Team:CAU China/Data

NOTEBOOK January Form our team and find our instructors Registry for a CAU iGEM team. Congratulations~ Brainstorm for our projects Journal clubs for scientific issue February Engaged in extensive reading of references Drafting team execution rules Design our logo: the first draft is awesome~ (～ o ～) March Redesign our logo Discuss our E-Periodicals April Brainstorm for how our projects carry out Literature review May Our first e-periodical comes out~o(≧v≦)o~ Yah ~ Recruit new members Open our project July Week 4 July 28th Wet lab Got Nadh1 (adh1 from Neurospora crassa) cDNA from He lab Got Sadh2 (adh2 from Saccharomyces cerevisiae) cDNA from Lou lab Lab meeting Journal club July 29th Wet lab Got expression vector pET-28a(+) from Novagen Preparing LB and LB-Kanamycin plates Autoclave basic materials July 30th Wet lab

 Got E.coli DH5α, JM109, BL21(DE3) from Chen lab
 Order the restriction endonuclease and DNA polymerase

Dry lab

Primers design for Nadh1, Sadh2 and ta0841 (Thermoplasma acidophilum)

August Week 1 August 1st Wet lab Transformation of pET-28a(+) for plasmid propagation Digestion of vector with BamHI and SalI PCR for Nadh1 and Sadh2 Gel electrophoresis of digested product and PCR product Plasmid DNA isolation for pET-28a(+) August 2ed Wet lab Gel purification of digested vector gel product and PCR product Gel check of plasmid extraction Digestion of Sadh2 PCR product with BamHI and SalI Ligation of Sadh2 with pET-28a(+) August 3rd Wet lab

Transformation of ligated pET-28a(+)-Sadh2
Clone PCR detection for transformation

Small inculation of pET-28a(+)-Sadh2(1st) Dry lab

 Calculation of the entropy evolution

Week 2 August 4th Wet lab Digestion of vector with BamHI and EcoRI Digestion of Nadh1 PCR product with BamHI and EcoRI Gel electrophoresis of digested vector product Gel purification of digested vector gel product Ligation of Nadh1 with pET-28a(+) August 5th Wet lab

Transformation of ligated pET-28a(+)-Nadh1(1st)
Colony PCR identification for transformation

Result:no positive clone Dry lab August 6th Wet lab

Project interim report
Brainstorming sessions for division of work and experiment

Dry lab August 7th Wet lab

Plasmid extraction for pET-28a(+)-Sadh2
 Restriction analysis and DNA sequencing of pET-28a(+)-Sadh2 for identification
 Result:positive!

Dry lab August 8th Wet lab

Ligation of Nadh1 with pET-28a(+)
Transformation of ligated pET-28a(+)-Nadh1(2nd)

Result:no clone(⊙o⊙)! Dry lab August 9th Wet lab

Ligation of Nadh1 with pET-28a(+)
Transformation of ligated pET-28a(+)-Nadh1(2nd)

Result:no clone o_O??? Dry lab Week 3 August 11th Wet lab Discussion for experiment PCR of Nadh1 Gel electrophoresis of PCR product Digestion of vector and PCR product with BamHI and EcoRI (for a longer time) Gel electrophoresis of digested vector product Gel purification of digested vector gel product Column- purification of digested gene product Ligation of Nadh1 with pET-28a(+) August 12th Wet lab

Transformation of ligated pET-28a(+)-Nadh1(3rd)
Colony PCR detection for transformation

Result:no positive clone (+﹏+)~ Dry lab

Calculation of the entropy evolution in another way

August 13th Wet lab

Discussion for experiment

Dry lab August 14th Wet lab

Inoculation of pET-28a(+): repeat Inoculation
Dry lab 

Primers redesign for Nadh1 August 15th Wet lab

Clean the lab  
Preparing IPTG for protein induction

Autoclave the materials Dry lab August 16th Wet lab

Small inoculation for pET-28a(+)-Sadh2

Large inoculation for pET-28a(+)-Sadh2 Protein induction with 0.1mM IPTG, 37℃ Cell lysis Enzyme activity assay(1st) Dry lab Week 4 August 18th Wet lab Informal lab meeting August 19th Wet lab

