Team:SJTU-BioX-Shanghai/Notebook







In the notebook, there are some simplifications we made in sample names:

Orignal Simplified
ADH1 promoter( pADH1)ADH
GAL1 promoter (pGAL1)GAL
CUP1 promoter (pCUP1)CU
Nano-lantern cAMP 1.6NLc
human adrenergic receptor beta 2 (ADRB2)hAR
yeast nucleotide binding alpha subunit (Gpa2) with 5 amino acids modificationGpa2M
Yeast TRP section markerTRP
Yeast HIS section markerHIS
Yeast LEU section markerLEU
6 x His-taghis

20160624

  1. Designed primers for amplifying the ADH1 promoter from yeast genome .
  2. Our oder (Nano-lantern cAMP 1.6) from addgene arrived!

20160625

  1. pick up colonies of pcDNA3-Nanolantern.
  2. Plasmid extraction of RS416、426、424、LD-BS-ADH1-COS1( plasmid backbones)

T--SJTU-BioX-Shanghai--notebook-1.jpg

20160626

  1. Plasmid extraction of pcDNA3-Nanolantern

T--SJTU-BioX-Shanghai--notebook-2.jpg

  1. ClaI-AvrII restriction digestion of LD-BS-ADH1-COS1

T--SJTU-BioX-Shanghai--notebook-3.jpg

  1. PCR amplification of Nano-lantern cAMP 1.6 from pcDNA3-Nanolantern

20160629

  1. Plasmid extraction of TRP-pADH1-NLc

T--SJTU-BioX-Shanghai--notebook-4.jpg

  1. PCR amplify the GAL1 and CUP1 promoter from yeast genomic DNA and cloned them into plasmid backbones.

20160630

  1. Pick up colonies of GAL1 and CUP1 promoter with ADH1 terminator (TRP-CU/TRP-GAL).

20160701

  1. Plasmid extraction of TRP-CU/TRP-GAL.-

T--SJTU-BioX-Shanghai--notebook-5.jpg

20160706

  1. Got strain DJ03 and 9060 from professor Zhiping Xie’s lab.
  2. Ordered coelenterazine-h from YEASEN BIOTECHNOLOGY.

20160707

  1. Transformation of DJ03 and 9060 with construct TRP-ADH-Nlc.
VolumeOD600Control/ExperimetnalDigested DNA
DJ033ml0.91ml/2ml0μl/10μl
90603ml0.81ml/2ml0μl/10μl

20160709

  1. Seeking for the cds for human adrenergic receptor beta 2 (ADRB2)

20160710

  1. Making modification plans for heterologous expression of ADRB2 in yeast.

T--SJTU-BioX-Shanghai--notebook-6.jpg

20160711~20160713

  1. Finishing the construct TRP-CU-NLc and TRP-CU-NLc

20160713

  1. Fluorescent microscopy of TRP-ADH1-NLc to detect the YFP signal

T--SJTU-BioX-Shanghai--notebook-7.jpg T--SJTU-BioX-Shanghai--notebook-8.jpg

20160714

  1. Colony PCR of TRP-GAL-NLc3 abc/TRP-CU-NLc3 abc

T--SJTU-BioX-Shanghai--notebook-9.jpg

  1. Plasmid extraction of TRP-GAL-NLc3 abc/TRP-CU-NLc3 abc

T--SJTU-BioX-Shanghai--notebook-10.jpg

20160719~20160720

  1. Obtained plasmid backbone LD-BS-HIS3 and LD-BS-LEU2 from Zhiping Xie’s lab and amplified them to make larger amount.

T--SJTU-BioX-Shanghai--notebook-11.jpg

20160723

  1. Tried to amplify the ADBR2 cds from genomic DNA of A549 cells

T--SJTU-BioX-Shanghai--notebook-12.jpg

20160726

  1. Amplify the ADRB2 gene from genomic DNA of A549 cells using different melting temperature and different polymerase

T--SJTU-BioX-Shanghai--notebook-13.jpg

  1. Gel extortion of ADRB2 gene

T--SJTU-BioX-Shanghai--notebook-14.jpg T--SJTU-BioX-Shanghai--notebook-15.jpg

  1. Enzyme digestion of LD-BS-TRP-pADH1-CDS1-3HA and LD-BS-TRP-GAL-3HA-DGA1-aid-6HA
  2. Amplify the ADRB2 gene from the fragment from gel extraction product
ReagentsVolume
KOD plus neo1 μl
10x Buffer for KOD -Plus- Neo5 μl
2mM dNTPs5 μl
25mM MgSO43 μl
hAR10 μl
10 pmol/µl Primer 1x2 μl
ddH2O24 μl

