Development of β-Lactamase as a Tool for Monitoring Conditional Gene Expression by a Tetracycline-Riboswitch in Methanosarcina acetivorans

Table 1

Plasmids and strains used in this study.

Plasmid	Constructiona/description/relevant genotype	Reference/source
pBR322	Source of bla gene	[37]
pET28a(+)	Source of 𝑘𝑎𝑛^𝑅 marker	Novagen
pAMG46	For integration into M. acetivorans  hpt  locus; it contains 𝑚𝑡𝑎𝐶1𝑃	[19]
pAMG48	For integration into M. acetivorans  hpt  locus; it contains 𝑚𝑐𝑟𝐵𝑃	Guss and Metcalf, unpublished
pWM321	E. coli/M. acetivorans shuttle vector	[38]
pMR56	1,176 kb AlwNI/XmnI 𝑘𝑎𝑛^𝑅 fragment from pET28a(+) blunted with Klenow, ligated into AhdI/ApaLI restricted and then blunted pAMG48	This study
pBlaN	806 bp NdeI/NotI restricted blaN PCR fragment (primers 1 and 3) ligated into NdeI/NotI restricted pMR56; bla without signal peptide encoding sequence (blaN)	This study
pBlaNFSP	845 bp NdeI/NotI restricted blaNFSP PCR fragment (primers 2 and 3) ligated into NdeI/NotI restricted pMR56;  bla  signal peptide encoding sequence of MA3061	This study
pMG3	1,007 bp 𝑚𝑡𝑎𝐶1𝑃 NdeI/BglII fragment from pAMG46 ligated into NdeI/BglII pBlaNFSP	This study
p0145NFSP	1,424 bp NdeI/BglII restricted 𝑚𝑡𝑚𝐶1𝑃 PCR fragment (primers 4 and 5) ligated into NdeI/BglII restricted, pBlaNFSP	This study
pSP64-tc-minimer	Source of tc-RS encoding sequence	[28]
pBlaN-prom	𝑚𝑐𝑟𝐵𝑃 in pBlaN deleted; used as negative control	This study
pBlaN_tcRS1	PCR fragment encoding tc-RS1 in the 𝑚𝑐𝑟𝐵𝑃 5′UTR ligated into BlgII/NdeI restricted pBlaN	This study
pBlaN_tcRS2	PCR fragment encoding tc-RS2 in the 𝑚𝑐𝑟𝐵𝑃 5′UTR ligated into BlgII/NdeI restricted pBlaN	This study
pBlaN_tcRS3	PCR fragment encoding tc-RS3 in the 𝑚𝑐𝑟𝐵𝑃 5′UTR ligated into BlgII/NdeI restricted pBlaN	This study
pBlaN_tcRS4	PCR fragment encoding tc-RS4 in the 𝑚𝑐𝑟𝐵𝑃 5′UTR ligated into BlgII/NdeI restricted pBlaN	This study
pBlaN_tcRS4a	PCR fragment encoding tc-RS4a in the 𝑚𝑐𝑟𝐵𝑃 5′UTR ligated into BlgII/NdeI restricted pBlaN	This study
pBlaN_tcRS4b	PCR fragment encoding tc-RS2 in the 𝑚𝑐𝑟𝐵𝑃 5′UTR ligated into BlgII/NdeI restricted pBlaN	This study
pBlaN_tcRS5	PCR fragment encoding tc-RS5 in the 𝑚𝑐𝑟𝐵𝑃 5′UTR ligated into BlgII/NdeI restricted pBlaN	This study

aDNA sequences and maps of all plasmids are available upon request; primers used are listed in Supplementary Table S1.