Synergy between tetA and rpsL Provides High-Stringency Positive and Negative Selection in Bacterial Artificial Chromosome Vectors

Stavropoulos, Tom A.; Strathdee, Craig A.

doi:10.1006/geno.2000.6481

Cited by 25 publications

(34 citation statements)

References 35 publications

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“…Dual selectable markers are particularly advantageous for generating gene replacements, scar-less genome editing and selection-coupled biosensors (7). Several dual selectable markers have been reported [ rpsL (8,9), galK (10), thyA (11), hsvTK (12), tetA (13) and tolC (14)], but as with counter-selectable markers, dual-selectable markers suffer from high counter-selection escape and/or reliance on minimal media for robust counter-selection (10). …”

Section: Introductionmentioning

confidence: 99%

Rational optimization of tolC as a powerful dual selectable marker for genome engineering

Gregg

Lajoie

Napolitano

et al. 2014

Nucleic Acids Research

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Selection has been invaluable for genetic manipulation, although counter-selection has historically exhibited limited robustness and convenience. TolC, an outer membrane pore involved in transmembrane transport in E. coli, has been implemented as a selectable/counter-selectable marker, but counter-selection escape frequency using colicin E1 precludes using tolC for inefficient genetic manipulations and/or with large libraries. Here, we leveraged unbiased deep sequencing of 96 independent lineages exhibiting counter-selection escape to identify loss-of-function mutations, which offered mechanistic insight and guided strain engineering to reduce counter-selection escape frequency by ∼40-fold. We fundamentally improved the tolC counter-selection by supplementing a second agent, vancomycin, which reduces counter-selection escape by 425-fold, compared colicin E1 alone. Combining these improvements in a mismatch repair proficient strain reduced counter-selection escape frequency by 1.3E6-fold in total, making tolC counter-selection as effective as most selectable markers, and adding a valuable tool to the genome editing toolbox. These improvements permitted us to perform stable and continuous rounds of selection/counter-selection using tolC, enabling replacement of 10 alleles without requiring genotypic screening for the first time. Finally, we combined these advances to create an optimized E. coli strain for genome engineering that is ∼10-fold more efficient at achieving allelic diversity than previous best practices.

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Section: Introductionmentioning

confidence: 99%

Rational optimization of tolC as a powerful dual selectable marker for genome engineering

Gregg

Lajoie

Napolitano

et al. 2014

Nucleic Acids Research

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“…Several such methods, utilizing different counter-selection cassettes, have been described (6,8–10). We reported previously (8) an approach utilizing a counter-selection cassette, the RT-cassette, containing positive, tet A(C), and negative, rps L + , markers generated by PCR from the plasmid pBAC-RT (11). The RT-cassette is inserted into the target by an initial recombineering step, using positive selection with tetracycline (Tc), and then subsequently replaced seamlessly with another cassette, containing the desired sequence, in a second recombineering step applying negative selection.…”

Section: Introductionmentioning

confidence: 99%

Counter-selection recombineering of the baculovirus genome: a strategy for seamless modification of repeat-containing BACs

Westenberg

Soedling

Mann

et al. 2010

Nucleic Acids Research

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Recombineering is employed to modify large DNA clones such as fosmids, BACs and PACs. Subtle and seamless modifications can be achieved using counter-selection strategies in which a donor cassette carrying both positive and negative markers inserted in the target clone is replaced by the desired sequence change. We are applying counter-selection recombineering to modify bacmid bMON14272, a recombinant baculoviral genome, as we wish to engineer the virus into a therapeutically useful gene delivery vector with cell targeting characteristics. Initial attempts to replace gp64 with Fusion (F) genes from other baculoviruses resulted in many rearranged clones in which the counter-selection cassette had been deleted. Bacmid bMON14272 contains nine highly homologous regions (hrs) and deletions were mapped to recombination between hr pairs. Recombineering modifications were attempted to decrease intramolecular recombination and/or increase recombineering efficiency. Of these only the use of longer homology arms on the donor molecule proved effective permitting seamless modification. bMON14272, because of the presence of the hr sequences, can be considered equivalent to a highly repetitive BAC and, as such, the optimized method detailed here should prove useful to others applying counter-selection recombineering to modify BACs or PACs containing similar regions of significant repeating homologies.

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“…If under conditions of low efficiency recombination, the occurrence of false-positive clones will be promoted. The advantage of rpsL marker gene is that it is a highly stringent negative selectable marker that can eliminate or at least severely limit the growth of background nonmutated clones (Stavropoulos and Strathdee, 2001). Furthermore, most of the commonly used E. coli strains, including DH10B, HS996 and TOP10 (Invitrogen) carry a mutation in the rpsL gene conferring streptomycin resistance (Heermann et al, 2008;Drecktrah et al, 2010;Pelchovich et al, 2013), which makes the "rpsLcat-cassette" more efficient and facilitated.…”

Section: Discussionmentioning

confidence: 99%

Seamless stitching of biosynthetic gene cluster containing type I polyketide synthases using Red/ET mediated recombination for construction of stably co-existing plasmids

Zhao

Haina

et al. 2015

Gene

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Synergy between tetA and rpsL Provides High-Stringency Positive and Negative Selection in Bacterial Artificial Chromosome Vectors

Cited by 25 publications

References 35 publications

Rational optimization of tolC as a powerful dual selectable marker for genome engineering

Rational optimization of tolC as a powerful dual selectable marker for genome engineering

Counter-selection recombineering of the baculovirus genome: a strategy for seamless modification of repeat-containing BACs

Seamless stitching of biosynthetic gene cluster containing type I polyketide synthases using Red/ET mediated recombination for construction of stably co-existing plasmids

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