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, and to prototype this protocol by creating mutant libraries of the LacI, TetR and λ cI repressible promoters.

Why Make Mutant Libraries?

When designing novel genetic circuits, synthetic biologists are limited to using parts that are already available or that they can manufacture themselves. The creation of new parts can be incredibly time-consuming, since they must be extracted from existing natural genes and converted to a DNA part standard (like BioBricks) or modified from existing parts.

The parts registry contains many useful parts, but in cases where fine control over the function of a part is required for a genetic circuit to function properly, the existing selection may not offer enough variation in expression strength, chemical response, or other characteristics.

Fine-tuning the control of a part can be achieved by random mutation of the bases in the DNA sequence of a part to change some characteristic -- say, transcription initiation, fluorescence intensity, or protein binding affinity -- whilst maintaining the basic functionality of the part. This is generally done in such away that many mutants are created at once which are then screened for their activity levels.

This allows the creation of large numbers of mutants which are viable (that is, maintaining the core function of the part) in a relatively short period of time. By performing this process on popular parts, one team can produce not only parts that are of a suitable activity for their circuits, but also parts that might fall outside of their usage range but be useful to other teams. Submitting these mutant libraries to the registry allows other scientists access to a broader range of parts with a broader activity range and familiar functionality, preventing wasted man-hours and reagents.

Why mutate repressible promoters?

R0010, R0040 and R0051 (the LacI, TetR, and λ cI repressible promoters) are among the most commonly used promoters in the registry. They are useful because they allow control control over gene expression that can be regulated using their corresponding repressor proteins, C0012, C0040 and C0051. This permits the construction of complex circuits that take advantage of the ability to switch, oscillate, or otherwise control the expression of genes by combining these parts in different ways.

J23101, another extremely popular promoter, is a member of a constitutive promoter mutant library based on the wild-type J23119. This family offers mutants with similar functionality at different expression activity levels, making it an ideal choice for designing circuits where fine control over gene expression is beneficial. The creation of promoter mutant libraries for R0010, R0040 and R0051 makes the same kind of fine expression control possible within repressible systems. Extensive characterization of these parts allows synthetic biologists to pick and choose mutants with an ideal level of repressor binding affinity or overall promoter activity, which is vital for the efficient design of complex circuits.

Because promoters are integral to the function of all genetic circuits, it is especially important that they be available in a broad selection and that their activity is well-documented and predictable.

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.