16th	1. Inoculation of pIB184 2. Inoculation of Streptococcus Brevis ATCC367
17th	1. Prepare LB solid medium 2. Prepare LB liquid medium 3. Prepare MRS medium
18th	1. Amplification of genes thl, hbd, crt and ter by PCR 2. Agarose gel electrophoresis detection 3. Gel Extraction 4. One-step cloning 5. Plasmid pLY15-opt were transformed into competent cells, and LB plates were screened to obtain transformants, identify and preserve bacteria.
19th	1. Select the seed liquid of monoclonal culture 2. Plasmid extraction 3. Configure MRS liquid medium 4. Prepare 1mM magnesium chloride solution 5. Configure SM buffer
20th	Preparation and electroporation of natural competence
21th	1. Culture of Streptococcus Brevis ATCC367 2. Plasmid extraction and nucleic acid detection
23rd-30th	Butanol fermentation and detection