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CRISPR-associated transposons (CASTs) are RNA-guided mobile genetic elements that are widespread in bacterial genomes. Here, we describe the UltraCAST, a suicide vector with the Vibrio cholerae Type I-F CAST system and Golden Gate assembly sites with fluorescent protein gene dropouts for guide RNA and a mini-transposon cargo cloning. We show an example of UltraCAST genome editing by disrupting a gene in the chromosome of Serratia symbiotica CWBI-2.3T, a culturable relative of aphid endosymbionts. The UltraCAST can be used to flexibly insert DNA into specific genomic sites and facilitates testing this genome editing platform in non-model bacterial species that lack genetic tools.
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Standardizing cassette‐based deep mutagenesis by Golden Gate assembly
Abstract Protocols for the construction of large, deeply mutagenized protein encoding libraries via Golden Gate assembly of synthetic DNA cassettes employ disparate, system‐specific methodology. Here we present a standardized Golden Gate method for building user‐defined libraries. We demonstrate that a 25 μL reaction, using 40 fmol of input DNA, can generate a library on the order of 1 × 106members and that reaction volume or input DNA concentration can be scaled up with no losses in transformation efficiency. Such libraries can be constructed from dsDNA cassettes generated either by degenerate oligonucleotides or oligo pools. We demonstrate its real‐world effectiveness by building custom, user‐defined libraries on the order of 104–107unique protein encoding variants for two orthogonal protein engineering systems. We include a detailed protocol and provide several general‐use destination vectors.
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- Award ID(s):
- 2030221
- PAR ID:
- 10465325
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Biotechnology and Bioengineering
- Volume:
- 121
- Issue:
- 1
- ISSN:
- 0006-3592
- Format(s):
- Medium: X Size: p. 281-290
- Size(s):
- p. 281-290
- Sponsoring Org:
- National Science Foundation
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