The BIP-300 plasmid allows you to compare your pAN or pAC AviTagTM fusion gene’s protein expression against a confirmed AviTagTM fusion product under the same growth and induction conditions. When transformed into the same E. coli host strain as your own pAN or pAC AviTagTM fusion plasmid construct, and induced in parallel, the BIP-300 Positive Control Plasmid will provide a measure by which to compare your transformation efficiency, growth and antibiotic maintenance conditions, and induction of your plasmid’s gene-fusion construct. The BIP-300 construct consists of a modified β-galactosidase gene (8 amino acids were deleted from its N-terminus) cloned into Avidity’s pAN5 AviTagTM fusion vector at the Multiple Cloning Site. The resulting AviTagTM-protein fusion (AviTag-β-gal), as are all the pAN and pAC fusion products, is under the tight control of the Trc promoter system and expressed upon IPTG induction. The plasmid is maintained with ampicillin. An example induction protocol is given under Protocols. This protocol uses the Avidity AVB101 E. coli strain containing the BirA over-expressing pACYC-184 plasmid construct (pBirAcm) as the host strain. It is initially requires chloramphenicol for maintenance in addition to the ampicillin required to maintain the pAN or pAC plasmid construct. If you are using a different host strain please adjust the protocol accordingly.
Supplied as 1 vial of Positive Control Plasmid DNA; 2µg at 1mg/mL in nuclease-free water (2µL). Store at -20°C.
Do your vectors work off the T7 promoter expression system?
No. The BIP-300 positive control plasmid (and the pAN /pAC vectors) works off the Trc promoter.
Why is chloramphenicol not added to the growth/induction media to maintain the BIP-300 plasmid when it is used in the initial overnight culture? (relevant only if AVB101 E. coli is used as the host strain)
We find that the chloramphenicol is not necessary at this point to maintain the pBirAcm BirA over-expression plasmid. It is well enough maintained because of its low copy number.