Efficient Biotinylation E. coli XL1-Blue Strain AVB99

Strain AVB99 is an E.coli strain (XL1-Blue) containing a pACYC184 plasmid with an IPTG-inducible birA gene to overexpress biotin ligase (pBirAcm). The plasmid should be maintained with chloramphenicol at 10 µg/mL concentration (this is a low copy-number plasmid).

Expression of both biotin ligase and the AviTag protein are induced with IPTG (1 mM). Biotin should be added at the time of induction to a concentration of 50 µM. The pAN and pAC vectors require ampicillin for maintenance and the recommended concentration is 100 µg/mL.

Features of plasmid pBIRAcm

• Backbone is pACYC184.
• 1.3 Kb fragment contains lacIq inserted at EcoRV site (thus disrupting tetR gene).
• BirA is expressed from the tac promoter operator.
• rrnB terminator construct is between the laqIq region and destroys XbaI site of pACYC184.