Fig 2.
Construction of plasmids pGKE11T, pGAM46, pTK19, pΔpyrF, and pΔpyrR. pGKE11T was constructed from pUC19 by introduction of oriT and restriction sites. pGAM46 was constructed from pGKE11T by introduction of the pyrF marker and the GK0707 upstream (GK0707′) and downstream (′GK0707) fragments. pTK19 was constructed by introduction of the TK101 marker into pGKE11T and was used for construction of pΔpyrF and pΔpyrR. pΔpyrF contains pyrF upstream (pyrF up) and downstream (pyrF dw) regions to form the ΔpyrF sequence. pΔpyrR contains pyrR upstream (pyrR up) and downstream (pyrR dw) regions to form the ΔpyrR sequence. The ampicillin resistance gene (Ampr), the replicon from pUC19 (ori), and the conjugative transfer origin from pRK2013 (oriT) are shown, along with the relevant restriction sites. “MCS” denotes the multiple cloning sites of pUC19.