Wiki/Team:SJTU-BioX-Shanghai/Notebook/log2

From 2012.igem.org

July
Sun Mon Tue Wed Thu Fri Sat
1 2 3 4 5 6 7
8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30 31
August
Sun Mon Tue Wed Thu Fri Sat
1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30 31
September
Sun Mon Tue Wed Thu Fri Sat
1
2 3 4 5 6 7 8
9 10 11 12 13 14 15
16 17 18 19 20 21 22
23 24 25 26 27 28 29
30

August 1st, 2012

Fig7.1 Digestion results

Plasmid Extraction

lgt-pUC19 *4
GFP-pUC19 *4
lgt-GFP-pUC19

Digestion identification

Result: GFP-pUC19(3) successful
Correct sequence

Colony Picking

lac-pUC19, lgt-GFP-pUC19


August 2nd, 2012

Fig8.2 Digestion result

Digestion identification

Reconstructed plasmid

  • pET
  • pACYC
  • pRSF

System

Enzyme 1μl
10×FD Buffer or FD Green buffer2μl
DNA ≤1μg
Sterile waterup to 20μl




August 3rd, 2012

Fig8.3 Digestion result

Digestion identification

  • Fl3-lgt
  • Fl3-GFP

System

Enzyme 1μl
10×FD Buffer or FD Green buffer2μl
DNA ≤1μg
Sterile waterup to 20μl




August 4th, 2012

Fig8.4 ligation result

Ligation

  • LLG with pUC
  • LLG with pRSF

Ligation System

10X T4 DNA Ligase Buffer 2 μl
Vector DNA 0.025 pmol
Insert DNA 0.076 pmol
T4 DNA Ligase 1μl
Nuclease-free water to 20 μl



August 5th, 2012

Fig8.5 PCR result

PCR

  • fl3 with rbsABCE
  • fl3 with rbsABDE

System

kod enzyme

Transformation

  • fl3-rbsABCE
  • fl3-rbsABDE



August 6th, 2012

Fig8.6 Digestion result

Digestion

  • Llg with Ecor1
  • Llg with prst1

System

Ecor1,prst1 1μl
10×FD Buffer or FD Green buffer2μl
DNA ≤1μg
Sterile waterup to 20μl

Colony Picking

  • fl3-rbsABCE
  • fl3-rbsABDE



August 7th, 2012

Fig8.7 Digestion identification

Digestion identification

  • Llg

System

Ecor1 1μl
10×FD Buffer or FD Green buffer2μl
DNA ≤1μg
Sterile waterup to 20μl

Plasmid extraction

  • fl3-rbsABCE
  • fl3-rbsABDE



August 8th, 2012

Fig8.8 PCR result

PCR

  • VioC with FL3
  • Reaction temperature:
            Three-step method:
        Predenature
          94℃,2min
        Denature,annealing and extension
          98℃ 10sec   
         {
         (56)℃  30sec
          68℃  30sec/kb
          }
                       × 34 cycles
          4℃

Transformation

  • PCR product



August 9th, 2012

Colony Picking

  • vioC(fl3 rbs)*8

Plasmid Extraction

  • vioC(fl3 rbs)*8

August 10th, 2012

Fig8.10 PCR result

PCR

  • fl3 with vio A*2
  • with Vio B

Transformation

  • MFL3-vioA1
  • MFL3-vioA2
  • MFL3-vioB1



August 11th, 2012

Ligation

  1. dsbAss-mFL3-F with lgt-R
  2. 1mFL3-SH3d with SH3-SP-R
  3. dsbASS-PD2L-mFL3-F with lgt-R
  4. dsbASS-PD2d-F with PD2d-SP-R
  5. 1mFL3-GBDd-F with GBDd-SP-R

system

10X T4 DNA Ligase Buffer 2 μl
Vector DNA 0.025 pmol
Insert DNA 0.076 pmol
T4 DNA Ligase 1μl
Nuclease-free water to 20 μl

Colon picking

  • MFL3-vioA1
  • MFL3-vioA2
  • MFL3-vioB1

August 12th, 2012

Plasmid extracting

  • MFL3-vioA1
  • MFL3-vioA2
  • MFL3-vioB1

PCR identification

  • MFL3-vioA1
  • MFL3-vioA2
  • MFL3-vioB1

August 13th, 2012

Fig8.13 PCR result

PCR

  • Vio A
  • Vio B
  • Vio D
  • Vio E

Ligation

  • Vio A with fl3
  • Vio B with fl3
  • Vio D with fl3
  • Vio E with fl3



August 14th, 2012

Fig8.14 Confocal imaging

Confocal imaging fluroescent protein expression with concentration gradient test

  • 0.2% Ara induction
  • 2% Ara induction


Transformation

  • fl3-VioA
  • fl3-VioB
  • fl3-VioD
  • fl3-VioE


August 15th, 2012

Double antibody screening

  • AMP 50ug/mL Ara 0%
  • AMP 50ug/mL Ara 0.02 %
  • AMP 0ug/mL Ara 0.02 %

Colon picking

  • fl3-VioA
  • fl3-VioB
  • fl3-VioD
  • fl3-VioE

August 16th, 2012

Fig8.16 PCR results

PCR identification

control group of fatty acid synthesis acceleration system

  • rbs-TestA
  • rbs-FabZ
  • rbs-FabG

Reaction temperature:

        Three-step method:
        Predenature
          94℃,2min
        Denature,annealing and extension
          98℃ 10sec   
         {
         (56)℃  30sec
          68℃  30sec/kb
          }
                       × 34 cycles
          4℃




August 17th, 2012

Fig8.17 Digestion results

Digestion identification

control group of fatty acid synthesis acceleration system

  • rbs-G

Restriction enzyme: EcoRI, XbaI

system

  • Enzyme 1μl
  • 10×FD Buffer or FD Green buffer 2μl
  • DNA ≤1μg
  • Sterile water up to 20μl







August 18th, 2012

Ligation

fatty acid synthesis acceleration system

  • M-TestA:rbs-dsbAss-FL3-lgt-FL3-SH3domain-FL3 with TestA
  • M-FabG:rbs-dsbAss-PDZligand-FL3-lgt-FL3-SH3ligand-FL3 with FabG
  • M-FabI:rbs-dsbAss-PDZdomain-FL3-lgt-FL3-GBDdomain-FL3 with Fab I
  • M-FabZ:rbs-dsbAss- FL3-lgt-FL3-GBDligand-FL3 with FabZ

system

10X T4 DNA Ligase Buffer 2 μl
Vector DNA 0.025 pmol
Insert DNA 0.076 pmol
T4 DNA Ligase 1μl
Nuclease-free water to 20 μl

August 19th, 2012

Fig8.19 digestion results

Digestion identification for ligation production

fatty acid synthesis acceleration system

  • Restriction enzyme: EcoRI, XbaI

system

  • Enzyme 1μl
  • 10×FD Buffer or FD Green buffer 2μl
  • DNA ≤1μg
  • Sterile water up to 20μl




August 20th, 2012

Fig8.20 digestion results

Digestion identification

control group of fatty acid synthesis acceleration system

  • rbs-G
  • rbs-1
  • rbs-A
  • rbs-Z

Restriction enzyme: EcoRI, XbaI

system

  • Enzyme 1μl
  • 10×FD Buffer or FD Green buffer 2μl
  • DNA ≤1μg
  • Sterile water up to 20μl



August 21st, 2012

Fig8.21 digestion results

Digestion identification

  • m-A
  • m-G
  • m-Z

Restriction enzyme: EcoRI, XbaI

system

  • Enzyme 1μl
  • 10×FD Buffer or FD Green buffer 2μl
  • DNA ≤1μg
  • Sterile water up to 20μl


August 22nd, 2012

Ligation

  • M1 with EGFP1
  • M2 with EGFP2
  • M3 with EGFP1
  • M4 with EGFP2

System: 5ul solutionI + 0.5ul fusion membrane + 4.5ul according EGFP

August 23rd, 2012

transformation

System: 2ul ligation products with 30ul competent cells

Colon picking

  • M1 with EGFP1 *4
  • M2 with EGFP2 *4
  • M3 with EGFP1 *4
  • M4 with EGFP2 *4

August 24th, 2012

Fig78.24 PCR results

Confocal imaging

fluroescent protein expression

PCR identification

  • rbsvioB-pACYC
  • rbsvioA-pET
  • Reaction temperature:
        Three-step method:
        Predenature
          94℃,2min
        Denature,annealing and extension
          98℃ 10sec   
         {
         (58)℃  30sec
          68℃  30sec/kb
          }
                       × 36 cycles
          4℃



August 25th, 2012

Fig7.4.2 digestion result

Digestion identification

  • rbsvioB-pACYC

Restriction enzyme: EcoRI, XbaI system

  • Enzyme 1μl
  • 10×FD Buffer or FD Green buffer 2μl
  • DNA ≤1μg
  • Sterile water up to 20μl



August 26th, 2012

Fig8.26 PCR products

PCR identification

  • rbsvioA-pET


  • Reaction temperature:
        Three-step method:
        Predenature
          94℃,2min
        Denature,annealing and extension
          98℃ 10sec   
         {
         (56)℃  30sec
          68℃  30sec/kb
          }
                       × 32 cycles
          4℃

August 27th, 2012

Fig8.27 digestion result

Digestion identification

  • rbsvioA-pET

Restriction enzyme: EcoRI, XbaI

System

Enzyme 1μl
10×FD Buffer or FD Green buffer2μl
DNA ≤1μg
Sterile waterup to 20μl



August 28th, 2012

Transformation

  • rbsvioA-pET
  • M1-EGFP1
  • M2-EGFP2

August 29th, 2012

colon picking

  • rbsvioA-pET
  • M1-EGFP1
  • M2-EGFP2

Plasmid extracting

  • rbsvioA-pET

August 30th, 2012

Fig8.30 ligation result

Ligation

  • rbsvioA-pET with VioC

Transformation

  • rbsvioAC-pET

system

10X T4 DNA Ligase Buffer 2 μl
Vector DNA 0.025 pmol
Insert DNA 0.076 pmol
T4 DNA Ligase 1μl
Nuclease-free water to 20 μl



August 31st, 2012

Confocal imaging

fluroescent protein expression

  • M1-EGFP1
  • M2-EGFP2

Colon picking

  • rbs-VioAC-pET