The parts registry contains many parts that could be more useful if they were available in a range of activity levels. Because of this, we set out to develop a rugged, easy-to-use mutagenic PCR protocol for the rapid production of mutant libraries of any BioBrick part using standard primers.

To test our protocol's ability to produce functional protein mutants, we ran E0240 (GFP) through one round of our error-prone PCR procedure and performed visual inspections of the resulting colonies for their variation from wild-type GFP.

To show that our protocol can also be used to diversify the application of promoters and other regulatory gene sequences, we performed the same procedure on parts R0010, R0040 and R0051 (the LacI, TetR and c1 Lambda promoters), changing only the number of successive rounds of error-prone PCR. We screened these mutants for promoter activity compared to wild type, and characterized their response to changes in repressor concentration. We also characterized the response of wild-type and mutant LacI to IPTG at various repressor concentration levels.

General Mutant Screening Constructs

The specific order in which the parts are depicted below allows the user to swap in any promoter/repressor or promoter/activator pair using our regulatory characterization plasmid, K611018.



We designed this construct for characterizing promoter mutants. When pBAD is induced with arabinose, the repressor of choice is transcribed leading to decreased levels of the reporter, GFP.



The mutant repressor characterization construct is nearly identical to its promoter-based counterpart. The user simply ligates the wild-type promoter to the 5' end and a mutant repressor or activator to the 3' end.