RACRE Variant Libraries

Variant libraries are created using the RACRE technology, and delivering large libraries of mutant promoters to host cells to measure transcriptional activity of variant promoters. The commercial libraries include thousands of novel promoters that cover an extremely wide range of transcriptional activity, with a sub-sampling of promoters to characterize protein translation.

The libraries are available in whole and in segments, or as part of a service to determine promoter activity under your conditions. The commercial libraries are available to provide a low cost evaluation of promoters before committing to a commercial license.

 

hCMV Variant Library

To help increase production yields of biologics driven by the CMV promoter in CHO cells, we generated a variant library of the consensus human CMV promoter (which we refer to as the wild type promoter) with a complexity of approximately 9 million sequences that were cloned into our proprietary plasmid backbone. CHO cells were transfected using a lipid based transfection reagent, and incubated for 48hours. Cells were lysed and the remaining library population was purified and the transcripts prepped for deep sequencing.

The graph to the left represents data from 184,416 novel mutants that cover a range of transcriptional activity from 1% to over 9700% of the wild type activity!  The three data sets represent single, double, and triple mutants. Red data points indicate activity below wild type, and green data points indicate activity greater than wild type. To construct a catalog of commercially available promoters from this data set, we analyze the location of the mutations, the expression activities, and the synergy between single, double, and triple mutants.

The library currently includes 4100 individual promoters organized into the expression levels in the chart below. In addition to covering a wide range of transcriptional activity, the point mutations also cover the entire length (221 nt) of the promoter sequence with single, double, and triple mutants. This breadth of coverage provides a wide range of choices to ensure you can find the promoter you need to optimize your protein production.

Activity range of the novel promoters that are in the CMV library. Each stacked bar in the chart indicates the number of novel promoters that are in the category along the x-axis. Colored data points indicate the contribution from single, double, and triple mutants. For example, there are approximately 200 novel promoters that are in the 200-300% expression range.

Protein expression with CMV promoters

Mutant promoters were cloned into a GFP expression plasmid, transfected into CHO cells, and incubated for 48 hours. Measurements were taken on a fluorescent microscope at 20X, and individual well images were tiled together. Quartile data in the box plots represent the background subtracted fluorescence from the cells in each well, and each individual well included cells transfected with a single promoter. The images below show mosaic images of CHO cells transfected with different promoters driving GFP. All quantitative data in the box plot was generated by segmented image analysis in ImageJ. The CMV library can be used to isolate novel promoters that control a wide range of protein expression.

T7 Variant Library

The T7 RNA polymerase enhanced promoter library contains thousands of novel T7 mutant promoters that cover a wide range of expression activity compared to the wildtype T7 promoter. The library is organized into 5 expression levels as depicted in the diagram below. This product structure gives researchers a low cost entry into testing novel T7 promoters for research use before committing to a production facility. We’ve structured our library this way to facilitate transferring protein production from the lab to large scale facilities. For details and culturing conditions, please read our white paper.

This library is the most comprehensive tool set available to researchers that enable control of essentially any gene in E. coli at any practical expression level. The promoters can be used to drive expression for low yield proteins or optimize complex metabolic pathways, making the library ideal for:

  • Bio manufacturing facilities with a need to increase titers and yields
  • Synthetic biology and metabolic engineering researchers optimizing production chassis
  • Small molecule producers who need to increase their production output

T7-1

This product contains 5 novel T7 mutant promoters that represent expression levels evenly spread out between 1-100% (relative to the wild type T7 promoter). Weaker promoters are invaluable tools that allow researchers to easily and efficiently direct carbon flow in certain metabolic pathways and optimize their host chassis for small molecule production.

T7-2

This product contains all our promoters with transcriptional activity below the wild type T7 promoter. With almost 2800 novel promoters, this library provides exceptionally fine expression control for activity from 1-100%.

T7-3

This product contains 5 novel T7 mutant promoters that represent expression levels evenly spread out between 100-200% (relative to the wild type T7 promoter). This library is an excellent low cost entry to facilitate testing of promoters with increased activity. The T7-3 library currently includes 96 individual promoters.

T7-4

This product includes any single promoter with activity above 200% (relative to the wild type T7 promoter). The T7-4 library currently includes 35 individual promoters that range in activity from 202-553% (relative to the wild type T7 promoter). We are continually testing the thousands of additional promoter candidates from our original experiment, so check back to see if we’ve added any new promoters since the last time you checked.

Depending on the availability of transcriptional machinery (ie available ribosomes, transcription factors, chaperones, etc.), your host may not have the capacity to reach some of these higher expression levels. For that reason, we recommend trying the T7-3 library first, to confirm your host has the capacity to support higher protein expression levels.

T7-5

This product includes all the mutant T7 promoters in our library. The library currently includes 2678 individual promoters organized into the expression levels in the chart below.

Contact Us

For more details regarding prices and availability, please contact us.