Team:CAU China/Data

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 la b

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

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

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

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

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

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

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

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.