Experimental Materials
1) Strains
- Wild-type Bacillus HMBY (Hubei WEL-SAFE Biotechnology Co., Ltd.)
- Xanthomonas QKHT-5 (Hubei WEL-SAFE Biotechnology Co., Ltd.)
- Mutant strain HMBY-106 (obtained by mutagenesis of HMBY)
2) Media
LB medium: Peptone 10 g/L, sodium chloride: 5 g/L, yeast extract: 10 g/L, pH 7.0-7.2; solid medium supplemented with 15 g/L agar.
3) Gel electrophoresis
Agarose, TAE buffer, nucleic acid Marker (from Thermo Scientific)
4) Software and Tools
- BLAST tool: NCBI BLAST - Nucleotide BLAST - rRNA/ITS databases
- Constructing phylogenetic trees: MEGA 11
- Genome sequence annotation: Prokka (v.1.14.6)
- Gene sequence editing: Snap Gene (v6.2)
- Domain prediction: InterPro database
- Fluorescence quantitative reverse transcription PCR software: CFX Maestro Software (v2.3)
- High-performance liquid chromatography analysis software: Chromeleon (v7.1)
- Mass spectrometry analysis software: Xcalibur (v4.0)
- Transcriptome data filtering software: Cutadapt (v.3.4)
- Reference sequence alignment software: STAR (v.2.7.8a)
- Differential expression analysis software: DESeq2 (v.1.30.0)
- GO annotation and KEGG annotation software: Blast2Go (v.6.0.1)
- GO enrichment analysis software: cluster Profiler package
- KEGG enrichment analysis software: KEGG Mapper
- BioPython (Bio.Seq, Bio.SeqIO): FASTA parsing, sequence translation (inc. genetic code table 11), stop codon handling
- MAFFT (v7.526): Multiple sequence alignment (--auto/--localpair modes) for evolutionary/conservation analysis
- BLAST (ncbi-blast-2.17.0+): Local DB searches (makeblastdb/blastn/blastp) for homology identification & annotation
- IQ-TREE (v2.2.0 / v3.0.1): ML phylogenetic trees (-m MF/-bb) for evolutionary relationship inference
- FigTree (v1.4.4): Phylogenetic tree visualization, annotation, and export
Specification
1. Plasmid Extraction
2. SpeedyCut BsaI
Plasmid DNA system
| Component | Volume |
|---|---|
| ddH₂O | 15 μL |
| 10×SpeedyOne Buffer | 2 μL |
| Substrate DNA | 2 μL |
| SpeedyCut BsaI | 1 μL |
Enzymatic digestion system of genomic DNA
| Component | Volume |
|---|---|
| ddH₂O | 30 μL |
| 10×SpeedyOne Buffer | 5 μL |
| Substrate DNA | 10 μL |
| SpeedyCut BsaI | 5 μL |
3. Green Taq Mix
Using Green Taq Mix from Vazyme
Reaction system (20 μL)
| Component | Volume |
|---|---|
| Green Taq mix | 12.5 μL |
| Primer1 | 1 μL |
| Primer2 | 1 μL |
| Template | 4 μL |
| ddH₂O | 6.5 μL |
Reaction system (50 μL)
| Component | Volume |
|---|---|
| Green Taq mix | 25 μL |
| Primer1 | 2 μL |
| Primer2 | 2 μL |
| Template | x μL |
| ddH₂O | To 50 μL |
Reaction condition
| Steps | Temperature | Time |
|---|---|---|
| Initial denaturation | 95 ºC | 30 min |
| Denaturation | 95 ºC | 15 sec |
| Annealing | 55 ºC | 15 sec |
| Extension (35 cycles) | 72 ºC | 30 sec |
| Final extension | 72 ºC | 5 min |
| Temporary storage | 4 ºC | forever |
4. One Step Cloning
5. SpeedyCut SfiI
Using SpeedyCut SfiI from Vazyme
Plasmid DNA system
| Component | Volume |
|---|---|
| ddH₂O | 15 μL |
| 10×SpeedyOne Buffer | 2 μL |
| Substrate DNA | 2 μL |
| SpeedyCut SfiI | 1 μL |
Genomic DNA system
| Component | Volume |
|---|---|
| ddH₂O | 30 μL |
| 10×SpeedyOne Buffer | 5 μL |
| Substrate DNA | 10 μL |
| SpeedyCut SfiI | 5 μL |
6. Plus One Step Cloning
7. Thermolabile Alkaline Phosphatase
Round1-Random mutagenesis
Round2-Targeted gene engineering
Round3-Metabolic engineering
Round4-NRPS reprogramming
