User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/07/16

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Characterization B: Expression of PHIX174 promoters fused to UTR1-deGFP.

  • Continuing with minicultures and sequencing of the transformants specified on 07/11/2012.

Characterization C: Expression of PHIX174 promoters fused to UTRX-deGFP.

  • Continuing with minicultures and sequencing of the transformants specified on 07/11/2012.

Hypothesis 2: Gene L is necessary for phage propagation.

  • On hold until new primers arrive.

Hypothesis 3: Gene L codes for a ~6 kDA protein.

  • Experiment 3: Simga28 cascade cell free expression batch mode of +L and -L (no plasmid) followed up by high density SDS page.
  • Constructions in stock:
    • pBEST-Ptar-UTR1-L-T500 (110 nM)
  • Constructions needing amplification:
    • pBEST-OR1OR2Pr-UTR1-simga28-T500
  • Constructions needing to be made:
    • pBEST-Ptar-UTR1-L-deGFP-T500
  • Gave pBEST-OR1OR2Pr-UTR1-simga28-T500 to Mark for amplification.

Hypothesis 4: Gene L complexes with cell membrane.

Experiment 4: Simga28 cascade cell free expression in vesicles of L-deGFP and L. Followup with in vivo expression in cells observed by microscope (use PT7 promoter on pIvex2.3d). Constructions:

    • pBEST-Ptar-UTR1-L-eGFP (109 nM)
  • Constructions needing amplification:
    • pBEST-OR1OR2Pr-UTR1-simga28-T500 (same as H3 above)
  • Constructions needing to be made or obtained:
    • pBEST-Ptar-UTR1-L-deGFP
    • pIvex2.3d-L
    • pIvex2.3d-L-deGFP
    • pIvex2.3d-deGFP
  • Result: Preliminary results show binding of L-eGFP fusion to vesicle membrane.