Hneap and pSB1A2_T7_sRNA:pfkA (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands not as expected → Showed in all cases a band 400 bp over the expected value. We tried to extract and transform the promoter from another distribution plate (2013)
- Colony PCR of pSB1K3_pTet_prkA, pSB1K3_Tet_Hneap and pSB1K3_Tet_sRNA:pfkA (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands not as expected → We tried it again.
- Colony PCR of pSB1A2_T7 from the 2013 distribution plate (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~300 bp)
- Plasmid isolation of pSB1C3_ptac_prkA, pSB1C3_ptac_Hneap, pSB1C3_ptac_sRNA_pfkA and pSB1A2_T7
- csoS1-4 (shell proteins csoS4AB and csoS1CAB)
- We used the amplified products to assemble and transform them to get the shell protein construct.
- Restriction digestion with DpnI
- Gibson Assembly with csoS1-4 and pSB1C3
- Transformation with electrocompotetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~2100 bp)
- Plasmid isolation
- Restriction digestion with SpeI and XbaI
- Bands as expected (~1800 bp and ~2200 bp)
- can and csoS1-4
- We tried to assemble the shell proteins and the carbonic anhydrase for the carboxysome.
- BioBrick Assembly (Suffix)
- Transformation with electrocompotetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~3600 bp)
- Plasmid isolation of pSB1C3_can_csoS1-4
- glpX
- We tried to assemble and transform the glpX parts again.
- Restriction digestion with DpnI
- Gibson Assembly with glpX_1, glpX_2 and pSB1K3
- Transformation with electrocompotetent cells
- Colony PCR on one part of the fragment (fw-pSB1C3-BBa_B0034-SBPase, rv-SBPase-Schnittstelle)
- Annealing temperature: 55 °C
- Bands as expected (~500 bp)
- Plasmid isolation of glpX
- prkA and pHnCBscoS1D
- We tried to amplify prkA again and to assemble it with the plasmid pHnCBcsoS1D.
- Plasmid isolation of DH5α prkA
- PCR amplification on the isolated prkA (prkA_pHn_fwd, prkA_pHn_rev)
- Annealing temperature: 55 °C
- Bands as expected (~1000 bp)
- PCR products were purified out of the gel
- Gibson Assembly with prkA and pHnCBcsoS1D
- RuBisCO
- We tried to assemble both RuBisCO with pSB1C3_ptac_prkA
- BioBrick Assembly (Suffix)
- Backbone (digested with SpeI, PstI)
- ptac_prkA
- Inserts (digested with XbaI, PstI)
- Hneap
- SELAN
- We assembled pSB1C3_ptac_prkA with Hneap respectively SELAN
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- pSB1C3_ptac_prkA_Hneap
- Bands not as expected (too short). → We will try to ligate pSB1C3_ptac_prkA and Hneap again.
- pSB1C3_ptac_prkA_SELAN
- Bands as expected (~4200 bp)
- Plasmid isolation of ptac_prkA_Hneap and ptac_prkA_SELAN
- can_csoS1-4 and csoS1D
- We tried to assemble pSB1C3_can_csoS1-4 and csoS1D and transform the construct.
- BioBrick Assembly Suffix:
- Transformation with electrocompotetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~4300 bp)
Agarose gel from the restriction digestion with SpeI and XbaI. As a Ladder we used GeneRuler™ 1 kb DNA Ladder from Thermo Scientific.
- glpX
- This week we tried to amplify the backbone pSB1C3 for glpX again. We took the pSB1C3_RFP as a template.
- PCR amplification of the pSB1C3_RFP backbone (fw_SBPase_pSB1C3, rv_SBPase_pSB1)
- Annealing temperature: 54 °C
- Bands as expected (~2100 bp)
- PCR products were purified
- Restriction digestion with DpnI
- Gibson Assembly with glpX_1, glpX_2 and pSB1C3
- Transformation with electrocompotetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~1300 bp)
- Plasmid isolation of pSB1C3_glpX
- csoS1-4
- We tried to isolate the right plasmid again.
- Plasmid isolation of pSB1C3_csoS1-4
- Restriction digestion with PstI and EcoRI as a control
- Bands as expected (~2100 bp (backbone) and ~1700 bp (insert))
- can (BBa_K1465205)
- Purification of the carboxysome
- We want to express and purify the carboxysome based on the plasmid pHnCBcsoS1D, which we received from addgene. The procedure is based on methods described by Bonacci et al., 2011 and So et al., 2004.