Small inoculation for pET-28a(+)-Sadh2

Large inoculation for pET-28a(+)-Sadh2 Protein induction with 0.5mM IPTG ,16℃ Cell lysis SDS-PAGE for protein induced assay Result:no protein induced Dry lab

August 20th Wet lab

Small inoculation for pET-28a(+)-Sadh2

Large inoculation for pET-28a(+)-Sadh2 Protein induction with 0.1mM and 0.5mM IPTG,4℃ Cell lysis SDS-PAGE for protein induced assay Result: protein in precipitation not separate well Dry lab August 21st Wet lab

SDS-PAGE again for induction yesterday
Result:no protein induced in precipitation
Dry lab 

August 22nd Wet lab

New primers for Nadh1 arrival \(^o^)/YES！

PCR for Nadh1 with new primers Gel electrophoresis of PCR product Digestion of vector and PCR product with BamHI and NotI Gel electrophoresis of digested vector product Gel purification of digested vector gel product Column-purification of digested gene product Ligation of Nadh1 with pET-28a(+) Dry lab August 23rd Wet lab

Transformation of ligated pET-28a(+)-Nadh1(4th)

Result:no clone Ligation of Nadh1 with pET-28a(+) Dry lab August 24th Wet lab Transformation of ligated pET-28a(+)-Nadh1 (5th) Result:no clone o(︶︿︶)o Dry lab Week 4 August 25th Lab meeting and discuss for experiment August 26th Wet lab

Get the ta0841 (commercially synthesized CDS, BGI Crop)

PCR for ta0841 amplification Gel electrophoresis of PCR product Dry lab August 27th Wet lab Digestion of vector and PCR product with BamHI and SalI Gel electrophoresis of digested vector product Gel purification of digested vector gel product Column-purification of digested gene product Ligation of ta0841 with pET-28a(+) Dry lab August 28th Wet lab Transformation of ligated pET-28a(+)-ta0841 (1st)

Preparing of LB and autoclave
LB-Kanamycin, ampicillin and chloramphenicol plates
Order restriction endonuclease(NEB) and DNA Marker 
Result:no colone
Dry lab 

August 29st Wet lab Ligation of ta0841 with pET-28a(+) Ligation of Nadh1 with pET-28a(+) Transformation of ligated pET-28a(+)-ta0841 (2nd) and pET-28a(+)-Nadh1 (6st) Dry lab August 30th Wet lab

Colony PCR for transformation product identification

Small inoculation of pET-28a(+)-ta0841 (2st) and pET-28a(+)-Nadh1 (6st) for restriction anlysis Dry lab August 31st Wet lab Plasmid extraction from inoculation yesterday Result:none plasmid isolation Supercompetent cell Preparation for E.coli DH5αand JM109 Dry lab September Week 1 September 1st Lab meeting Discussion for experiment and wiki September 2nd Wet lab PCR for ta0841 and Nadh1 amplification Gel electrophoresis of PCR product Gel purification of PCR product Nanoview: the concentration is OK. \(^o^)/YEH~ Digestion of pET-28a(+) by BamH1-HF and Sal1-HF for ta0841; Not1-HF and BamH1-HF for Nadh1 Nanoview: the concentration is not high (⊙_⊙?) Digestion of ta0841 with BamH1-HF and Sal1-HF Digestion of Nadh1 with Not1-HF and BamH1-HF Column-purification of digested gene Nanoview: the concentration is OK O(∩_∩)O~ Ligation of ta0841 with pET-28a(+) Ligation of Nadh1 with pET-28a(+) Dry lab

Building enzyme kinetic equations to describe the enzyme catalysis reactions

September 3rd Wet lab Transformation of ligated pET-28a(+)-ta0841 (3rd) and pET-28a(+)-Nadh1 (7st) Colony PCR for transformation product identification Gel check for colony PCR Result:positive results! ( ⊙o⊙ ) Small inoculation of pET-28a(+)- (3rd) and pET-28a(+)-Nadh1 (7st) for restriction anlysis Dry lab

