MOESM1 of Development of a novel strategy for robust synthetic bacterial promoters based on a stepwise evolution targeting the spacer region of the core promoter in Bacillus subtilis
Creators: name: Han, Laichuang
name: Cui, Wenjing
name: Suo, Feiya
name: Miao, Shengnan
name: Hao, Wenliang
name: Chen, Qiaoqing
name: Guo, Junling
name: Liu, Zhongmei
name: Zhou, Li
name: Zhou, Zhemin
Publisher: name: figshare
Year: 2019
Subjects: subject: Biochemistry
subject: Microbiology
subject: FOS: Biological sciences
subject: Genetics
subject: Molecular Biology
subject: Biotechnology
subject: Evolutionary Biology
subject: Biological Sciences not elsewhere classified
subject: Infectious Diseases
subject: FOS: Health sciences

description: Additional file 1: Figure S1. Transcriptional level analysis of strong and weak promoters. The transcriptional level of the 50 strongest promoters (A) and the 50 weakest promoters (B) were analyzed and shown by heatmap. Data were obtained from Gene Expression Omnibus (GEO) of National Center for Biotechnology Information Search database (NCBI) (GEO accession: GSE19831). Names of genes were labeled on the left of figures, and the serial numbers above means various growth time of samples. Figure S2. Construction and optimization of high-efficient transformation system in Bacillus subtilis. The competence transcription factor ComK was under control of xylose operon, which was integrated into the lacA site through double cross (A). Three factors: OD600 when added the xylose (B), concentration of xylose (C) and induction time (D), that influenceing transformation efficiency were optimized. Error bars are the s.d. of three independent experiments. Figure S3. Screening of pPML of PsrfA. (A) One hundred and eighty-five transformants were screened from the MPL. The fluorescence intensity (FI) of all the mutants was ranked from the highest level to the lowest level. Fourteen mutants were denoted on the corresponding columns, which were selected for furthure verification and characterization. (B) Sequence alignment of the mutants from the first round of evolution. (C) Characterization of the FI of the parental promoter and 14 mutants from the first round of evolution. The dotted line stands for the 2-fold threshold of the FI compared to the parental promoter. Figure S4. Screening of sPML of PsrfA. Fabrication and characterization of the four sMPLs, including MPL1, MPL2, MPL3, and MPL4, based on the random mutagenesis of the adjacent four nucleotides immediately upstream of the positions in the first-round evolution. The transformants obtained from the four MPLs based on the second round of evolution were screened in 96-well plates, and the gradient FI of these mutants in each MPL was ranked from the highest level to the lowest. The results were arranged by their templates, B2 (A), D11(B), F2 (C) and E9 (D), which were obtained from the first round of evolution. (E) Sequence alignment of the selected mutants from the MPL1 to MPL4 based on the second-round evolution. (F) Determination of the FI of the 12 mutants from the four sMPLs based on the second-round evolution. The dotted line stands for the 3-fold FI compared to the WT. Figure S5. Preliminary screening of sPML and pPML of PylbP. (A) Screening of one hundred and eighty-five mutants of PylbP from MPL generated by the first round of mutagenesis targeting the 3 bp sequence adjacent to the upstream of -10 box. The fluorescent intensity (FI) of all the mutants was ranked from the highest level to the lowest level. (B) and (C) Fabrication and screening of the two sMPLs generated by random mutagenesis targeting the 4 bp adjacent to the upstream of first-round mutated region of PylbP-G1 and PylbP-F9 by SETarSCoP. Figure S6. Engineering of PxylA through SETarSCoP. (A) One hundred and eighty-five transformants were screened from the MPL of PxylA. The FI of all the mutants was ranked from the highest level to the lowest level. (B) Characterization of the FI of the parental promoter and 6 mutants from the first round of evolution. (C) Sequence alignment of the mutants from the first round of evolution. (D) and (E) Fabrication and screening of the two sMPLs from PxylAâ D7 and PxylAâ D5. Table S1. Primers.