Mobile-crispri plasmids and related methods

A method of constructing a knockdown library of bacterial cells, comprising:

• (a) cloning a plurality of nucleotide sequence encoding single guide RNA (sgRNAs) targeting a plurality of genes of interest into a plurality of vectors having at least 90% identity to SEQ ID NO:19, thereby generating a vector library;
• (b) transforming a plurality of bacterial cells comprising RP4 transfer machinery with the vector library, wherein the bacterial cells are auxotrophic and require presence of a nutritional substance for growth, and wherein the bacterial cells comprise a gene whose expression permits a conditional original of replication of a vector having at least 90% identity to SEQ ID NO:19 to be functional;
• (c) contacting under conditions allowing for mating (i) the plurality of transformed bacterial cells from step (b), (ii) a plurality of engineered bacterial cells comprising a transposase plasmid, wherein the engineered bacterial cells are auxotrophic and require the presence of the nutritional substance for growth, and (iii) a plurality of recipient bacterial cells not requiring the presence of the nutritional substance for growth;
• and
• (d) growing the contacted bacterial cells of step (c) in or on a medium comprising ampicillin and not including the nutritional substance thereby generating the knockdown library of bacterial cells.

A method of constructing a knockdown library of bacterial cells, comprising:

• (a) cloning a plurality of nucleotide sequences encoding single guide RNAs (sgRNAs) into a plurality of vectors having at least 90% identity to SEQ ID NO:18, thereby generating a vector library;
• (b) transforming a plurality of bacterial cells with the vector library, wherein the bacterial cells comprise conjugation genes and a gene whose expression permits a conditional original of replication of a vector having at least 90% identity to SEQ ID NO:18 to be functional;
• (c) inducing expression of the conjugation genes in the transformed bacterial cells, thereby promoting excision of vector modules comprising the nucleotide sequences encoding the sgRNAs from the vector library;
• (d) after the inducing, contacting transformed bacterial cells with a plurality of recipient bacterial cells under conditions allowing for mating of the transformed bacterial cells and the recipient bacterial cells, thereby resulting in transfer of the sgRNAs into the recipient bacterial cells;
• and
• (e) growing the contacted bacterial cells in or on a medium comprising ampicillin, thereby generating the knockdown library of bacterial cells.