Additional file 1 of Use of the mCherry fluorescent protein to optimize the expression of class I lanthipeptides in Escherichia coli
Creators: name: Van Zyl, Winschau F
name: Van Staden, Anton D.
name: Dicks, Leon M T.
name: Trindade, Marla
Publisher: name: figshare
Year: 2023
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: Figure S1. Growth curves of E. coli BL21 strains NisSTldhSTldh and NisSTldhT7 expressing mCherry-NisA. Each vector with the respective promoter combinations was co-transformed with pACYCNisCi. The negative non-fluorescent control expression strain (Control) was transformed with empty pRSF-Duet 1 and pACYCNisCi. STldhSTldh = STldh promoter used for expression of mCherry-NisA and NisB; STldhT7 = STldh used for expression of mCherry-NisA and T7 promoter for NisB. Figure S2. Growth profiles of strain NisSTldhT7 expressing mCherry-NisA under various culturing and induction conditions. Cultures were grown in terrific broth supplemented without (A) and with (B) glucose (1% v/v) for 24 h. Control cultures were transformed with empty pRSF Duet-1 (without mCherry) and pACYC Duet-1 plasmids, while recombinant E. coli BL21 were transformed with pRSTldhCherryPrenisinNisB and pACYC-NisC plasmids. Culturing and induction conditions included: (−IPTG; −GlyM; −GlyIn) no induction; (−IPTG; +GlyM; −GlyIn) no IPTG induction with glycerol (0.5% v/v) added to pre-growth media; (−IPTG; −GlyM; +GlyIn) no IPTG induction but induced with glycerol (0.5% v/v); (+IPTG; +GlyM; −GlyIn) induced with 0.15 mM IPTG and glycerol added to pre-growth media; (+IPTG; −GlyM; +GlyIn) induced with 0.15 mM IPTG and glycerol; (+IPTG; −GlyM; −GlyIn) induced with 0.15 mM IPTG. IPTG = Thio-B-d-galactopyranoside; GlyM = glycerol presence in pre-growth media; GlyIn = glycerol used as inducer agent; +: added and −: not added. Figure S3. Growth profiles of strain NisSTldhT7 expressing mCherry-NisA in M9 minimal media, LB, BHI, TBG (TB containing 0.5% glycerol), TB-G (TB without 0.5% glycerol), A induced with 0.15 mM IPTG and 0.5% glycerol (v/v) and B un-induced. Figure S4. A pRSTc*nB backbone plasmid map. B Close up view of gene fragments. Relevant plasmid elements, promoters, genes and restriction sites are shown. Lanthipeptide genes are inserted at PstI/NotI restriction enzyme sites. Figure S5. A pRSSc*nB backbone plasmid map. B Close up view of gene fragments. Relevant plasmid elements, promoters, genes and restriction sites are shown. Lanthipeptide genes are inserted at PstI/NotI restriction enzyme sites. The T7 promoter in MCS2 was replaced with a second STldh promoter using NotI/BglII restriction enzyme sites. Figure S6. A pRSTcnA*nB and B pRSScnA*nB mCherry-nisA heterologous expression plasmids. C, D Linearized views of gene fragments shown in A and B, respectively. Relevant plasmid elements, promoters, genes and restriction sites are shown. Figure S7. A pRSTc*cB backbone plasmid map. B Close up view of gene fragments. Relevant plasmid elements, promoters, genes and restriction sites are shown. Lanthipeptide genes are inserted at PstI/NotI restriction enzyme sites. Figure S8. A pRSTccA*cB mCherry-PreClausA heterologous expression plasmid, B pClausCi clausC cyclase expression plasmid and C pClausDi clausD expression plasmid. D, E and F Close-up and linearized views of gene fragments shown in A, B and C, respectively. Relevant plasmid elements, promoters, genes and restriction sites are shown. Figure S9. A pRSTceA*cB backbone plasmid map. B Close-up view of gene fragments. Relevant plasmid elements, promoters, genes and restriction sites are shown. Lanthipeptide genes are inserted at PstI/NotI restriction enzyme sites. Figure S10. Differentiation of mCherry-NisA production based on color produced by host cells in spent expression media carrying the promoter arrangement described for strains (1) NisSTldhSTldh and (2) NisSTldhT7, respectively, after 20 h incubation. Figure S11. Mass spectrum of hNisP cleaved mCherry-NisA produced in E. coli. Activity zones are representative of cleaved and extracted samples before ESI–MS analysis. Figure S12. Visual representation of color differentiation of cell pellets produced when cultures are kept uninduced (off) and when induced (on). Table S1. Bacterial strains and plasmids used in this study. Table S2. Oligonucleotides utilized in this study.