Team:CAU China/J Sep

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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~

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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^)/


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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.


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September 22st

Wet lab

Wild-type enzyme activity assay of yeast alcohol dehydrogenase


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