7.1-7.9 Conducting laboratory safety training and safety assessments
all wet team members
7.10-7.14 Construstion of plasmid pMTL-Pthl-adhE2-dac
1. Fragment dac was amplified by PCR on the genome of Clostridium tyrobutyricum L319.
2. The linearized vector pMTL-Pthl-adhE2 was amplified by PCR using pMTL82151-Pthl-adhE2 as the template.
3. Plasmid pMTL-Pthl-adhE2-dac was obtained by Gibson assembly
(Experimenter: Xiaojing Tong, Zilai Chen, Yinong Fu, Yuyang Fu, Mingzhe Zhao)
Experiment records：
7.10：Preparation of media (solid & liquid), making gels; PCR amplification of fragments Dac.The linearized vector pMTL-Pthl-adhE2 was amplified by PCR using pMTL82151-Pthl-adhE2 as the template；7.11：The gel was recovered to obtain the target DNA; the vector system was configured and the vector was ligated to the fragment:pMTL-Pthl-adhE2-Dac;
7.12：Transformation, 12 hours culture at 37 degrees Celsius;
7.13：Colony PCR validation, #2,4,5 were successful;
7.14：Purity testing; SDS-page; Bacterial preservation；
7.17-7.21 Construstion of plasmid pMTL-Pthl-adhE2-Pcat1-cat1
1. The fragment cat1 and the cat1 promoter was amplified by PCR on the genome of Clostridium tyrobutyricum L319.
2. The linearized vector pMTL-Pthl-adhE2 was amplified by PCR using pMTL82151-Pthl-adhE2 as the template.
3. Plasmid pMTL-Pthl-adhE2-Pcat1-cat1 was obtained by Gibson assembly
(Experimenter: Xiaojing Tong, Zilai Chen, Yinong Fu, Yuyang Fu, Mingzhe Zhao, Tianyunran Huang)
7.17-7.18: The fragment cat1, cat1 promoter and the vector pMTL-Pthl-adhE2 were obtained by PCR。The linearized vector pMTL-Pthl-adhE2 was amplified by PCR using pMTL82151-Pthl-adhE2 as the template.
7.19: Fragment and vector ligation；Transformation
7.20: Colony PCR validation
7.21: SDS-page; Bacterial preservation;
7.24-7.28: Construstion of plasmid pMTL-Pthl-adhE2-bcd-crt
1. The linearized vector Vp-adhE2 was amplified by PCR using Pthl-Pthl-adhE2(BBa_K4408008) as the template.
2. The fragment bcd, crt was amplified by PCR on the genome of Clostridium tyrobutyricum L319;then bcd gene fragment and crt gene fragment were fused into bcd-crt fragment by fusion PCR.
3.Gibson assembly method was used to link bcd-crt gene fragment to Vp-adhE2 linearized vector
(Experimenter:Yangyang Liu, Minmo Zhu, Hongchang Lv, Lingyi Chen, Kewei Li, Jian Zhou)
Experiment records：
7.24 The fragment bcd was amplified by PCR
7.25 The fragment crt was amplified by PCR; Linear pMTL-Pthl-adhE2
7.26 The target plasmid verified by colony PCR was pMTL-Pthl-adhE2-bcd
7.27 The template plasmid was pMTL-Pthl-adhE2-bcd
7.28 The target plasmid verified by colony PCR was pMTL-Pthl-adhE2-bcd-crt；SDS-page; Bacterial preservation;
7.31-8.4: Promoter engineering: Replace the promoter in Pthl-adhE2 to build Ptkt-adhE2
(Experimenter:Yangyang Liu, Minmo Zhu,Kewei Li,Yuyang Fu,Yinong Fu)
1. pMTL-Pthl-adhE2 plasmid (BBa_K4408008) was used as a template, and the linearized vector Vtkt was amplified.
2. Clostridium tyrobutyricum genome was used as template to amplify the fragment tkt.
3. Gibson Assembly ligated the linearized vector Vtkt with the fragment tkt to obtain the recombinant plasmid pMTL-Ptkt-adhE2;
4. Colony PCR validation; SDS-PAGE assay.
5. Bacterial preservation;
8.6-8.18:Product determination
（Experimenter:Yangyang Liu, Minmo Zhu, Hongchang Lv）
8.6 All of the preserved strains were recovered.
8.7 After 12 hours of cultivation, add CALB lipase and hexadecane to the culture medium. And then continue to cultivate for 203 hours.
8.17-8.18 HPLC analysis of acetate, butyrate and butanol; GC analysis of butryl butyrate. Data analysis.
