Team:HUST-China/Notebook

iGEM 2018 HUST-China

NoteBook

April 1-30

Ziyang Xiao, Junhao Xiong, Jingyu Ren, Yan chen, Qian Li
Pre-experiment of electrogenesis: tested and optimized the electrogenesis condition for MFC (Microbial Fuel Cell)
May 1-31

Yiyan Yu, Ziyi Li, Junhao Xiong
Basic design of molecular experiments: design sequences, primers and the construction of Shewanella
July 1-7

Junhao Xiong, Ziqi Yin, Jiayi Liu
Preparation for following experiments including preparing LB culture medium, antibiotics and optimizing culture condition of Shewanella oneidensis MR-1.

Bo Peng
Construction of pSB1C3-RBS-mleS

Ziyang Xiao, Haotian Ren, Yan Chen, Hao Qiu
synechocystis Group took the responsibility of doing the Interlab. Preparation for the experiment.
July 8-21

Junhao Xiong, Ziqi Yin, Jiayi Liu
Construction of pYYDT-pflB: PCR, double enzyme digestion (with NdeI & SalI), ligation, transformation (into E.coli TOP10).

Qian Li
Construction of pSB1C3-RBS-lldp-RBS-ldhA-TT

Ziyang Xiao, Haotian Ren, Yan Chen, Hao Qiu
Finishing the cell cultures cultivation and using some machines to measure some related data.
July 22-29

Junhao Xiong, Ziqi Yin, Jiayi Liu
Construction of pYYDT-gapA-mdh, pYYDT-lldEFG: PCR, double enzyme digestion (with NdeI & SalI), ligation, transformation (into E.coli TOP10).

Haibo Huang
Construction of pSB1C3-RBS-lldP and pSB1C3-RBS-ldhA.

Haotian Ren
Restriction enzyme digestion of psb1c3
Ziyang Xiao, Yan Chen, Haotian Ren
Testing the cultivation ways of synechocystis PCC6803
July 30 - August 5

Ziyi Li, Jiayi Liu, Yizhe Zeng
Construction of pYYDT-gapA, pYYDT-mdh
Standardization of gapA, mdh

Kaiwen Liu
Construction of pmg105-PpckA-RBS-lldP-RBS-ldhA-TT

Yan Chen, Sijie Xu
Interlab Experiment 4
August 6-12

Ziyi Li, Jiayi Liu, Yizhe Zeng
Construction of pYYDT-dld
Standardization of dld, lldEFG

Haibo Huang
Construction of pSB1C3-RBS-ldhA-RBS-lldP-TT

Yan Chen, Yuanda Huang
Restriction enzyme digestion of psb-lldEFG
Yan Chen, Sijie Xu, Yuanda Huang, Ziyang Xiao
Constructing ldhDc-lldP, ldhDARSDR-lldP, ldhDnARSDR-lldPT
August 13-19

Ziyi Li, Yizhe Zeng
Overlap extension PCR of dld and lldEFG: We got the right band but the sequencing result revealed a mutation of it.

Making competent cells of E.coli WM3064: we need the deficiency type WM3064 to conjugate with Shewanella oneidensis MR-1.
Bo Peng
Construction of pmg105-PpckA-RBS-lldP-TT, pmg105-PpckA-RBS-ldhA-RBS-lldP-TT and pmg105-PpckA-RBS-mleS-TT.

Yuanda Huang, Yan Chen, Sijie Xu, Tao Jian, Ziyang Xiao
PCR of ldhDnARSdR, PCR of ldhDARSdR,
Overlap extension PCR of ldhDnARSdR and lldp (Dn-L)
Overlap extension PCR of ldhDARSdR and lldp (DA-L)
Overlap extension PCR of ldhDC and lldp (DC-L)
August 20-26

Ziyi Li, Junhao Xiong, Yizhe Zeng
Conjugation of E.coli WM3064 and Shewanella oneidensis MR-1: we transferred the targeted plasmid into E.coli WM3064 and conjugated WM3064 and Shewanella oneidensis MR-1 so that the Shewanella oneidensis MR-1 could finally get the targetted plasmid.
Conjugation pYYDT and pYYDT-lldEFG this week.

Using device to produce electricity with Shewanella. We proved that Shewanella could produce more electricity with the existence of lactate.

Yuanda Huang, Haotian Ren, Ziyang Xiao
PCR of ldhDnARSdR, PCR of ldhDARSdR,
PCR of ldhDnARSdR, extraction enzyme digestion of ldhDnARSdR,
Ligation of ldhDnARSdR and psb1c3, transformation
Hao Qiu
Constructing the pathway of TH-glad-lldp.
Insert sequence TH-glad-lldp to vector psb1c3.
August 27 – September 2

Ziyi Li, Junhao Xiong, Yizhe Zeng
Redesigning about connecting dld and lldEFG: We decided to drop the idea about overlap extension PCR to connect dld and lldEFG and started to use isocaudamer (XbaI and SpeI) to connect these genes.

Conjugation of pYYDT-gapA and pYYDT-mdh.
By constructing Rhodopseudomonas palustris-Shewanella oneidensis mutualism system, we found that Shewanella could produce more electricity if Rhodopseudomonas palustris exists.
Qian Li
Construction of pSB1C3-RBS-mleS-RBS-lldp.

