NotI-HF®

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	The extracted plasmid and M13-NotI-Kan vector were digested by KpnI-HF and NotI-HF.	The desired products were isolated by gel electrophoresis and purified with QIAquick Gel Extraction Kit. The g3p-N homologue was ligated into the M13-NotI-Kan vector using T4 DNA ligase.

Protocol tips
The extracted plasmid and M13-NotI-Kan vector were digested by KpnI-HF and NotI-HF.
Downstream tips
The desired products were isolated by gel electrophoresis and purified with QIAquick Gel Extraction Kit. The g3p-N homologue was ligated into the M13-NotI-Kan vector using T4 DNA ligase.

Publication protocol

The RBP of M13 (g3p) was engineered to replace its N-terminal domain (g3p-N) with the homologous domain from a phage having specificity toward the target bacterial species (Table 1). A plasmid containing the g3p-N homologue of the source phage flanked by KpnI and NotI restriction sites (see the Supporting Information) was synthesized (IDT) and transformed into Mix and Go competent E. coli cells. The cells were grown in LB media with ampicillin (10 μg mL–1), and the plasmid was isolated using the QIAprep Spin Miniprep Kit. The M13-NotI-Kan phage vector, in which a NotI restriction site was introduced between the N- and C-terminal domains of M13, was prepared previously.12 The extracted plasmid and M13-NotI-Kan vector were digested by KpnI-HF and NotI-HF. The desired products were isolated by gel electrophoresis and purified with QIAquick Gel Extraction Kit. The g3p-N homologue was ligated into the M13-NotI-Kan vector using T4 DNA ligase. The recombinant plasmid was then transformed into Mix and Go competent cells, which were plated on LB with kanamycin (50 μg mL–1) and IPTG (0.1 M) and incubated at 37 °C overnight. A single colony was selected and cultured in LB media with kanamycin (50 μg mL–1) and IPTG (0.1 M) in a shaking incubator at 37 °C overnight. The recombinant plasmid was isolated using a QIAprep Spin Miniprep Kit. The inserted gene in the phage vector was amplified by PCR (forward primer: 5′- TTTGGAGCCTTTTTTTTGGAGATTTTCAAC-3′; reverse primer: 5′- CACCACCAGAGCCTGC-3′; PCR conditions: 95 °C for 3 min, then 11 cycles of 95 °C for 30 s, 52.3 °C for 30 s and 72 °C for 60 s) and Sanger sequenced (UC Berkeley core facility) to confirm the sequence of the chimeric phage genome. To produce chimeric phages, a colony containing the chimeric phage genome was used to inoculate a liquid culture and shaken overnight at 37 °C. The phages were precipitated and purified by the double-polyethylene glycol (PEG) precipitation method described above.

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