addgene 71 addgene plasmid  (Addgene inc)

Journal: Nucleic acids research

Article Title: Defining serine tRNA knockout as a strategy for effective repression of gene expression in organisms with a recoded genome.

doi: 10.1093/nar/gkae1266

Figure Lengend Snippet: Figure 1. serU (tRNA Ser(CGA) ) decodes TCA codons. The tRNA–codon interaction map of ( A ) wild-type E. coli (dashed orange line represents the proposed serU decoding of TCA described here), ( B ) Syn61 T , with serT (encodes tRNA Ser(UGA) ) knocked out, ( C ) Syn61 U , with serU (encodes tRNA Ser(CGA) ) knocked out, and ( D ) Syn61 3 , with both serT and serU knocked out. In all Syn61 strains, every TCA and TCG codon has been removed from the genome via synonymous mutations, indicated by the lack of codon boxes. ( E ) A plasmid encoding a KanR gene is transformed into different Syn61 tRNA knockout variants (vertical lines represent the presence of TCA codons in the KanR CDS). Given the widely reported interaction map in panel (A), transformation of this plasmid into Syn61 T and Syn61 3 is expected to not yield transformants. ( F ) Mean + standard deviation of colony forming units (transformants) resulting from this transformation reaction ( n = 3). ( G ) Representative images of the transformant plates quantified in panel (F). Syn61 3 containing ( H ) helper plasmids that encode wild-type serU (with anticodon CGA) or a form of serU where the anticodon has been mutated to that of serV (GCU) were transformed with the same KanR plasmids as in panels (F)–(G). ( I ) Mean + standard deviation of transformants resulting from this transformation reaction ( n = 3). Created in BioRender. Voorhees, P. (2025) https:// BioRender.com/ v18p889 .

Article Snippet: If he flanked sequence is not present and the attL sequence is ligned with ≥99% identity, the read is considered to have ndergone recombination. esources table Reagent or resource Source Identifier acterial and virus strains Syn61 Addgene Cat#174513 Syn61 3 Addgene Cat#174514 Syn61 T This paper Syn61 U This paper hemicals, peptides and recombinant proteins Streptomycin sulfate Sigma–Aldrich Cat#S6501 Chloramphenicol Sigma–Aldrich Cat#R4408-10ML l -(+)-Arabinose Sigma–Aldrich Cat#A3256-100G Kanamycin sulfate Sigma–Aldrich Cat#60615-5G ritical commercial assays NEBuilder HiFi DNA Assembly Master Mix NEB Cat#E2621L NEBridge Golden Gate Assembly Kit (BsaI-HF v2) NEB Cat#E1601L Monarch Genomic DNA Purification Kit NEB Cat#T3010L Nano-Glo Luciferase Assay System Promega Cat#N1130 Zyppy Plasmid Miniprep Zymo Research Cat#D4036 eposited data Raw whole plasmid sequencing reads This paper PRJNA1066256 ecombinant DNA pKD46 NovoPro Bioscience V007013 See Supplementary Table S3 for plasmids generated in this study This paper See Supplementary Table S4 for synthesized DNA sequences This paper oftware and algorithms Recombination Analyzer This paper doi:10.5281 / zenodo.10525174 esults erU (tRNA Ser(CGA) ) is a weak decoder of TCA odons t is widely reported that serU (tRNA Ser(CGA) ) only decodes CG and that TCA is only decoded by serT (tRNA Ser(UGA) ) 11–15 ).

Techniques: Plasmid Preparation, Transformation Assay, Knock-Out, Standard Deviation

Journal: Nucleic acids research

Article Title: Defining serine tRNA knockout as a strategy for effective repression of gene expression in organisms with a recoded genome.

doi: 10.1093/nar/gkae1266

Figure Lengend Snippet: Figure 2. Compressed codons mediate repression in select GROs only if sufficient codons are present. ( A ) Mean log 10 luminescence intensity across a panel of Syn61 tRNA knockout variants ( y -axis) transformed with a NanoLuc gene containing varying numbers of TCA and TCG codons in the coding sequence ( x -axis) ( n = 2). ( B ) Ov ervie w of the recombination assay. A panel of plasmids encoding (i) the Bxb1 site-specific recombinase with varying numbers of TCA and TCG codons in the coding sequence and (ii) the Bxb1 attP and attB sites, which are oriented such that their recombination by the SSR (which forms an attL site) e x cises ∼800 bp from the plasmid, is transformed into Syn61 tRNA knockout variants. Plasmid DNA is purified from the entire population of transformants and sequenced by long-read nanopore sequencing. The reads are then analyzed for signatures of recombination and the percent of sequenced plasmid molecules that ha v e undergone recombination is calculated. ( C ) Mean + standard deviation of percent recombination from the protocol described in panel (B) ( n = 2). Created in BioRender. Voorhees, P. (2025) https:// BioRender.com/ v26h108 .

Article Snippet: If he flanked sequence is not present and the attL sequence is ligned with ≥99% identity, the read is considered to have ndergone recombination. esources table Reagent or resource Source Identifier acterial and virus strains Syn61 Addgene Cat#174513 Syn61 3 Addgene Cat#174514 Syn61 T This paper Syn61 U This paper hemicals, peptides and recombinant proteins Streptomycin sulfate Sigma–Aldrich Cat#S6501 Chloramphenicol Sigma–Aldrich Cat#R4408-10ML l -(+)-Arabinose Sigma–Aldrich Cat#A3256-100G Kanamycin sulfate Sigma–Aldrich Cat#60615-5G ritical commercial assays NEBuilder HiFi DNA Assembly Master Mix NEB Cat#E2621L NEBridge Golden Gate Assembly Kit (BsaI-HF v2) NEB Cat#E1601L Monarch Genomic DNA Purification Kit NEB Cat#T3010L Nano-Glo Luciferase Assay System Promega Cat#N1130 Zyppy Plasmid Miniprep Zymo Research Cat#D4036 eposited data Raw whole plasmid sequencing reads This paper PRJNA1066256 ecombinant DNA pKD46 NovoPro Bioscience V007013 See Supplementary Table S3 for plasmids generated in this study This paper See Supplementary Table S4 for synthesized DNA sequences This paper oftware and algorithms Recombination Analyzer This paper doi:10.5281 / zenodo.10525174 esults erU (tRNA Ser(CGA) ) is a weak decoder of TCA odons t is widely reported that serU (tRNA Ser(CGA) ) only decodes CG and that TCA is only decoded by serT (tRNA Ser(UGA) ) 11–15 ).

Techniques: Knock-Out, Transformation Assay, Sequencing, Recombination Assay, Plasmid Preparation, Purification, Nanopore Sequencing, Standard Deviation