10/7-14/7 Construction of recombinant plasmid pMTL-Pthl-F/Xpk(BD)

 

1. PCR amplification on the genome of Clostridium acetobutylicum to obtain the fragment F/Xpk(BD).

2. Linearized vector pMTL-Pthl was obtained by PCR amplification using plasmid pMTL82151-Pthl-adhE2 as template;

3. pMTL-Pthl-F/Xpk(BD) was obtained by Gibson assembly

 

Experimenter：Pengfei Bai,Huaize Zhu,Ruige Li,Junjie Zheng,Youchang Zhang,Yuyao Cao,Yuntian Yang,Shufan Wu

Recorder：Huaize Zhu

 

PDF1- Construction of recombinant plasmid pMTL-Pthl-F/Xpk(BD)

 

17/7-20/7 Construction of recombinant plasmid pMTL-Pthl-F/Xpk(QS)

 

1. The fragment F/Xpk(QS) was obtained by PCR amplification on Bifidobacterium adolescentis genome

2. Linearized vector pMTL-Pthl was obtained by PCR amplification using plasmid pMTL82151-Pthl-adhE2 as template;

3. pMTL-Pthl-F/Xpk(QS) was obtained by Gibson assembly

 

Experimenter：Pengfei Bai,Huaize Zhu,Junjie Zheng,Youchang Zhang,Yuyao Cao

Recorder：Huaize Zhu

 

PDF2-Construction of recombinant plasmid pMTL-Pthl-F/Xpk(QS)

 

21/7-1/8 Construction of recombinant plasmids ：pMTL-Pfba-F/Xpk、pMTL-Ptkt-F/Xpk

 

1. PCR amplification on the genome of Clostridium tyrobutyricum to obtain the fragment Pfba、Ptkt).

2. Linearized vector pMTL-F/Xpk(BD) was obtained by PCR amplification using plasmid pMTL-Pthl-F/Xpk(BD) as template;

3. pMTL-Pfba-F/Xpk(BD)、pMTL-Ptkt-F/Xpk(BD) was obtained by Gibson assembly

 

Experimenter：Leyi Zhang,Yijiu Lu,Xiaoran Zhao,Yixuan Liu,Qingyue Feng,Ruoxi Liu,Jiale Li

Recorder：Leyi Zhang

 

PDF3-Construction of recombinant plasmids ：pMTL-Pfba-F/Xpk、pMTL-Ptkt-F/Xpk

 

7/8-17/8 Replacement of carbon sources and determination of butyric acid

 

1. Culture of Clostridium tyrobutyricum engineered bacteria Ct (Pthl F/Xpk-BD) using TGY medium supplemented with different carbon sources

2. Fermentation broths of Ct (Pthl F/Xpk-BD) in media containing different carbon sources were collected and assayed for product content using HPLC

3. Analysis of HPLC data

 

Experimenter：Yijiu Lu,Pengfei Bai,Huaize Zhu,Leyi Zhang，Shuyan Zhou,Keyu Chen,Zexi Zheng,Yixuan Liu

Recorder:Yijiu Lu

 

PDF4-Replacement of carbon sources and determination of butyric acid

 

18/8-25/8 Ancestral sequence reconstruction (ASR) and Construction of recombinant plasmids pMTL-Pthl-F/Xpk(ASR)

1. Using PUC57 vector plasmid as the template, F/Xpk(ASR) fragment (2436 bp) was amplifie;

2.Linearized vector pMTL-Pthl was obtained by PCR amplification using plasmid pMTL-Pthl-F/Xpk(BD) as template;

3.The F/Xpk (ASR) gene fragment and the X-pMTL-Pthl linearized vector were ligated by Gibson assembly.

 

Experimenter：Yijiu Lu,Pengfei Bai,Huaize Zhu

Recorder:Pengfei Bai

PDF5-Construction of recombinant plasmids ：pMTL-Pthl-F_Xpk-ASR.pdf