Primer: hAR : hAR-R/hAR-F TRP-ADH-hAR : TRP-ADH-hAR-R/ TRP-ADH-hAR-F TRP-GAL-hAR : TRP-GAL-hAR-R/ TRP-GAL-hAR-F

  1. Gel electrophoresis of the PCR product in 5 and endonuclease digested product in 4

T--SJTU-BioX-Shanghai--notebook-16.jpg

  1. Gel extraction of endonuclease digested product

T--SJTU-BioX-Shanghai--notebook-17.jpg

20160727

  1. PCR amplification of ADRB2 gene.

450px

  1. PCR cleaning

T--SJTU-BioX-Shanghai--notebook-19.jpg

  1. Gel electrophoresis of product in 3.

T--SJTU-BioX-Shanghai--notebook-20.jpg

  1. Gel extraction

T--SJTU-BioX-Shanghai--notebook-21.jpg

20160728

  1. ADRB2 gene fragment PCR
NamePrimerTmTemplate(μl)ddH2O (μl)Total(μl)
hARhAR-R/hAR-F48℃(48℃) 2.411.220
ADH-hARTRP-ADH-hAR-R/ TRP-ADH-hAR-F48℃(48℃) 62850
ADH-hAR 58℃(58℃) 33150
ADH-hAR 65℃(48℃) 62850

Cycle:

NameTemperature Duration
Pre-denaturation95℃3 min
Denaturation95℃40s39x
Anealing48/58/65℃30s 
Extension68℃1min 30s 
Final extension72℃10min

T--SJTU-BioX-Shanghai--notebook-22.jpg

  1. PCR cleaning

Mixed the parallel groups in step 1, then added equal volume of Buffer CP to them, and follow the standardized protocol provided by the manufacturer. The final elution volume is 30 μl. T--SJTU-BioX-Shanghai--notebook-23.jpg

  1. Gel recovery

Ran the samples obtained in Step.3 and recovered the gel. The elution volume is 30 μl.

GelDNA sample volume (after PCR cleaning)6 x Loading bufferDL2000
1% agarose30 μl6 μl10 μl

T--SJTU-BioX-Shanghai--notebook-24.jpg T--SJTU-BioX-Shanghai--notebook-25.jpg

  1. Insert the fragment into the plasmid TRP-ADH/TRP-GAL

The ligation was achieved using ClonExpress II One Step Cloning Kit following the manufacturer’s protocol.

  1. Transformation of DH5a competent cells
DNA volumeCompetent cells LB mediaSpread on plate
10 μl100 μl600 μl350 μl

20160729

  1. Pick up positive colonies
StrainsPositive colonies
TRP-ADH-hAR (48)5/5
TRP-ADH-hAR (58)2/2
TRP-GAL-hAR (58)5/N

20160730

  1. Colony PCR

20 μl system:

ReagentsVolume
Taq1 μl
Buffer2 μl
5μM dNTPs1.76 μl
Culture1 μl
10 pmol/µl Primer 0.4x2 μl
ddH2O14.04 μl

Primer: hAR : hAR-R/hAR-F TRP-ADH-hAR : TRP-ADH-hAR-R/ TRP-ADH-hAR-F PCR condition was set as follows:

NameTemperature Duration
Pre-denaturation94℃3 min
Denaturation94℃40s35x
Anealing60℃30s 
Extension72℃2min 
Final extension72℃7min
  1. Gel electrophoresis of PCR products in Step.1
GelPCR product6 x Loading bufferDNA ladder
1% agarose20μl4μl5μl

