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 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) using multiple 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.

Generating Mutant Libraries with Error-Prone PCR

The process by which we produce our mutant libraries is both fast and simple. We select a BioBrick part and prepare an error-prone PCR reaction using a small amount of template, standard VF2 and VR primers, the error-prone PCR mix listed on our protocols page and Taq polymerase enzyme. The products of this reaction may be diluted and run through another round of PCR to introduce more mutations.

Mutant parts are then ligated directly into a screening construct like those listed below to help assess part activity. For example, mutant promoters can be placed in front of GFP to estimate their strength by relative fluorescence, whereas repressors can be paired with their corresponding promoter in front of a reporter. Transformation of these ligation products into competent DH5-α cells yields plates with many colonies, many containing mutants of the selected BioBrick part.

Colonies with the desired level of reporter expression are transferred to replica plates, grown up in liquid culture, and assayed in a 96-well plate reader. Consecutive screens are performed to narrow down the number of potential mutants so that they may be run in triplicate in our plate reader, using Octave scripts to select mutants with the desired range of activity.

Once a final group of mutants has been picked, they are carefully characterized for their activity. You can read more about the characterization process for our promoter mutants here.

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.