Generating Site Saturation Mutagenesis Libraries and Transferring Them to Broad Host-Range Plasmids Using Golden Gate Cloning

Overview

Journal Methods Mol Biol

Specialty Molecular Biology

Date 2024 Oct 3

PMID 39363076

Authors

Niels N Oehlmann

Johannes G Rebelein

Affiliations

Soon will be listed here.

Abstract

Protein engineering is an established method for tailoring enzymatic reactivity. A commonly used method is directed evolution, where the mutagenesis and natural selection process is mimicked and accelerated in the laboratory. Here, we describe a reliable method for generating saturation mutagenesis libraries by Golden Gate cloning in a broad host range plasmid containing the pBBR1 replicon. The applicability is demonstrated by generating a mutant library of the iron nitrogenase gene cluster (anfHDGK) of Rhodobacter capsulatus, which is subsequently screened for the improved formation of molecular hydrogen.

References

Shortle D, DiMaio D, Nathans D . Directed mutagenesis. Annu Rev Genet. 1981; 15:265-94. DOI: 10.1146/annurev.ge.15.120181.001405. View

Smith M . In vitro mutagenesis. Annu Rev Genet. 1985; 19:423-62. DOI: 10.1146/annurev.ge.19.120185.002231. View

Brannigan J, Wilkinson A . Protein engineering 20 years on. Nat Rev Mol Cell Biol. 2002; 3(12):964-70. DOI: 10.1038/nrm975. View

Pourmir A, Johannes T . Directed evolution: selection of the host organism. Comput Struct Biotechnol J. 2014; 2:e201209012. PMC: 3962113. DOI: 10.5936/csbj.201209012. View

Wang Y, Xue P, Cao M, Yu T, Lane S, Zhao H . Directed Evolution: Methodologies and Applications. Chem Rev. 2021; 121(20):12384-12444. DOI: 10.1021/acs.chemrev.1c00260. View

Reetz M, Carballeira J . Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes. Nat Protoc. 2007; 2(4):891-903. DOI: 10.1038/nprot.2007.72. View

Liu H, Naismith J . An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. BMC Biotechnol. 2008; 8:91. PMC: 2629768. DOI: 10.1186/1472-6750-8-91. View

Pullmann P, Ulpinnis C, Marillonnet S, Gruetzner R, Neumann S, Weissenborn M . Golden Mutagenesis: An efficient multi-site-saturation mutagenesis approach by Golden Gate cloning with automated primer design. Sci Rep. 2019; 9(1):10932. PMC: 6662682. DOI: 10.1038/s41598-019-47376-1. View

Stein A, Chen D, Igareta N, Cotelle Y, Rebelein J, Ward T . A Dual Anchoring Strategy for the Directed Evolution of Improved Artificial Transfer Hydrogenases Based on Carbonic Anhydrase. ACS Cent Sci. 2021; 7(11):1874-1884. PMC: 8620556. DOI: 10.1021/acscentsci.1c00825. View

10.

Engler C, Kandzia R, Marillonnet S . A one pot, one step, precision cloning method with high throughput capability. PLoS One. 2008; 3(11):e3647. PMC: 2574415. DOI: 10.1371/journal.pone.0003647. View

11.

Couturier M, Bex F, Bergquist P, Maas W . Identification and classification of bacterial plasmids. Microbiol Rev. 1988; 52(3):375-95. PMC: 373151. DOI: 10.1128/mr.52.3.375-395.1988. View

12.

Jain A, Srivastava P . Broad host range plasmids. FEMS Microbiol Lett. 2013; 348(2):87-96. DOI: 10.1111/1574-6968.12241. View

13.

Antoine R, Locht C . Isolation and molecular characterization of a novel broad-host-range plasmid from Bordetella bronchiseptica with sequence similarities to plasmids from gram-positive organisms. Mol Microbiol. 1992; 6(13):1785-99. DOI: 10.1111/j.1365-2958.1992.tb01351.x. View

14.

Potapov V, Ong J, Kucera R, Langhorst B, Bilotti K, Pryor J . Comprehensive Profiling of Four Base Overhang Ligation Fidelity by T4 DNA Ligase and Application to DNA Assembly. ACS Synth Biol. 2018; 7(11):2665-2674. DOI: 10.1021/acssynbio.8b00333. View

15.

Geddes B, Mendoza-Suarez M, Poole P . A Bacterial Expression Vector Archive (BEVA) for Flexible Modular Assembly of Golden Gate-Compatible Vectors. Front Microbiol. 2019; 9:3345. PMC: 6339899. DOI: 10.3389/fmicb.2018.03345. View

16.

Katzke N, Bergmann R, Jaeger K, Drepper T . Heterologous high-level gene expression in the photosynthetic bacterium Rhodobacter capsulatus. Methods Mol Biol. 2011; 824:251-69. DOI: 10.1007/978-1-61779-433-9_13. View

17.

Klipp W, Masepohl B, Puhler A . Identification and mapping of nitrogen fixation genes of Rhodobacter capsulatus: duplication of a nifA-nifB region. J Bacteriol. 1988; 170(2):693-9. PMC: 210710. DOI: 10.1128/jb.170.2.693-699.1988. View

18.

Figurski D, Helinski D . Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A. 1979; 76(4):1648-52. PMC: 383447. DOI: 10.1073/pnas.76.4.1648. View

19.

SantaLucia Jr J . A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc Natl Acad Sci U S A. 1998; 95(4):1460-5. PMC: 19045. DOI: 10.1073/pnas.95.4.1460. View

20.

Mansoury M, Hamed M, Karmustaji R, Hannan F, Safrany S . The edge effect: A global problem. The trouble with culturing cells in 96-well plates. Biochem Biophys Rep. 2021; 26:100987. PMC: 8024881. DOI: 10.1016/j.bbrep.2021.100987. View