Constructing Suppressor Plasmids for recA/polA

The 3.4 kb DNA fragment of srp, a suppressor of recA polA lethality^{6 (link)}, was cloned into the BamHI site on pHSG576^{30 (link)}. The resultant plasmids were termed pSRO1. pSRO1, pSROΔhslO, and pSrpC were described previously^{6 (link)}. To construct pSROΔyrfG, pAQ10917 was digested with NsiI and BsmBI and self-ligated. In self-ligations, 1 μg digested DNA was blunted using 1 unit of T4 DNA polymerase (New England Bio Labs, Ipswich, MA, USA) supplemented with 1 mM dNTPS in NEBbuffer 1.1 at 12 °C for 20 min in 20 μL reaction, followed with heat inactivation at 75 °C for 30 min. Ethanol-precipitated blunted DNA was then ligated with 250 units of T4 DNA ligase (Nippon gene, Tokyo, Japan) in manufacturer-supplemented reaction buffer at 16 °C for 12 h. Then, a BamHI DNA fragment with a yrfG deletion was cloned into pHSG576. Subsequently, an spc cassette was cloned into the EcoRI site. The resultant plasmid was called pSROΔyrfG. To construct pSROΔhslRO, pAQ10917 was digested with BsmBI and BstEII and self-ligated. Subsequently, a BamHI DNA fragment containing a hslR to hslO deletion was cloned into pHSG576. Then, an spc cassette was cloned into the EcoRI site. The resultant plasmid was called pSROΔhslRO.