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Table 2 Strains and plasmids used in this study

From: Establishment of a functional system for recombinant production of secreted proteins at 50 °C in the thermophilic Bacillus methanolicus

Strain

Description

Reference

Escherichia coli DH5α

General cloning host, F-thi-1 endA1 hsdR17(r-, m-) supE44 _lacU169 (_80lacZ_M15) recA1 gyrA96 relA1

Stratagene/Genomics Agilent

Bacillus methanolicus MGA3

Wild type strain

ATCC53907

B. licheniformis MW3

Bacillus licheniformis DSM13 (ΔhsdR1, ΔhsdR2)

[100]

B. subtilis 168

 

Wild type strain

ATCC23857

Geobacillus stearothermophilus 10

Wild type strain

DSM13240

Plasmid

Strain abbreviationa

Description

Reference

pBV2xp

EV

KmR and ApR; pHCMC04 derivative, gene expression under the control of the inducible xylose promoter from B. megaterium, theta replicating

[83]

Protease gene carrying strains/plasmids

 pBV2xp-aprBl

spPBl-aprBl

KmR and ApR; pBV2xp derivative for expression of apr from B. licheniformis with native signal peptide (locus tag BLI_RS05490)

This study

 pBV2xp-aprBs

spPBs-aprBs

KmR and ApR; pBV2xp derivative for expression of aprE from B. subtilis with native signal peptide (locus tag GP2222_11200)

This study

 pBV2xp-aprGs

spPGs-aprGs

KmR and ApR; pBV2xp derivative for expression of aprGs from G. stearothermophilus with native signal peptide (locus tag GT50_00190)

This study

α-amylase gene carrying strains/plasmids

 pBV2xp-spBl-amyBl

spBl-amyBl

KmR and ApR; pBV2xp derivative for expression of amyL from B. licheniformis with native signal peptide (locus tag BL00499)

This study

 pBV2xp-spBs-amyBs

spBs-amyBs

KmR and ApR; pBV2xp derivative for expression of amyE from B. subtilis with native signal peptide (locus tag BSU03040)

This study

 pBVxp-spGs-amyGs

spGs-amyGs

KmR and ApR; pBV2xp derivative for expression of amyS from G. stearothermophilus with native signal peptide (locus tag GT50_07800)

This study

 pBV2xp-spBm-amyBm

spBm-amyBm

KmR and ApR; pBV2xp derivative for expression of amy from B. methanolicus with native signal peptide (locus tag BMMGA3_04345)

This study

 pBV2xp-spGs-amyBs

spGs-amyBs

KmR and ApR; pBV2xp derivative for expression of amyE from B. subtilis with α-amylase signal peptide from G. stearothermophilus

This study

 pBVxp-spGs-amyBl

spGs-amyBl

KmR and ApR; pBV2xp derivative for expression of amyL from B. licheniformis with α-amylase signal peptide from G. stearothermophilus

This study

sfGFP gene carrying strains/plasmids

 sfGFP-pBAD

 

AmR; pBAD/His derivative for expression of sfGFP under the control of ParaBAD

sfGFP-pBAD was a gift from Michael Davidson and Geoffrey Waldo (Addgene plasmid # 54519) [82]

 pBV2xp-sfGFP

sp0-sfGFP

KmR and ApR; pBV2xp derivative for expression of sfGFP from sfGFP-pBAD under control of the xp promoter

This study

 pBV2xp-spGs-sfGFP

spGs-sfGFP

KmR and ApR; pBV2xp-sfGFP derivative with amylase signal peptide from G. stearothermophilus

This study

 pBV2xp-spBl-sfGFP

spBl-sfGFP

KmR and ApR; pBV2xp-sfGFP derivative with amylase signal peptide from B. licheniformis

This study

 pBV2xp-spBs-sfGFP

spBs-sfGFP

KmR and ApR; pBV2xp-sfGFP derivative with amylase signal peptide from B. subtilis

This study

 pBV2xp-spBm-sfGFP

spBm-sfGFP

KmR and ApR; pBV2xp-sfGFP derivative with amylase signal peptide from B. methanolicus

This study

  1. aAbbreviation used for B. methanolicus strain MGA3 carrying the listed plasmid. spP: signal peptide preceding protease; sp: signal peptide preceding α-amylase. Example: Strain spBl-amyBl carries plasmid with (α-amylase) signal peptide from B. licheniformis, and α-amylase gene from B. licheniformis
  2. KmR kanamycin resistance marker, ApR ampicillin resistance marker