Yuanda Huang, Ziyang Xiao
Overlap extension PCR of ldhDnARSdR-lldp

Tao Jian
Adding Flag tag to ldhD-lldP.
Yan Chen
Adding Flag tag to ldhDc-lldP
September 3-9

Ziyi Li, Junhao Xiong, Yizhe Zeng, Ziqi Yin
Conjugation of pYYDT-dld and pYYDT-Ptac-pflB.

New method to connect dld and lldEFG. However, we failed at the construction of pSB1C3-ptac-lldEFG because of the biobrick prefix on pYYDT.
We proved that Shewanella could produce more electricty under anaerobic condition.
Yuanda Huang
Adding tags to ldhDnARSdR-lldp
Yan Chen
Adding his tag to ldhD-lldP and ldhDc-lldP
Hao Qiu
Amplifying the gene and transforming in E. coli.
Ziyang Xiao
synechocystis culture.
September 10-16

Ziyi Li, Junhao Xiong, Yizhe Zeng, Ziqi Yin
Conjugation of pYYDT-gapA-mdh

Haibo Huang
Adding tags to ldhDnARSdR-lldp
Yan Chen
Construction of pmg105-PpckA-RBS-ldhA-TT.

Yuanda Huang
Adding tags to ldhDARSdR-lldp
Yan Chen
Recombination of ldhD-lldP and ldhDc-lldP to shuttle plasmid PCK306
Hao Qiu
Checking the finial consequence by gene sequencing.
Ziyang Xiao
synechocystis culture.
September 17-23

Ziyi Li, Junhao Xiong, Yizhe Zeng, Ziqi Yin
Application of the electrogenesis: we used the device to charge an LED (1.7V), a cell phone (5V) and a small engine (5V). The success proved that we can put our device into practice.
We proved that carbon cloth has better effect than carbon rods.
Kaiwen Liu
Construction of pmg105-PpckA-RBS-mleS-RBS-lldP-TT

Haibo Huang
Inserting ldhDnARSdR-lldp biaoda into the plasmid called pck306 through recombinase,
Inserting ldhDARSdR-lldp biaoda into the plasmid called pck306 through recombinase

Yan Chen
Transforming recombinant shuttle plasmid into synechocystis
Hao Qiu
Transforming ldhd sequence into synechocystis.
Ziyang Xiao
synechocystis culture.
September 24-30

Ziyi Li, Junhao Xiong, Yizhe Zeng, Ziqi Yin
Construction of pSB1C3-pflB-fdh:we found that there was no tac promotor before.

We proved that the electricity produced by Rhodopseudomonas palustrls could be ignored in the electrogenesis system.
At the same time, it was demonstrated that the lactate could be the only carbon source in the culture medium.
Qian Li
Construction of pSB1C3- RBS-mleS-RBS-lldP-RBS-ldhA-TT

Hao Qiu
Using DNS to detect the condition of producing lactate by synechocystis.
Yuanda Huang, Yan Chen
Transforming recombinant shuttle plasmid into synechocystis.
Ziyang Xiao
synechocystis culture.
October 1-7

Ziyi Li, Junhao Xiong, Yizhe Zeng, Ziqi Yin
Construction of pYYDT-pflB-fdh

Conjugation of pYYDT-pflB-fdh
We proved that the engineering bacteria with pYYDT-gapA-mdh could produce more electricity than the bacteria with pYYDT.
Bo Peng
Construction of RBS-mleS-RBS-lldP-TT pmg105-PpckA-RBS-mleS-RBS-lldP-RBS-ldhA-tt.

Yuanda Huang
Overlap extension PCR of ldhDnARSdR and lldp.
Hao Qiu
Expansion culture of synechocystis. DNS detection of sugar production of synechocystis, YFP fluorescent protein detection of synechocystis.
Yan Chen
Transforming recombinant shuttle plasmid into synechocystis.
October 8-14

Ziyi Li, Junhao Xiong, Yizhe Zeng, Ziqi Yin
1. Construction of pSB1C3-Ptac-pflB-fdh
2. Reconjugation of pYYDT-lldEFG
3. We did two groups of electrogenesis experiments. One of them included the engineering synechocystis and Shewanella, the other included the engineering Rhodopseudomonas palustris and Shewanella. It turned out that the later one worked better than the former one.
Yuanda Huang
Add the first tag to ldhDnARSdR-lldp /ldhDARSdR-lldp.
Hao Qiu
Detection of YFP fluorescent protein of synechocystis.
Yan Chen
Detection of YFP fluorescent protein of synechocystis. Detection of Flag tag.
Ziyang Xiao
Detection of YFP fluorescent protein of synechocystis. Detection of 6× His tag and lactate.
Conjugation of pYYDT-pflB-fdh
We proved that the engineering bacteria with pYYDT-gapA-mdh could produce more electricity than the bacteria with pYYDT.
Yuanda Huang
Overlap extension PCR of ldhDnARSdR and lldp.
Hao Qiu
Expansion culture of synechocystis. DNS detection of sugar production of synechocystis, YFP fluorescent protein detection of synechocystis.
Yan Chen
Transforming recombinant shuttle plasmid into synechocystis.