We comprehensively assessed the contribution of the Shine-Dalgarno sequence to protein expression and used the data to develop EMOPEC (Empirical Model and Oligos for Protein Expression Changes; http://emopec.biosustain.dtu.dk). EMOPEC is a free tool that makes it possible to modulate the expression level of any Escherichia coli gene by changing only a few bases. Measured protein levels for 91% of our designed sequences were within twofold of the desired target level.
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We thank H. Genee, A. Wallin, S. Cardinale and H. Wang for discussions and suggestions regarding this manuscript, and we thank A. Koza for assistance with DNA sequencing. The research leading to these results received funding from the Novo Nordisk Foundation through the Novo Nordisk Foundation Center for Biosustainability and the European Union Seventh Framework Programme (FP7-KBBE-2013-7-single-stage) under grant agreement 613745, Promys.
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 RBS Calculator values versus estimated expression.
Flow-seq estimated RBS strength compared with RBS Calculator estimated RBS strength. The highlighted data points (open red circles) are the 106 sequences measured as additional controls in fig. 1e and f. The downloadable version 1 of the RBS Calculator was used to estimate RBS strength.
Supplementary Figure 2 Random Forest prediction of missing sequences: cross-validation.
5-fold cross validation of Random Forest model for predicting SD strength of sequences not identified with >50 reads in the Flow-seq dataset. The out-of-bag estimate is R2 = 0.90, the five-fold cross-validation R2 = 0.89.
Supplementary Figure 3 Change in secondary structure for optilib oligos.
(a) Change in secondary structure for the transcripts of all the 40,526 targetting E. coli genes. The average change is 0.51 kcal/mol. * denotes previously observed changes in free energy that led to a significant change in expression levels19. (b) Distribution of oligos with 1-6 nucleotides changed from the wild-type in optilib.
Supplementary Figure 4 Distribution of predicted expression levels in the constrained optilib.
The 40,526 oligos designed to change expression of all E. coli genes. The oligos were designed with the constraint to not modify the coding sequence of overlapping genes. Even with the constraints, it was possible to design sequences with predicted expression close to the intended target value for most sequences.
Supplementary Figure 5 mCherry mRNA levels.
mRNA levels measured by Real Time PCR. No notable difference was found between mRNA levels of the different transcripts.
Supplementary Figure 6 Predicted versus measured protein levels for EMOPEC and RBS Calculator.
Measured expression values of the mCherry, LacZ, Ppc, AspC, Can, and AceA validation strains compared with the EMOPEC predicted values and RBS Calculator (V2.0) predicted values.
Supplementary Figure 7 Pooled measured expression values compared to EMOPEC and RBS Calculator predictions.
Measured expression levels for mCherry and the native E. coli genes LacZ, AceA, Can, Ppc and AspC for EMOPEC (left) and the RBS calculator (right). Linear regression, p < 0.001 for both plots.
Supplementary Figure 8 Signal distributions for each flow-sorted bin.
Flow cytometry signal distributions for each bin and Gaussian fit to the signal.
Supplementary Figure 9 Read-count distribution.
Distribution of merged read counts across all bins for the SD sequences.
Supplementary Text and Figures
Supplementary Figures 1–9 (PDF 1113 kb)
Supplementary Table 1
All 3,864 estimated GFP expression values. (XLSX 162 kb)
Supplementary Table 2
106 individually verified single colonies from the main library. (XLSX 14 kb)
Supplementary Table 3
EMOPEC designed oligos, ten expression levels for every annotated gene in E. coli K12 MG1655. (XLSX 8970 kb)
Supplementary Table 4
Strains, plasmids and nucleotide oligos used in this study. (XLSX 15 kb)
Supplementary Table 5
Barcoded primers used for sequencing the flow-sorted bins. (XLSX 9 kb)
Supplementary Table 6
Sequence counts from the flow-sorted bins. (XLSX 465 kb)
EMOPEC Python library. Source code for the EMOPEC algorithm as a Python library including the web server. (ZIP 1131 kb)
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Bonde, M., Pedersen, M., Klausen, M. et al. Predictable tuning of protein expression in bacteria. Nat Methods 13, 233–236 (2016). https://doi.org/10.1038/nmeth.3727
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