lentiviral crispr cas9 expression plasmid pcrispr paxillin  (Addgene inc)

Journal: bioRxiv

Article Title: ESBL plasmid compatibility with the surrounding microbial community influences ESBL gene survival under CRISPR-antimicrobial targeting

doi: 10.1101/2025.04.15.648872

Figure Lengend Snippet: (a) the CRICON delivery strains used in this study. A control strain (HB-CC) harbors a mobilized CRISPR plasmid without a spacer, while a spacer-containing strain (HB-S) harbors a mobilized CRISPR plasmid with a spacer targeting bla CTX-M-15 gene. Both strains harbor RP4-Λι bla TEM plasmid as delivery plasmid mediating the conjugation. (b) The experimental design of the microcosm setups. Four CRICON-targeted E. coli -ESBL strains (E4, E6, E8, and E11) were tested individually in four different treatments (CC = CRICON control – without migration, S = CRICON spacer – without migration, CCM = CRICON control – with migration, and SM = CRICON spacer – with migration). CRICON strain was added daily into the communities. In the migration (M) treatments, plasmid-free E. coli HB101 was added daily. The microcosms also contained three other bacterial strains ( Citrobacter freundii 4B24, Klebsiella sp. 4B41, and K. pneumoniae DSM681) as a core community. The cultures were refreshed daily (1:100) for 10 cycles (N = 3 replicates/treatment) with an antibiotic pulse of cephalothin after cycle 5 to select for the ESBL gene.

Article Snippet: The CRISPR plasmid (pCRISPR) was previously constructed from plasmid pCas9 (a gift from Luciano Marraffini, Addgene plasmid # 42876), encoding chloramphenicol resistance gene for selection and a CRISPR-Cas system including Cas9 endonuclease and a programmable CRISPR array site, mobilized via cloning of the oriT site ( ).

Techniques: Control, CRISPR, Plasmid Preparation, Conjugation Assay, Migration

Journal: Biotechnology for biofuels

Article Title: CRISPR-mediated multigene integration enables Shikimate pathway refactoring for enhanced 2-phenylethanol biosynthesis in Kluyveromyces marxianus.

doi: 10.1186/s13068-020-01852-3

Figure Lengend Snippet: Fig. 2 Markerless CRISPR-Cas9-mediated multigene integration in K. marxianus CBS 6556. a Schematic representation of the three-primer colony PCR test and example gels for single and multiple gene integrations into the ABZ1 locus. The length of the up- and down-stream homology regions is 700 bp. b Integration efficiency of single (EGFP), dual (EGFP, DSRED), and triple (EGFP, YFP, and DSRED) genes. c Relative fluorescence intensity of EGFP expressed from different loci as measured by fluorescence microplate reader. d Normalized expression level of fluorescent proteins when produced individually or simultaneously with one or two additional fluorescent proteins. All experiments were performed in biological triplicate. Bars represent the mean, while error bars represent the standard deviation

Article Snippet: CRISPR plasmids (pCRISPR) were constructed using pIW601 (Addgene ID 98907) linearized at PspXI and reassembled with a 60 bp insert containing 20 bp upstream and downstream homology as well as the 20 bp target sequence by Gibson assembly.

Techniques: CRISPR, Fluorescence, Expressing, Produced, Standard Deviation