CB414 cells were initially transformed with three compatible plasmids: a plasmid encoding a variant of FnCas12a, a plasmid encoding a CRISPR array or no-spacer control, and a plasmid encoding GFP under the control of the lacZ, lacIQ, or araB promoter. Overnight cultures of cells harboring the three plasmids were back-diluted to ABS600 ~0.01 in LB medium with ampicillin, kanamycin and chloramphenicol and the promoter’s inducer and shaken at 250 rpm at 37 °C until ABS600 reach ~0.2. Cultures were then diluted 1:25 in 1x phosphate-buffered saline (PBS) and analyzed on an Accuri C6 flow cytometer with C6 sampler plate loader (Becton Dickinson) equipped with CFlow plate sampler, a 488-nm laser, and a 530+/− 15-nm bandpass filter. GFP fluorescence was measured similar to prior work55 (link). Briefly, forward scatter (cutoff of 15,500) and side scatter (cutoff of 600) were used to eliminate non-cellular events. The mean value within FL1-H of 30,000 events within a gate set for E. coli were used for data analysis56 (link).
C6 sampler plate loader
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4 protocols using c6 sampler plate loader
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CRISPR-Cas12a Mediated Gene Expression Control
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Synthetic Promoters Drive deGFP Expression
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Evaluating CRISPR-Mediated Gene Repression in E. coli
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To demonstrate the gene repression efficiencies of the CRISPR arrays to be tested, the E. coli MG1655 derived strain CL471 was transformed with three compatible plasmids encoding a dCas9, a gfp-targeting CRISPR array and a deGFP reporter gene. For normalization purposes a positive control strain harbouring a non-spacer array and a negative control strain lacking the degfp encoding reporter plasmid was included. Overnight cultures of cells harbouring the above mentioned plasmids were back-diluted to OD600 of ~ 0.01 in LB medium with ampicillin, chloramphenicol and/or kanamycin and were incubated with shaking at 250 rpm, at 37°C until reaching an OD600 of 1. Cultures were then diluted 1:25 in 1× phosphate-buffered saline (PBS) and analysed on an Accuri C6 flow cytometer with C6 sampler plate loader (Becton Dickinson) equipped with CFlow plate sampler, a 488-nm laser, and a 530 ± 15-nm bandpass filter. Briefly, forward scatter (cut-off of 15,500) and side scatter (cut-off of 600) were used to eliminate non-cellular events. The mean value within FL1-H of at least 25,000 events within a gate set for E. coli was used for data analysis. For each experiment, triplicate or quadruplicate cultures were measured, and their standard deviation was indicated as the error bar.
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Synthetic Promoters Drive deGFP Expression
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Plasmids encoding deGFP driven by different synthetic constitutive promoters in the J23100 series (J23119, J23100, J23116 and J23103) along with the base editor, and their corresponding target plasmids were transformed into E. coli MG1655-derived strain CB414. For the flow cytometry analysis, after 3 h induction, cultures were sampled and diluted 1:100 into 1× PBS and analyzed on an Accuri C6 flow cytometer with C6 sampler plate loader (Becton Dickinson) equipped with CFlow plate sampler, a 488-nm laser and a 530 ± 15-nm bandpass filter. Lower cutoff values of 11,500 and 500 were used for forward scatter (FSC-H) and side scatter (SSC-H), respectively. At least 50,000 gated events were collected for each measurement. Bulk sequencing, colony sequencing and single-cell sequencing were conducted as described in the section Heterologous transcript recording with slight modifications. For bulk sequencing, the induction was performed for 7 h. For bulk sequencing and colony sequencing, primers CJpr1376 and CJpr0299 were used to amplify the edited regions, and CJpr1376 was used for sequencing. For single-cell sequencing, primers CJpr2355 and CJpr2356 were used to amplify the edited regions, and CJpr2355 was used for sequencing.
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