Composability of regulatory sequences controlling transcription and translation in Escherichia coli.
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.
Wyss Institute for Biologically Inspired Engineering, Boston, MA 02115, USA.
Sriram
S
Kosuri
Daniel B
DB
Goodman
Guillaume
G
Cambray
Vivek K
VK
Mutalik
Yuan
Y
Gao
Adam P
AP
Arkin
Drew
D
Endy
George M
GM
Church
D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D015723:Gene Library; D005818:Genetic Engineering / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing; D008957:Models, Genetic; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics*; D050436:Regulatory Elements, Transcriptional / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012270:Ribosomes / Q000235:genetics; D049490:Systems Biology / Q000379:methods*
D017931:DNA Primers; D012333:RNA, Messenger
next-generation sequencing; synthetic biology; systems biology