Screening antimicrobial peptides and probiotics using multiple deep learning and directed evolution strategies
The raw data contains the antibacterial activity test, peptide screening, peptide purification, PANCE for SrtA, structure prediction and whole genome sequencing.
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Activity assay of AMPs pBAD18-AMPs were transformed into was transferred in to Escherichia coli DH5α. The single colony strain was inoculated with 100 mg/L ampicillin and divided into two parts equally, one of which containing 10 mM L- arabinose. The two parts were cultured at the temperature of 37 °C and at the rotate speed of 200 rpm until that the OD600 value of one of them reached 0.6. OD600 value of them was detected and the value ratio w/o L- arabinose was calculated. Random mutant plasmid library construction We used two random PCRs to obtain a random DNA library of 150 bp in pBAD18-GFP and pTET-GFP. The first round of random PCR contains 75 random bases downstream of the start codon ATG. The second round of random PCR contains 75 random bases downstream of the first round of 75 random bases, with 20 bases isolated in the middle for primer binding. Random mutant plasmid library was amplified by Hieff Canace® Gold High Fidelity DNA Polymerase (Yeasen Biotechnology Shanghai co. ltd, Cat.No. 10148ES60) using pBAD18-GFP and pTET-GFP as template. The amplified PCR product was digested by DpnI and recovered using MolPure Gel Extraction Kit (Yeasen Biotechnology Shanghai co. ltd, Cat.No. 19101ES70). Total product was transformed into DH5α by electro-conversion to repair DNA. Total plasmid library was extracted using MolPure® Plasmid Mini Kit (Yeasen Biotechnology Shanghai co. ltd, Cat.No. 19001ES70) and was used as the template in the next round of Random PCR. The next round PCR product was treated and recovered using the same processes. Final plasmid library was extracted and used as random mutant plasmid library. Next generation sequencing for distinguishing AMPs Random mutant plasmid library was transferred into Escherichia coli DH5α. The strain pools were cultured with or without the inducer at the temperature of 37 °C and at the rotate speed of 200 rpm until that the OD600 value reached 0.6. The plasmid pool was extracted and amplified using primers contained illumina universal sequence and index. The PCR product was recovered and sequenced by illumina NovaSeq 6000 platform by PE150 model. After sequencing, the adaptor was trimmed by Trimmomatic. Reads 1 and Reads 2 were assembled. The fastaq data was translated from the start codon and the amino acid sequences were obtained. The abundance of these peptides was calculated and the ratio of peptide abundance with or without inducer was calculated.