T--SJTU-BioX-Shanghai--notebook-26.jpg

  1. Plasmid extraction

58GAL1,2,3,4,5/48ADH1,2/58ADH1 Elution: 30μl in ddH2O;

  1. Endonuclease digestion
Plasmid12μl
Endonuclease1μl HindIII (HF)
1μl ApaI
Buffer5μl Cutsmart
ddH2O31μl

Gel electrophoresis

GelDigestion product6 x Loading bufferDL2000/15000
1% agarose50μl10μl10μl

T--SJTU-BioX-Shanghai--notebook-27.jpg

  1. Gel recovery

Elution: 15μl in ddH2O T--SJTU-BioX-Shanghai--notebook-28.jpg

20160731

  1. Ligation

Ligation process were done followed the standardized protocol from the manufacturer, the plasmids and DNA volume were set as follows:

Construct PlasmidDNA Total volume
HIS2-GAL26.7 μl HIS2 9 μl GAL220 μl
HIS3-GAL37.4 μl HIS39.6 μl GAL320 μl

22℃ 1h

  1. Transformation of DH5a competent cells
DNA volumeCompetent cells LB mediaSpread on plate
10 μl50 μl300 μl360 μl

20160801

  1. colony pcr of HIS3-GAL3-hAR a/b/c +HIS2-GAL2-hAR a/b/c transformed cells

20 μl system:

ReagentsVolume
Taq1 μl
Buffer2 μl
5μM dNTPs1.76 μl
Culture1 μl
10 pmol/µl Primer 0.4x2 μl
ddH2O14.04 μl

Primer:TRP-GAL-hAR-R/ TRP-GAL-hAR-F PCR condition was set as follows:

NameTemperature Duration
Pre-denaturation94℃3 min
Denaturation94℃40s30x
Anealing60℃30s 
Extension72℃2min 
Final extension72℃7min
  1. Gel electrophoresis of PCR products in Step.1
GelPCR product6 x Loading bufferDNA ladder
1% agarose20μl4μl5μl

10μl/well T--SJTU-BioX-Shanghai--notebook-29.jpg

  1. HIS3-GAL3-hAR a/b/c +HIS2-GAL2-hAR a/b/c +TRP-ADH a/b plasmid extraction

20160804

  1. hAR gene fragment PCR(ADH )

PCR system: 50 μl x 3 tubes

Namevolume(μl)
templateGAL-hAR0.5
primerTRP-ADH-Har1

PCR conditions:

NameTemperature Duration
Pre-denaturation95℃3 min
Denaturation95℃40s39x
Anealing48℃30s 
Extension68℃1min 30s 
Final extension72℃10min

electrophoresis:

GelPCR product6 x Loading bufferDL2000
1% agarose5μl1μl5μl

T--SJTU-BioX-Shanghai--notebook-30.jpg

  1. PCR cleaning

Elution volume: 30μl;3 in 1 tube T--SJTU-BioX-Shanghai--notebook-31.jpg

  1. Gel recovery

electrophoresis: T--SJTU-BioX-Shanghai--notebook-32.jpg

GelPCR cleaning product6 x Loading bufferDL2000
1% agarose30μl6μl10μl

gel recovery: T--SJTU-BioX-Shanghai--notebook-33.jpg

20160805

  1. Ligation using recombinase

Ligation process were done followed the standardized protocol from the manufacturer, the plasmids and DNA volume were set as follows:

Construct PlasmidDNA Total volume
TRP-ADH-hAR1.6 μl
TRP-ADH(切)0.7 μl
ADH-hAR20 μl

37℃ 0.5h

  1. Transformation of DH5a competent cells
construct nameDNA volumeCompetent cells LB mediaSpread on plate
TRP-ADH-hAR10 μl50 μl600 μl300 μl
GAL-3HA-UG276 μl50 μl600 μl300 μl

20160806

  1. Pick up colonies

TRP-ADH-hAR: 5 GAL-3HA-UG27: 2

  1. Colony PCR of construct TRP-ADH-hAR

20 μl system:

ReagentsVolume
Taq1 μl
Buffer2 μl
5μM dNTPs1.76 μl
Culture1 μl
10 pmol/µl Primer 0.4x2 μl
ddH2O14.04 μl

