A synthetic system links FeFe-hydrogenases to essential E. coli sulfur metabolism

Table 1 Plasmids used in this study

Name	Constructs		Backbone	Resistance	Source
Hydrogenase activity *in vivo* ^A
pET.mp1	ca HydE	ca HydA	pETDuet-1	Ampicillin	Matthew Posewitz[31]
pCDF.mp2	ca HydF	ca HydG	pCDFDuet-1	Spectinomycin	Matthew Posewitz[31]
pACYC.ew3	da PFOR		pACYCDuet-1	Chloramphenicol	This work
pACYC.ew4	da PFOR	so FD	pACYCDuet-1	Chloramphenicol	This work
pACYC.ew5	da PFOR	zm FD	pACYCDuet-1	Chloramphenicol	This work
pACYC.ew6	da PFOR	cr FD	pACYCDuet-1	Chloramphenicol	This work
pACYC.ew7	da PFOR	ca FD	pACYCDuet-1	Chloramphenicol	This work
pACYC.ew8	so FD		pACYCDuet-1	Chloramphenicol	This work
pACYC.ew9	zm FD		pACYCDuet-1	Chloramphenicol	This work
pACYC.ew10	cr FD		pACYCDuet-1	Chloramphenicol	This work
pACYC.ew11	ca FD		pACYCDuet-1	Chloramphenicol	This work
FNR-supported growth ^B
pCDF.ew12	zm FNR		pCDFDuet-1	Spectinomycin	This work
pACYC.ew13	so FD	zm SIR	pACYCDuet-1	Chloramphenicol	This work
pACYC.ew14	zm FD	zm SIR	pACYCDuet-1	Chloramphenicol	This work
pACYC.ew15	cr FD	zm SIR	pACYCDuet-1	Chloramphenicol	This work
pACYC.ew16	ca FD	zm SIR	pACYCDuet-1	Chloramphenicol	This work
pACYC.ew17	zm SIR		pACYCDuet-1	Chloramphenicol	This work
Hydrogenase-supported growth and selection ^C
pACYC.ew18	cr HydEF	cr HydG	pACYCDuet-1	Chloramphenicol	This work
pET.ew19	so FD	zm SIR	pETDuet-1	Ampicillin	This work
pCDF.ew20	cr HydA		pCDFDuet-1	Spectinomycin	This work
pCDF.ew21	ca HydA		pCDFDuet-1	Spectinomycin	This work
pCDF.ew22	cs HydA		pCDFDuet-1	Spectinomycin	This work

Duet vector backbones (Novagen) were used for all protein expression. Complete vector sequences are provided in additional file 1. A) Plasmids mp1-ew11 were used to generate PFOR-driven hydrogenase activity data for Figure 4. B) Plasmids ew12-ew17 were used to assess knockout strain insulation for Figure 2. They were also used to measure ferredoxin performance in Figure 4. C) Plasmids ew18-ew22 facilitated the hydrogenase O₂ tolerance measurements in Figure 3. They were also used in the hydrogenase-supported growth curves in Figure 5 and for the genetic selection.

ISSN: 1754-1611