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

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: mBio

Article Title: A new role for lipoproteins LpqZ and FecB in orchestrating mycobacterial cell envelope biogenesis

doi: 10.1128/mbio.02119-25

Figure Lengend Snippet: CRISPR/Cas9 mutants of conserved M. marinum lipoproteins. ( a ) The mycobacterial cell envelope and its main components: long-chain mycolic acids form the unique outer membrane (mycomembrane). Mycolic acids of the inner leaflet are covalently linked to a layer of arabinogalactan, which in turn is linked to the peptidoglycan. This macropolymer can be referred to as the mycolyl-arabinogalactan-peptidoglycan (mAGP) complex. The two most abundant glycolipids, lipoarabinomannan and its precursor lipomannan, are synthesized at the plasma membrane and can be transported to the outer membrane. Lipoproteins are a key group of membrane proteins that we believe play important roles for the integrity of the cell envelope. ( b ) Putative lipoprotein genes in M. tuberculosis and M. marinum and their conserved fraction. ( c ) Wild-type ( M. marinum WT pCRISPRx-Sth1-Cas9 L5 empty) and 74 successfully sequence-verified M. marinum lipoprotein CRISPR/Cas9 frameshift mutants were adjusted to OD 0.1 and spotted on 7H10 kanamycin plates in tenfold serial dilutions. This assay was performed once for all strains and repeated for strains showing abnormal growth.

Article Snippet: Similarly, construction of pCRISPRx-Sth1-Cas9-L5 (kana R , Addgene 140993), containing sgRNAs for the respective target genes , was carried out as described in ( ).

Techniques: CRISPR, Membrane, Synthesized, Clinical Proteomics, Sequencing

Journal: mBio

Article Title: A new role for lipoproteins LpqZ and FecB in orchestrating mycobacterial cell envelope biogenesis

doi: 10.1128/mbio.02119-25

Figure Lengend Snippet: Pooled susceptibility testing of lipoprotein mutants. ( a ) 74 lipoprotein CRISPR/Cas9 frameshift mutants and M. marinum wild-type strain (WT pCRISPRx-Sth1-Cas9-L5 empty) were pooled together and incubated for 7–14 days on 7H10 plates with vancomycin, rifampicin, augmentin (amoxicillin and clavulanic acid), or without antibiotics. Bacterial DNA was extracted, and the gRNA locus was amplified by PCR for next-generation sequencing (NGS). General growth ability was assessed by comparing gRNA counts of input pool and 7H10 plates without antibiotics. Antibiotic susceptibility was assessed by comparing gRNA counts from 7H10 plates and plates with the respective antibiotics. ( b ) Heatmap and clustering analysis of relative gRNA counts (log2 fold change) of 74 lipoprotein mutants and the WT strain. Data represent the mean of two independent experiments. Red indicates gRNA depletion, and blue indicates enrichment. Asterisks denote P -values as determined by a drugZ test (* P < 0.05, ** P < 0.01, *** P < 0.001). ( c ) Individual strains from ( b ) were adjusted to OD 0.1 and spotted on 7H10 plates containing different concentrations of the respective antibiotics. ( d ) Genetically complemented lpqZ fs mutant ( lpqZ fs + lpqZ HA) and complemented fecB fs mutant ( fecB fs + fecB ) were compared to WT and fs mutants containing the empty pSMT3 vector (EV) for antibiotic susceptibility as shown in panel ( c ). Representative pictures from two independent experiments.

Article Snippet: Similarly, construction of pCRISPRx-Sth1-Cas9-L5 (kana R , Addgene 140993), containing sgRNAs for the respective target genes , was carried out as described in ( ).

Techniques: CRISPR, Incubation, Amplification, Next-Generation Sequencing, Mutagenesis, Plasmid Preparation