<p>A) Murine DHFR is cloned into pET-Duet as two independent fragments consisting of the carboxyl-terminal region fused to three amino-terminal glycines in one open reading frame and amino terminus fused to an LPETG sort-tag in a second open reading frame. Endogenous methionine aminopeptidase (MAP) cleaves the initiating methionine to expose the terminal glycines. B) SrtA enzymatically ligates the two fragments to generate an active DHFR. Endogenous bacterial DHFR is inhibited by the prokaryotic specific trimethoprim. C) Initial culture conditions testing the requirement for SrtA in the DHFR complementation assay. D) Overnight growth of bacteria containing the various assay components in the presence and absence of trimethoprim was monitored using optical density at 600nm, OD<sub>600</sub>. Each trial was completed in duplicate. Cells carrying either the pET vector expressing only the split DHFR, or the pRSF vector driving expression of SrtA fail to grow in the presence of trimethoprim. Alternatively, bacteria expressing a positive control mDHFR (mDHFR-(PC)) with the internal LPETGG sequence, grow robustly in the absence of SrtA, but in the presence of trimethoprim. Similarly, bacteria expressing the split mDHFR gene along with SrtA also grow robustly following an overnight growth in trimethoprim. E) Growth of bacteria on LB-Agar plates containing Ampicillin, Kanamycin, IPTG, and trimethoprim. I) and III) BL21(DE3) cells containing pET-Duet C/N-mDHFR with empty pRSF vector, or II) and IV) BL21(DE3) cells containing pET-Duet C/N-mDHFR with pRSF-SrtA.</p
