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Table 1 Plasmids used in this study

From: The length of ribosomal binding site spacer sequence controls the production yield for intracellular and secreted proteins by Bacillus subtilis

Name Description Source
pBSMul1 E. coliB. subtilis shuttle vector, PHpaII, secretion signal (SPlipA), ColE1 repB KmrAmpr [22]
pET22-sfGFP1-10 pET22b(+) containing the truncated sfGFP1-10 gen under control of PT7 [31]
pEBP41 E. coliB. subtilisP. putida shuttle vector, PT7, Pxyl, Gmr, GFPmut3 [29]
pEBP41-NdeI pEBP41 with deleted NdeI site within the GFPmut3 gene by QuikChange PCR® using the primers P1 and P2 This study
pBSMul1(SPBox)-SPxxx-cut-11 Signal peptide (SP) library based on pBSMul1 containing HindIII–XbaI inserts of the cutinase cut of F. solani pisi fused to a GFP11 tag and with different signal peptides (SPepr, SPpel, SPbsn) [31]
pBSMul1(SPBox)-SPxxx-EXLX1-11 Signal peptide (SP) library based on pBSMul1 containing HindIII–XbaI inserts of the swollenin EXLX1 of B. subtilis fused to a GFP11 tag and with different signal peptides (SPepr, SPpel, SPbsn) [31]
Standard expression plasmids (based on pBSMul1 with a 4 nucleotide spacer)
 pBS4nt-GFPmut3 pBSMul1 containing a 723 bp NdeI–XbaI GFPmut3 fragment amplified from pEBP41-NdeI with primers P3/P4 This study
 pBS4nt-GUS pBSMul1 containing a 1828 bp NdeI–XbaI (uidA) fragment amplified from E. coli DH5α genome with primers P5/P6 This study
 pBS4nt-SPepr-cut-11 pBSMul1 containing an 845 bp NdeI–XbaI SPepr-cut-11 fragment amplified from SP library using primers P7/P10 This study
 pBS4nt-SPpel-cut-11 pBSMul1 containing an 827 bp NdeI–XbaI SPpel-cut-11 fragment amplified from SP library using primers P8/P10 This study
 pBS4nt-SPbsn-cut-11 pBSMul1 containing an 848 bp NdeI–XbaI SPbsn-cut-11 fragment amplified from SP library using primers P9/P10 This study
 pBS4nt-SPepr-EXLX1-11 pBSMul1 containing an 824 bp NdeI–XbaI SPEXLX1-cut-11 fragment amplified from SP library using primers P7/P10 This study
 pBS4nt-SPpel-EXLX1-11 pBSMul1 containing an 806 bp NdeI–XbaI SPpel-EXLX1-11 fragment amplified from SP library using primers P8/P10 This study
 pBS4nt-SPbsn-EXLX1-11 pBSMul1 containing an 827 bp NdeI–XbaI SPbsn-EXLX1-11 fragment amplified from SP library using primers P9/P10 This study
Expression plasmids with extended spacers
 pBSxnt-Y Spacers ranging in length from 5 to 12 nucleotides (xnt) by insertion of adenosines for all above mentioned plasmids (Y: -GFPmut3, -GUS, -SPepr-cut-11, -SPpel-cut-11, -SPbsn-cut-11, -SPepr-EXLX1-11, -SPpel-EXLX1-11, -SPbsn-EXLX1-11) This study
 pBSxnt-SPepr-cut-11_start1 pBSxnt-SPepr-cut-11 with the second putative translational start codon ATG exchanged by ACG This study
 pBSxnt-SPepr-cut-11_start2 pBSxnt-SPepr-cut-11 with the first translational start codon ATG exchanged by ACG This study
Spacer sequence library
 pBS7nt-4N-GFPmut3 Spacer library based on pBS7nt-GFPmut3 containing the 7nt long spacer sequence 5′-NNNNCAT-3′ inserted by QuikChange PCR® and primers P27/P28 This study
 pBS7nt-4N-SPpel-cut-11 Spacer library based on pBS7nt-SPpel-cut-11 containing the 7nt long spacer sequence 5′-NNNNCAT-3′ inserted by QuikChange PCR® and primers P29/P30 This study