- Grow cells containing the pHnCBcsoS1D in TB medium containing antibiotic at 20 °C overnight
- Dilute the overnight culture 1:50 in TB medium containing antibiotic. A culture volume of 500 ml was used
- Grow cells until they reach an OD600 of 0.9-1.2
- Induce protein expression by adding IPTG to a final concentration of 50 µM. Grow cells overnight
- Harvest cells by centrifugation with 4500 x g for 20 min at 4 °C
- Resuspend cells in 50 mL TEMB buffer. Use protease inhibitors, for example PMSF
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TEMB buffer:
- 5 mM Tris-HCl (pH 8.0)
- 1 mM EDTA
- 10 mM MgCl2
- 20 mM NaHCO3
- Cell lysis via sonification (6 x 1 min with 1 min cooling interval between every cycle). Therefore a Bandelin Sonopuls HD 2070 with a SH70G with a power of 70 W was used. The power was adjust to 70 %
- Centrifuge the disruptet cell material with 12,000 x g for 30 min at 4 °C to spin down cell debris. The cellular proteins should be in the supernatant.
- Collect the supernatant and centrifuge with 40,000 x g for 30 min at 4 °C
- Discard supernatant. Resuspend the resulting pellet in 20 mL of a 33 % (vol/vol) solution of CellLytic B (Sigma) in TEMB
- Centrifuge again with 40,000 x g for 30 min at 4 °C
- Discard supernatant. Resuspend pellet in 3 mL TEMB buffer. Clarify by centrifugation with 3,000 x g for 1 min
- Load the supernatant onto 10 to 50 % (wt/vol) linear sucrose gradient. Sucrose gradients are made by slowly dribbling high % to low % sucrose down the side of the tube. For ultracentrifugation Polyallomer tubes with a volume of 13,2 mL were used. Ultraspin the sample in a Beckman Coulter OptimaTM LE 80 K Ultracentrifuge with a SW41 Ti Swinging Rotor Bucket with 105,000 x g for 30 min
- After centrifugation, the carboxysome should be seen as a band near the middle of the gradient → The purification was not succesful, as you could not recognize a visible band in the gradient
-
→ We made another sequencing but got these five mutations again at the same positions as before. Maybe we got the wrong accession number, so we will further work with our can construct.
- pHnCBcsoS1D_prkA
- This week we tried to transform the pHnCBcsoS1D_prkA construct.
- Transformation with electrocompotetent cells
- Colony PCR (prkA_pHn_rev, prkA_pHn_fwd)
- Annealing temperature: 55°C
- Bands not as expected → We got a lot of bands and could not interpret the result.
- csoS1-4
- This week we tried to amplify and transform the shell proteins again.
- 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)
- All PCR products were purified
- Restriction digestion with DpnI
- Gibson Assembly with csoS1-4 and pSB1C3
- Transformation with electrocompotetent cells
- prkA and pTet
- This week we tried to assemble the pTet promoter with the prkA.
- BioBrick Assembly (Suffix)
- Transformation with electrocompotetent cells → Only a few colonies did grow. Maybe the TetR (repressor) is not strong enough so the prkA is too toxic.
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~2200 bp)
- Hneap and pTet
- This week we tried to assemble the pTet promoter with the Hneap.
- BioBrick Assembly (Suffix)
- Transformation with electrocompotetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~3000 bp)
- sap_2
- This week we tried to bring the synthesized sap_2 construct in the pJet vector with blunt end cloning. We also amplified the backbonefor an assembly.
- Blunt-End cloning of sap_2 in the pJet vector
- Colony PCR (Primer of pJet set)
- Annealing temperature: 55 °C
- Bands as expected (~1500 bp)
- PCR amplification of the backbone (pSB1C3) (pSB1C3_pre_sap_1, pSB1C3_suf_sap2)
- Annealing temperature: 55 °C
- Bands as expected (~2100 bp)
- GFP BioBrick (BBa_E0040)
- This week we tried isolate the green fluorescent protein GFP (pSB1A2_GFP) of the parts distribution 2014.
