Pbluescript sk

For production of pME4591, 1.2 kb of the flbB 5’ region (primers kt515/516), 1 kb of the flbB 3’ (primers kt517/518) and the phleoRM cassette were cloned into pBluescript SK(+), employing the Seamless Cloning and Assembly Kit (Invitrogen). The ΔflbB cassette was excised from the pME4591 with MssI and integrated into AGB551, AGB1007 and AGB1008, resulting in AGB1035, AGB1036 and AGB1037, respectively.

For production of pME4603, the vosA 5’ (primers SR11/12) and 3’ (primers SR13/14) regions and the natRM cassette were cloned into pBluescript SK(+), employing the Seamless Cloning and Assembly Kit (Invitrogen). The ΔvosA construct was excised from pME4603 with MssI and transformed into AGB551 and AGB1007, resulting in AGB1057 and AGB1058, respectively. pME4578 was integrated into AGB1057, resulting in AGB1059.

For production of pME4574, the veA 5’ (primers JG863/985) and 3’ (primers JG865/866) regions and the natRM cassette were cloned into pBluescript SK(+), employing the Seamless Cloning and Assembly Kit (Invitrogen). The ΔveA construct was excised from pME4574 with MssI and transformed into AGB551 resulting in AGB1066. The ΔsclB cassette from pME4575 was integrated into AGB1066, resulting in AGB1067.

For single, soluble enzymatic domains: a modified tACE construct, tACEΔ36NJ, that lacks the transmembrane region and unique 36 amino acid N-terminus (and therefore identical to the sACE C-domain; hereon referred to as C-domain) had been generated previously52 (link). A soluble form of the N-domain, consisting of amino acids 1 to 629 of somatic ACE (hereon referred to as N-domain), in vector pECE was a kind gift from Dr. Sergei Danilov UIC, Chicago) and was cloned into sequencing vector pBlueScript SK+ (Invitrogen) as previously described29 (link)53 (link).
The full length domain knock outs of sACE were a kind gift from Dr. Vincent Dive6 (link). A full length molecule with only an active N-domain active site (N-sACE) has zinc binding residues His361 and His365 converted to Lys. Similarly a full length molecule with only an active C-domain active site (C-sACE) possesses conversions of the corresponding His residues. The CC-sACE molecule consists of two C-domains joined by the sACE inter-domain linker region and was constructed as described previously50 (link). All full length constructs have the complete signal, transmembrane and stalk region corresponding to wild type sACE.

For production of pME4587, 1.5 kb of the abaA 5’ region (primers kt354/355), the phleoRM cassette and 1.4 kb of the abaA 3’ region (primers kt356/363) were cloned into pBluescript SK(+), employing the Seamless Cloning and Assembly Kit (Invitrogen). The ΔabaA cassette was excised from pME4587 with MssI and integrated into AGB551 and AGB1007, resulting in AGB1028 and AGB1029, respectively.

For production of pME4589, 1.7 kb of the brlA 5’ region (primers kt487/488), 1.2 kb of the brlA 3’ region (primers kt489/490) and the phleoRM cassette were cloned into pBluescript SK(+), employing the Seamless Cloning and Assembly Kit (Invitrogen). The ΔbrlA cassette was excised from pME4589 with MssI and integrated into AGB551 and AGB1007, resulting in AGB1031 and AGB1032, respectively.

For production of pME4595, 1.1 kb of the flbD 5’ region (primers kt523/524), 1.2 kb of the flbD 3’ region (primers kt525/526) and the phleoRM cassette were cloned into pBluescript SK(+), employing the Seamless Cloning and Assembly Kit (Invitrogen). The ΔflbD cassette was excised from pME4595 with MssI and integrated into AGB551, AGB1007 and AGB1008, resulting in AGB1043, AGB1044 and AGB1045, respectively.