Team:Bielefeld-CeBiTec/Notebook/Journal/CO2-fixation/Aug

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August

  • 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)

  • can and csoS1-4
    • We tried to assemble the shell proteins and the carbonic anhydrase for the carboxysome.
    • → After we got the sequencing results of pSB1C3_can we found out that we have some mutations so this construct has to be made again with the right sequence of can.

  • glpX

  • prkA and pHnCBscoS1D

  • 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
      → Because of the mutations in Selan we cannot use this construct. Also the restriction digestion of pSB1C3_ptac_prkA_Hneap was not successful.

  • can_csoS1-4 and csoS1D
    • We tried to assemble pSB1C3_can_csoS1-4 and csoS1D and transform the construct.
    • → After we got the sequencing results of pSB1C3_can we found out that we have some mutations so this construct has to be made again with the right sequence of can.
  • glpX

  • csoS1-4
    • We tried to isolate the right plasmid again.
    • → Sequencing not successful again.

    • can (BBa_K1465205)
      • → 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.

  • 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
          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