Genome-Wide Tuning of Protein Expression Levels to Rapidly Engineer Microbial Traits

doi:10.1021/acssynbio.5b00133

. 2015 Nov 20;4(11):1244-53.

doi: 10.1021/acssynbio.5b00133. Epub 2015 Nov 5.

Genome-Wide Tuning of Protein Expression Levels to Rapidly Engineer Microbial Traits

Emily F Freed¹, James D Winkler¹, Sophie J Weiss¹, Andrew D Garst¹, Vivek K Mutalik^{2

3}, Adam P Arkin^{2

3}, Rob Knight⁴, Ryan T Gill¹

Affiliations

¹ Department of Chemical and Biological Engineering, University of Colorado Boulder , Boulder, Colorado 80309, United States.
² Lawrence Berkeley National Laboratory , Physical Biosciences Division, Berkeley, California 94720, United States.
³ Department of Bioengineering, University of California, Berkeley , Berkeley, California 94720, United States.
⁴ Departments of Pediatrics and Computer Science & Engineering, University of California San Diego , La Jolla, California 92093, United States.

PMID: 26478262
DOI: 10.1021/acssynbio.5b00133

Genome-Wide Tuning of Protein Expression Levels to Rapidly Engineer Microbial Traits

Emily F Freed et al. ACS Synth Biol. 2015.

. 2015 Nov 20;4(11):1244-53.

doi: 10.1021/acssynbio.5b00133. Epub 2015 Nov 5.

Authors

Emily F Freed¹, James D Winkler¹, Sophie J Weiss¹, Andrew D Garst¹, Vivek K Mutalik^{2

3}, Adam P Arkin^{2

3}, Rob Knight⁴, Ryan T Gill¹

Affiliations

¹ Department of Chemical and Biological Engineering, University of Colorado Boulder , Boulder, Colorado 80309, United States.
² Lawrence Berkeley National Laboratory , Physical Biosciences Division, Berkeley, California 94720, United States.
³ Department of Bioengineering, University of California, Berkeley , Berkeley, California 94720, United States.
⁴ Departments of Pediatrics and Computer Science & Engineering, University of California San Diego , La Jolla, California 92093, United States.

PMID: 26478262
DOI: 10.1021/acssynbio.5b00133

Abstract

The reliable engineering of biological systems requires quantitative mapping of predictable and context-independent expression over a broad range of protein expression levels. However, current techniques for modifying expression levels are cumbersome and are not amenable to high-throughput approaches. Here we present major improvements to current techniques through the design and construction of E. coli genome-wide libraries using synthetic DNA cassettes that can tune expression over a ∼10(4) range. The cassettes also contain molecular barcodes that are optimized for next-generation sequencing, enabling rapid and quantitative tracking of alleles that have the highest fitness advantage. We show these libraries can be used to determine which genes and expression levels confer greater fitness to E. coli under different growth conditions.

Keywords: Illumina sequencing; directed evolution; genome-wide expression library; genotype-phenotype mapping; recombineering.

PubMed Disclaimer

Cited by

A versatile platform strain for high-fidelity multiplex genome editing.
Egbert RG, Rishi HS, Adler BA, McCormick DM, Toro E, Gill RT, Arkin AP. Egbert RG, et al. Nucleic Acids Res. 2019 Apr 8;47(6):3244-3256. doi: 10.1093/nar/gkz085. Nucleic Acids Res. 2019. PMID: 30788501 Free PMC article.
Dual-barcoded shotgun expression library sequencing for high-throughput characterization of functional traits in bacteria.
Mutalik VK, Novichkov PS, Price MN, Owens TK, Callaghan M, Carim S, Deutschbauer AM, Arkin AP. Mutalik VK, et al. Nat Commun. 2019 Jan 18;10(1):308. doi: 10.1038/s41467-018-08177-8. Nat Commun. 2019. PMID: 30659179 Free PMC article.
Pooled CRISPR interference screening enables genome-scale functional genomics study in bacteria with superior performance.
Wang T, Guan C, Guo J, Liu B, Wu Y, Xie Z, Zhang C, Xing XH. Wang T, et al. Nat Commun. 2018 Jun 26;9(1):2475. doi: 10.1038/s41467-018-04899-x. Nat Commun. 2018. PMID: 29946130 Free PMC article.
Prospects for engineering dynamic CRISPR-Cas transcriptional circuits to improve bioproduction.
Fontana J, Voje WE, Zalatan JG, Carothers JM. Fontana J, et al. J Ind Microbiol Biotechnol. 2018 Jul;45(7):481-490. doi: 10.1007/s10295-018-2039-z. Epub 2018 May 8. J Ind Microbiol Biotechnol. 2018. PMID: 29740742 Review.
CRISPR interference as a titratable, trans-acting regulatory tool for metabolic engineering in the cyanobacterium Synechococcus sp. strain PCC 7002.
Gordon GC, Korosh TC, Cameron JC, Markley AL, Begemann MB, Pfleger BF. Gordon GC, et al. Metab Eng. 2016 Nov;38:170-179. doi: 10.1016/j.ymben.2016.07.007. Epub 2016 Jul 29. Metab Eng. 2016. PMID: 27481676 Free PMC article.

See all "Cited by" articles

Publication types

Actions

MeSH terms

Actions
Actions
Actions
Actions

LinkOut - more resources

Full Text Sources
- American Chemical Society

Save citation to file

Email citation

Add to Collections

Add to My Bibliography

Your saved search

Create a file for external citation management software

Your RSS Feed

Genome-Wide Tuning of Protein Expression Levels to Rapidly Engineer Microbial Traits

Affiliations

Genome-Wide Tuning of Protein Expression Levels to Rapidly Engineer Microbial Traits

Authors

Affiliations

Abstract

Similar articles

Cited by

Publication types

MeSH terms

LinkOut - more resources

Full Text Sources