Dataset Information

A bacterial dual positive and negative selection system for dCas9 activity.

ABSTRACT: The engineering of switchable or activatable dCas9 proteins would benefit from a single system for both positive and negative selection of dCas9 activity. Most systems that are used to interrogate dCas9 libraries use a fluorescent protein screen or an antibiotic selection for active dCas9 variants. To avoid some of the limitations of these systems, we have developed a single system capable of selecting for either active or inactive dCas9 variants. E. coli expressing active dCas9 variants are isolated in the positive selection system through growth in the presence of ampicillin. The negative selection can isolate cells lacking dCas9 activity through two separate mechanisms: growth in M9 minimal media or growth in media containing streptomycin. This system is capable of enriching for rare dCas9 variants up to 9,000-fold and possesses potential utility in directed evolution experiments to create switchable dCas9 proteins.

SUBMITTER: Spisak S

PROVIDER: S-EPMC9165777 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Publications

A bacterial dual positive and negative selection system for dCas9 activity.

Spisak Shaun S O'Brien Brett B Ostermeier Marc M

PloS one 20220603 6

The engineering of switchable or activatable dCas9 proteins would benefit from a single system for both positive and negative selection of dCas9 activity. Most systems that are used to interrogate dCas9 libraries use a fluorescent protein screen or an antibiotic selection for active dCas9 variants. To avoid some of the limitations of these systems, we have developed a single system capable of selecting for either active or inactive dCas9 variants. E. coli expressing active dCas9 variants are iso ...[more]

PMID: 35657952

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Project description:Plasmids play an important role in bacterial evolution by transferring niche-adaptive functional genes between lineages, thus driving genomic diversification. Bacterial genomes commonly contain multiple, coexisting plasmid replicons, which could fuel adaptation by increasing the range of gene functions available to selection and allowing their recombination. However, plasmid coexistence is difficult to explain because the acquisition of plasmids typically incurs high fitness costs for the host cell. Here, we show that plasmid coexistence was stably maintained without positive selection for plasmid-borne gene functions and was associated with compensatory evolution to reduce fitness costs. In contrast, with positive selection, plasmid coexistence was unstable despite compensatory evolution. Positive selection discriminated between differential fitness benefits of functionally redundant plasmid replicons, retaining only the more beneficial plasmid. These data suggest that while the efficiency of negative selection against plasmid fitness costs declines over time due to compensatory evolution, positive selection to maximize plasmid-derived fitness benefits remains efficient. Our findings help to explain the forces structuring bacterial genomes: coexistence of multiple plasmids in a genome is likely to require either rare positive selection in nature or nonredundancy of accessory gene functions among the coexisting plasmids.IMPORTANCE Bacterial genomes often contain multiple coexisting plasmids that provide important functions like antibiotic resistance. Using lab experiments, we show that such plasmid coexistence within a genome is stable only in environments where the function they encode is useless but is unstable if the function is useful and beneficial for bacterial fitness. Where competing plasmids perform the same useful function, only the most beneficial plasmid is kept by the cell, a process that is similar to competitive exclusion in ecological communities. This process helps explain how bacterial genomes are structured: bacterial genomes expand in size by acquiring multiple plasmids when selection is relaxed but subsequently contract during periods of strong selection for the useful plasmid-encoded function.

Dataset Information

A bacterial dual positive and negative selection system for dCas9 activity.

Publications

A bacterial dual positive and negative selection system for dCas9 activity.

Similar Datasets

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