Sriram Kosuri, Daniel B. Goodman, Guillaume Cambray, Vivek K. Mutalik, Yuan Gao et al.
The inability to predict heterologous gene expression levels precisely hinders our ability to engineer biological systems. Using well-characterized regulatory elements offers a potential solution only if such elements behave predictably when combined. We synthesized 12,563 combinations of common promoters and ribosome binding sites and simultaneously measured DNA, RNA, and protein levels from the entire library. Using a simple model, we found that RNA and protein expression were within twofold of expected levels 80% and 64% of the time, respectively. The large dataset allowed quantitation of global effects, such as translation rate on mRNA stability and mRNA secondary structure on translation rate. However, the worst 5% of constructs deviated from prediction by 13-fold on average, which could hinder large-scale genetic engineering projects. The ease and scale this of approach indicates that rather than relying on prediction or standardization, we can screen synthetic libraries for desired behavior.
Sriram Kosuri, Daniel B. Goodman, Guillaume Cambray, Vivek K. Mutalik, Yuan Gao, Adam P. Arkin, Drew Endy, George M. Church. Composability of regulatory sequences controlling transcription and translation in Escherichia coli. 2013-08-07. https://doi.org/10.1073/pnas.1301301110
An episomal pGERC reporter library combined 114 promoters with 111 ribosome-binding sites upstream of superfolder GFP, with constitutive mCherry as an internal control. DNA-seq and RNA-seq quantified construct representation and transcript abundance, while FlowSeq measured GFP/RFP protein output across twelve FACS bins during basal growth of E. coli MG1655.
Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.
Download all 5 files (ZIP)1301301110_pnas.201301301SI.pdf1301301110_sd01.xls1301301110_sd02.xls1301301110_sd03.xlsREADME.txt