Construction of phr and argD Knockout PCR Products.
solfataricus argD gene with approximately 100 bp of the 5' and 3' flanking regions was amplified by PCR using the primers SsoargD-KpnI-F/PstI-R, which contain the KpnI and PstI restriction sites, respectively, and Premix Taq (Ex Taq Version 2.0; Takara Bio).
When pyrE or argD selectable marker was used for positive selection, colonies appearing on the plate were scored except for tiny colonies that might have been background.
To characterize the phenotypes of the DNA photolyase-deficient strain DP-1 ([DELTA]pyrE [DELTA]suaI [DELTA]phr) and argD deletion mutant SK-5 ([DELTA]pyrE [DELTA]suaI [DELTA]argD), UV sensitivity and agmatine auxotrophy were examined, respectively.
We disrupted the argD gene using the MONSTER to establish a robust unmarked gene disruption system, and a positive selectable marker in S.
To establish a selection marker system based on complementation of the argD gene, a S.
The usefulness of this technique was proven by unmarked gene knockout of the phr and argD genes.
Caption: Figure 5: In-frame deletion of argD via the MONSTER.