- Plasmid isolation of pSB1A2_GFP
- Purification vector
- This week we tried to amplify the T7_RBS promoter for the purification vector. Additionally we amplified the intein tag containing a chitin binding domain.
- PCR amplification (RBS_int_rev, pSB1C3_int_fw) of the promoter (T7_RBS)
- Annealing temperature: 55°C
- Bands as expected (~2000 bp)
- PCR amplification (int_RBS_fw, int_pSB1C3_rev) the intein tag with chitin binding domain (intCBD)
- Annealing temperature: 55°C
- Bands as expected (~1000 bp)
- PCR products were purified
- Gibson Assembly with T7_RBS and intCBD
- Transformation with electrocompotetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55°C
- Bands as expected (~3300 bp)
- Plasmid isolation of pSB1C3_T7_RBS_intCBD
- Purification of the carboxysome
- As the purification of the carboxysome showed no results, we decided to induce protein expression with higher concentrations of IPTG.
- Cultivation was carried out using a modified method of Cultivation for Expression of recombinant proteins . Cells were continous cultivated by a temperature of 20 °C, and protein expression was induced when the culture reaches an OD600 of 0.9 - 1.2. IPTG in different final concentrations of 0.5 mM, 2 mM and 5 mM was used to verify the expression of the carboxysomal proteins through SDS-PAGE and MALDI-TOF . Samples were generated using the protocol for Fast Cell Lysis for SDS-PAGE.
Proteinexpression of pHnCBcsoS1D
induced with 0.5 mM IPTG
Proteinexpression of pHnCBcsoS1D
induced with 2 mM IPTG
Proteinexpression of pHnCBcsoS1D
induced with 5 mM IPTG
- sap_2
- We tried to isolate pJet_sap_2 for further experiments.
- Plasmid isolation of pJet_sap_2
- glpX
- We tried to amplify the pSB1K3 backbone for an assembly with glpX.
- PCR amplification of the pSB1C3_RFP backbone (fw_SBPase_pSB1C3, rv_SBPase_pSB1)
- Annealing temperature: 54 °C
- Bands as expected (~2100 bp)
- PCR products were purified
- Restriction digestion with DpnI
- Gibson Assembly with glpX_1, glpX_2 and pSB1K3
- Transformation with electrocompotetent cells
- csoS1-4 (BBa_K1465206)
- We tried to find correct colonies of the psB1C3_csoS1-4 construct so another part of the carboxysome is complete.
- Colony PCR (fw_pSB1C3_csoS4A, rv_csoS1_pSB1C3)
- Annealing temperature: 65 °C
- Bands (not) as expected (~1600 bp)
- Plasmid isolation of pSB1C3_csoS1-4
- Restriction digestion with EcoRI and PstI
- Bands as expected (~2000 bp backbone, ~1800 bp insert)
- sRNA:pfkA and ptac
- We tried to assemble the sRNA:pfkA construct and pSB1C3_ptac
- BioBrick Assembly (Suffix)
- Backbone (digested with SpeI, PstI)
- pSB1C3_ptac
- Insert (digested with XbaI, PstI)
- sRNA:pfkA
- Transformation with electrocompotetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~1700 bp)
- Plasmid isolation of pSB1C3_ptac_sRNA:pfkA
- sRNA:pfkA and T7 (BBa_K1465227)
- We tried to assemble the sRNA:pfkA construct and pSB1A2_T7
- BioBrick Assembly (Suffix)
- Backbone (digested with SpeI, PstI)
- pSB1A2_T7
- Insert (digested with XbaI, PstI)
- sRNA:pfkA
- Transformation with electrocompotetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~900 bp)
- Plasmid isolation of pSB1A2_T7_sRNA:pfkA
- BioBrick Assembly (Suffix)
- Transformation with electrocompotetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~600 bp)
- Plasmid isolation of pSB1C3_T7_sRNA:pfkA
- GFP
- We tried to amplify GFP for a fusion with the shell proteins.
- PCR amplification (fw-csoS1A-GFP, rv-csoS1B-GFP)
- Annealing temperature: 54 °C
- Bands as expected (~750 bp)
- PCR products were purified out of the gel
- can and csoS1-4
- We tried to assemble can and csoS1-4 for the carboxysome.
- BioBrick Assembly (Prefix)
- Transformation with electrocompotetent cells
- 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.
