Construction of Fluorescent amilCP Reporter under recA Promoter

The recA promoter sequence was amplified from the wild-type MG1665 strain (gene accession number KEGG b2699) with the following primers recAamilXhoI_Fw (5′-ACTGCTCGAGAGAGAAGCCTGTCGGCAC-3′) and recAamilBamHI_Rv (5′-CACGGATCCCATTTTTACTCCTGTCATGCCG-3′) using the following amplification conditions: 30 s at 94 °C, 45 s at 50 °C and 50 s at 72 °C, 34 cycles. By using standard molecular biology techniques, the recA promoter fragment was ligated into the pQE30 plasmid (Qiagen, Hilden, Germany) which has the amilCP gene sequence (iGEM accession number BBa_K592009) cloned as BamHI-HindIII fragment (Tafoya-Ramírez et al. 2018 (link)). All restriction enzymes were purchased from New England Biolabs (Beverly, MA, USA). XL1-Blue MRF' (Agilent, Santa Clara, USA) and MG1665 E. coli strains were transformed with the resulting plasmid pRecA-AmilCP. The final cassette with the amilCP gene under the control of recA promoter was tested by colony PCR with the primers recAamilXhoI_Fw and amilColiHindIII_Rv (5′-ACCAAGCTTTCATTAAGCAACAACCGGC-3′) and the following amplification conditions: 30 s at 94 °C, 30 s at 47 °C and 1 min at 72 °C, 34 cycles. The PCR reactions were performed with GoTaq IX Green Master mix (Promega, WI, USA).