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Abstract
With only hundreds of genes contained within their genomes, mycoplasmas have become model organisms for precise understanding of cellular processes, as well as platform organisms for predictable engineering of microbial functions for mission-critical applications. Despite the availability of “whole genome writing” in Mycoplasma mycoides, some traditional methods for genetic engineering are underdeveloped in mycoplasmas. Here we demonstrate two facile transposon-mediated approaches for introducing genes into the synthetic cell based on M. mycoides. The marker-less approach involves preparing a fragment containing only a small genomic region of interest with flanking transposase-binding sites, followed by in vitro transposase loading and introduction into the cells. The marker-driven approach involves cloning an open reading frame (ORF) of interest into a vector containing a marker for mycoplasma transformation, as well as sites for transposase loading and random genomic integration. An innovative feature of this construct is to use a single promoter to express the transformation marker and the introduced ORF. The marker-driven approach can be conveniently applied to any exogenous or synthetic gene without any information on the effect of the gene on the strain, whereas the marker-less approach requires that the fragment has a recognizable effect. Using the marker-less method, we found that a region containing the nusG gene rescues a slow growth phenotype of a strain containing a larger deletion encompassing this gene. Using the marker-driven approach, we better defined this finding, thereby establishing that nusG is required for a normal growth rate in synthetic M. mycoides. These methods are suitable for complementation tests to identify genes responsible for assorted functions lacking in deletion mutants. These approaches are also expected to facilitate rapid testing of various natural and engineered genes or gene clusters from numerous sources in M. mycoides.
Highlights
The ability to insert, delete, and mutate genes forms the basis of genetic engineering
The concentration of DNA was adjusted to 100 ng/μL in Tris EDTA (TE) buffer. 2 μL of the digested and purified fragment was mixed with 2 μL of 100% glycerol and 4 μL of EZ-Tn5 Transposase and incubated for 30 min at room temperature to prepare Tn5 transposomes. 4 μL of the resulting mixture was used for mycoplasma transformation
THE MARKER-LESS APPROACH TO COMPLEMENTATION We explored the ability to restore one or a few genes in a deletion mutant mycoplasma strain without using whole genome construction, in order to accelerate the characterization of gene functions and the optimization of gene contents for engineering strains for various purposes
Summary
The ability to insert, delete, and mutate genes forms the basis of genetic engineering. A fragment containing the open reading frame (ORF) of nusG (642 bp), as well as flanking 40-bp sequences with homology to the pLS-Tn5-Puro vector, was generated using PCR with the primers described below and genomic DNA of M. mycoides JCVI-syn1.0 as a template (Supplementary Figure 3).
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