Building enzyme kinetic equations to describe the enzyme catalysis reactions

September 4st Wet lab

Plasmid extraction of pET-28a(+)-ta0841 (3rd) and pET-28a(+)-Nadh1 (7st)

Restriction analysis for identification Result:positive results!!! ~\(≧▽≦)/~ Bravo~

September 5th Wet lab DNA sequencing for pET-28a(+)-ta0841 (3rd) and pET-28a(+)-Nadh1 (7st) Result:positive! positive! positive! ~\(≧▽≦)/~ Dry lab September 6th Wet lab Digestion PSB1C3 backbone by PstI+EcoRI and gel check Gel purification of digested PSB1C3 Dry lab

 Design primers for biobricks

Week 2 September 8th Lab meeting Discussion for experiment and T-shirt September 9th Wet lab Primers for biobricks arrive Digestion of Sadh2with EcoR1 and Pst1 Digestion of Nadh1 with EcoR1 and Pst1 Column-purification of digested gene Nanoview: the concentration is OK O(∩_∩)O~ Ligation of Sadh2 with pET-28a(+) Ligation of Nadh1 with pET-28a(+) Dry lab September 10th Wet lab Transformation of ligated PSB1C3- Sadh2 (1st) andPSB1C3-Nadh1 (1st) Result: no colony! ( ⊙o⊙ ) Dry lab September 11th Transformation of ligated PSB1C3- Sadh2 (2st) andPSB1C3-Nadh1 (2st) Result: no colony! o(︶︿︶)o Analysis for the bad result Dry lab September 12th Wet lab Digestion of Sadh2 with EcoR1 and Pst1 Digestion of Nadh1 with EcoR1 and Pst1 Column-purification of digested gene Nanoview: the concentration is high O(∩_∩)O~ Ligation of Sadh2 with pET-28a(+) Ligation of Nadh1 with pET-28a(+) Dry lab September 13th Wet lab Transformation of ligated PSB1C3- Sadh2 (3rd) and PSB1C3-Nadh1 (3rd) Result: look! The plate of Sadh2 has colony! Yeh~ Small inoculation of PSB1C3-Sadh2 (3rd) Dry lab September 14th Wet lab Plasmid extraction of PSB1C3-Sadh2 (3rd) for identification Result:positive results!!! ~\(≧▽≦)/~ Bravo~ Dry lab Week 3 September 15th Lab meeting Presentation rehearsing September 16th Wet lab

Error-pone PCR of Sadh2, Nadh1 and ta0841 for random mutagenesis

Mailing our biobrick Plasmid maximum extraction(alkaline lysis method) Gel electrophoresis of Error-pone PCR product Gel purification of Error-pone PCR product Transformation of pET-28a(+)-Sadh2 into BL21(Rosetta) Dry lab September 17th Wet lab Inoculation for pET-28a(+)-Sadh2 Protein induction with 0.6mM IPTG,16℃,over night Dry lab September 18th Cell lysis Affinity chromatography with a Nickel column Purification by gel-filtration chromatography using the Superdex 200 High Performance column SDS-PAGE for protein induced assay Result:our protein has been induced! \(^o^)/

Dry lab September 19th Wet lab Digestion of Error-Pone Sadh2and ta0841with Bam1-HF and Sal1-HF Digestion of Error-Pone Nadh1 with BamH1-HF and Not1-HF Column-purification of digested gene Nanoview: the concentration is high O(∩_∩)O~ Ligation of purification products with pET-28a(+) Transformation of pET-28a(+)-Nadh1 and pET-28a(+)-ta0841 into BL21(Rosetta) Dry lab Mathematical modeling Information about the Judging Forms can be found in our Model September 20th Wet lab Transformation of conjunction into BL21 competent cells Inoculation for pET-28a(+)-ta0841 and pET-28a(+)-Nadh1 Protein induction with 0.6mM IPTG,16℃,over night Dry lab September 21st Cell lysis Affinity chromatography with a Nickel column Purification by gel-filtration chromatography using the Superdex 200 High Performance column SDS-PAGE for protein induced assay Result: ta0841 has been induced,but Nadh1 isn’t.