MOESM1 of A seamless and iterative DNA assembly method named PS-Brick and its assisted metabolic engineering for threonine and 1-propanol production
Creators: name: Liu, Shuwen
name: Xiao, Haihan
name: Zhang, Fangfang
name: Lu, Zheng
name: Zhang, Yun
name: Deng, Aihua
name: Li, Zhongcai
name: Yang, Cui
name: Wen, Tingyi
Publisher: name: figshare
Year: 2019
Subjects: subject: Biochemistry
subject: Microbiology
subject: FOS: Biological sciences
subject: Genetics
subject: Molecular Biology
subject: Biological Sciences not elsewhere classified
subject: Infectious Diseases
subject: FOS: Health sciences

description: Additional file 1: Figure S1. The cutting efficiency of BmrI (A) and MlyI (B) on vector pOB and pOM was tested through electrophoresis with cutting time ranging from 15 min to 180 min. Figure S2. The workflow of PS-Brick assembly. The insertion part F1 was PCR product amplified by Ex-Taq DNA Polymerase or KAPA high-fidelity polymerase. The PCR product was gel-purified, digested with IIP RE only and then column-purified for ligation. The base plasmids containing entrance REs site were firstly cleaved by Type IIS RE for 15 min, and the linearized vectors were separated by electrophoresis, gel purified, and then recovered for the second digestion by Type IIP RE for 15 min. The double REs-digested vectors and Type IIP RE-digested PCR products were heat-inactivated at 60°C for 20 min and then were column-purified for ligation together. After ligation reaction for 15 min, the ligation mix was transformed into E. coli DH5α competent cells. The chemically competent E. coli cells mixed with DNA were then placed on ice for 30 min, heat shock at 42°C for 90 s, chilled on ice for another 3 min, incubated at 37°C for 1 h. Finally, the mixture was spread onto selection plate and incubated overnight at 37 °C. Figure S3. Transcriptional analysis of the ppc, aspA, aspC, asd and pntAB genes by real-time RT-PCR. The control strain was E. coli K12 MG1655/ pACYC184-thrA433pheBC, and the engineering strains were E. coli K12 MG1655 strain harboring plasmids pACYC184-thrA433pheBC-ppc/aspA/aspC/asd/pntAB, respectively. Data shown are mean values from three biological replicates, and the standard deviations are presented. Symbol “*” denotes the relative expression level of the gene overexpressed in plasmid. Take Panel A, quantitative PCR of ppc gene for all the strains, as an example, “*” denotes the strain overexpressed ppc gene, and the relative expression level of ppc was predictively higher than other strains. Figure S4. Transcriptional analysis of the ppc, aspA, aspC, asd and pntAB genes by real-time RT-PCR. The control strain was E. coli K12 MG1655/ pACYC184-thrA433pheBC-asd, and the engineering strains were E. coli K12 MG1655 strain harboring plasmids pACYC184-thrA433pheBC-asd-rhtA/rhtB/rhtC/yeaS, respectively. Data shown are mean values from three biological replicates, and the standard deviations are presented. “*” denotes the relative expression level of the gene overexpressed in plasmid. Take Panel A, quantitative PCR of rhtA gene for all the strains, as an example, “*” denotes the strain overexpressed rhtA gene, and the relative expression level of rhtA was predictively higher than other strains. Figure S5. Construction of CRISPR arrays containing sequence repeats via TA clone/BciVI based PS-Brick assembly. The N20 sequence flanked with the same promoter pJ23119 and the sgRNA sequence located in pTargetET vector. The entrance site of HindIII/BciVI and editing template was introduced into pTargetF to generate original vector ptargetET for PS-Brick assembly. The N20 fragments of tdh, ilvA and tdcC fixed with the same promoter and sgRNA at each end were sequentially inserted into ptargetET through three rounds of PS-Brick reactions. Table S1. Strains and plasmids used in this study with relevant characteristics.