Annotated genes are indicated by rectangles in the upper graph shared genes are indicated by open rectangles, with coding strand indicated by position of the rectangle above (positive) or below (negative) the central line. A comparison of the annotations of ∼100 kb of homologous regions of the UTI89 and MG1655 chromosomes is shown. ( C) One transconjugant was assembled and compared to the UTI89 and MG1655 genome reference sequences. Blocks of consecutive SNPs relative to the MG1655 genome sequence are represented by red bars. Individual clones are plotted at different locations on the y axis. Genomic coordinate is plotted on the x axis. Sequencing data from individual transconjugants were analyzed using the MG1655 genome as a reference. ( B) Transferred SNPs for the SLC-H1 library of hybrids. Brackets enclose a representative single donor and recipient conjugation in the bottom left section. Experimental manipulations are indicated by the text adjacent to arrows. A kanamycin resistance gene (light blue box) was used as the positive selection marker for the donor libraries and a chloramphenicol resistance gene (dark blue box) was used for the recipient libraries. The negative selection module contained the tse2 toxin gene under the control of P rhaB promoter (for both the donor and recipient transposon libraries, gray box). The fluorescent protein used was mCherry (red box). Genetic markers are represented by colored squares, according to the legend at the top right. Each circle represents a single bacterial chromosome within a cell (for clarity, cell membranes are not drawn). coli strains, especially clinical isolates ( 15).įig. However, transformation was found to be too inefficient by several orders of magnitude, while transduction required strain-specific phages that are not generally available for many E. jejuni) or generalized transduction as methods to exchange DNA between strains using these, the donor strain would not need to be modified. Of note, we had initially considered using transformation of purified genomic DNA (as was done with C. Selection of the mixture on restrictive conditions for the negative selection cassette (i.e., growth on minimal media M9 with rhamnose to induce expression of the toxic tse2 gene) results in survival only of hybrid transconjugant strains the donor negative selection cassette is not transferred due to the position and orientation of the oriT, while the recipient negative selection cassette is removed by recombination with incoming donor DNA. Finally, the donor is transformed with a helper plasmid for conjugation, then a standard conjugation is performed. For tracking efficiency, the donor and recipient transposons also differ in their positively selectable antibiotic markers and a fluorescent protein (kanamycin resistance and mCherry fluorescence on the donor transposon, chloramphenicol resistance on the recipient transposon, and a plasmid-based green fluorescent protein fluorescence in recipients). The donor transposon additionally has an outward facing oriT (origin of transfer) sequence to initiate chromosomal transfer adjacent to (and not including) the custom transposon. Both strains are subjected to a Tn5-mediated transposon mutagenesis with a customized transposon which carries a stringent negative selection cassette. In this study the uropathogenic strain UTI89 is the donor while the nonpathogenic strain MG1655 is the recipient. coli strain as the donor and the other as the recipient. This technique is a targeted screen requiring engineering and mating of strains during each round ( 11).Ī conceptual schematic of MAE is shown in Fig. By leveraging a phenotypic difference between two strains, multiple rounds of conjugation are used to map the genetic locus responsible for the phenotype. The REGRES technique, in contrast, achieves genotype–phenotype linkage similar in concept to the original Hfr technique. Furthermore, chromosome-wide engineering requires high efficiency, which has usually only been demonstrated in well-characterized (and “well-behaved”) cloning strains of E. However, all of these technologies, except for REGRES, are designed for engineering defined chromosomal edits, albeit at the megabase scale. coli genome to remove all instances of three codons, as a precursor to their repurposing for other synthetic biology applications ( 13). These include CAGE, MAGE, REXER, GENESIS, and REGRES ( 9– 12) the power of REXER and GENESIS has been recently demonstrated with complete recoding of the E. Several advanced techniques have been recently developed to perform chromosome-wide engineering at a scale comparable to that achieved by the original Hfr-mediated E.
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