IGEM:Cambridge/2008/Notebook/Magnetic Bacteria/2008/07/23

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Biobrick Selection and Amplification

New standardised vector to be used for mms6 and mamC genes, along with an inducible promoter.

Promoter

Part:BBa_I0500 - Inducible pBad/araC

pBad is an E. coli promoter that is tightly controlled by: inducer: L-arabinose. repressor: AraC apparently acts as the repressor When grown with 0.2% arabinose, promoter is weak-medium. [jb, 5/24/04] Part may not be compatible with MC4100 as cell line is araD 139 MC4100 is not a good chassis for operating BBa_I0500 (pBad promoter). The feed-forward regulation of the endogenous promoter controlling expression of the arabinose transporter prevents linear induction with increasing arabinose concentration. ((Engineered strain from Keasling's lab, used by jrk for operation of the screening plasmid.))

Plasmid Vector

pSB3K3-1 is a medium copy plasmid with kanamycin resistance. It has a p15A pMR101-derived replication origin, copy number 20-30. (Lutz and Bujard, 1997) pSB3K3-1 has a terminator upstream of its MCS, which is oriented to prevent transcription from *inside* the MCS from reading out into the vectorl. A second terminator (E.coli His operon-derived) is downstream of the MCS, again insulating the vector from transcription reading out of the MCS. Ideally, future versions of standard biobrick vectors would have terminators bracketing the MCS that were 100% efficient in terminating transcription both into and out of the MCS region.


Work Done:

  • Made glycerol stock of TOP10 transformed with mms6 or mamC plasmids by picking single colonies from 1/10 agar plates prepared on 22/7/2008
  • Followed protocols on green folder for preparation of stock
  • PCR amplification of mms6 and mamC with primers VR and VF2
  • PCR amplification of I0500 and B0014 biobricks
  • Result of PCR: PCR failed as primers attachment sites are on plasmid vector backbone rather than biobrick prefix or suffix





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