Primer: TRP-ADH-hAR-R/ TRP-ADH-hAR-F PCR condition was set as follows:

NameTemperature Duration
Pre-denaturation94℃3 min
Denaturation94℃40s30x
Anealing60℃30s 
Extension72℃2min 
Final extension72℃7min
  1. Gel electrophoresis of PCR products in Step.1
GelPCR product6 x Loading bufferVolume loadedDNA ladder
1% agarose20μl4μl10 μl5μl

T--SJTU-BioX-Shanghai--notebook-34.jpg

20160807

  1. Plasmid extraction

TRP-ADH-hAR 1,2,3,4 Elution: 40μl in ddH2O

  1. Endonulease digestion

37℃, 4.5h

Plasmid
(TRP-ADH-hAR 1/LD-BS-HIS 3)12μl
Endonuclease1μl HindIII (HF)
1μl ApaI
Buffer5μl Cutsmart
ddH2O31μl

Gel electrophoresis

GelDigestion product6 x Loading bufferDL15000
1% agarose50μl10μl10μl

T--SJTU-BioX-Shanghai--notebook-35.jpg

  1. Gel recovery

Elution: 40μl in ddH2O T--SJTU-BioX-Shanghai--notebook-36.jpg

  1. Ligation using T4 ligase

Ligation process were done followed the standardized protocol from the manufacturer, the plasmids and DNA volume were set as follows:

Construct PlasmidDNA Total volume
HIS-ADH-hAR6.7 μl LD-BS-HIS3 13 μl TRP-ADH-hAR20 μl

22℃ 1h

  1. Transformation of DH5a competent cells
construct nameDNA volumeCompetent cells LB mediaSpread on plate
HIS-ADH-hAR10 μl50 μl600 μl300 μl

20160808

  1. Pick up colonies

HIS-ADH-hAR: 4 (1pm) + 2 (7:30pm)

  1. Colony PCR of construct TRP-ADH-hAR (4 picked up at 1 pm )

20 μl system:

ReagentsVolume
Taq1 μl
Buffer2 μl
5μM dNTPs1.76 μl
Culture1 μl
10 pmol/µl Primer 0.4x2 μl
ddH2O14.04 μl

Primer: TRP-ADH-hAR-R/ TRP-ADH-hAR-F PCR condition was set as follows:

NameTemperature Duration
Pre-denaturation94℃3 min
Denaturation94℃40s30x
Anealing60℃30s 
Extension72℃2min 
Final extension72℃7min

Gel electrophoresis of PCR products:

GelPCR product6 x Loading bufferVolume loadedDNA ladder
1% agarose20μl4μl10 μl5μl

T--SJTU-BioX-Shanghai--notebook-37.jpg

20160809

  1. Colony PCR of construct TRP-ADH-hAR (3 picked up at 1 pm, 1 picked up at 7:30 pm )

20 μl system:

ReagentsVolume
Taq1 μl
Buffer2 μl
5μM dNTPs1.76 μl
Culture1 μl
10 pmol/µl Primer 0.4x2 μl
ddH2O14.04 μl

Primer: TRP-ADH-hAR-R/ TRP-ADH-hAR-F PCR condition was set as follows:

NameTemperature Duration
Pre-denaturation94℃3 min
Denaturation94℃40s30x
Anealing60℃30s 
Extension72℃2min 
Final extension72℃7min

Gel electrophoresis of PCR products:

GelPCR product6 x Loading bufferVolume loadedDNA ladder
1% agarose20μl4μl10 μl5μl

T--SJTU-BioX-Shanghai--notebook-38.jpg

  1. Plasmid extraction

HIS-ADH-hAR: 2,3 Elution: 40μl in ddH2O T--SJTU-BioX-Shanghai--notebook-39.jpg

  1. Taq PCR of plasmid

20 μl system:

ReagentsVolume
Taq1 μl
Buffer2 μl
5μM dNTPs1.76 μl
Extracted plasmid1 μl
10 pmol/µl Primer 0.4x2 μl
ddH2O14.04 μl

Primer: TRP-ADH-hAR-R/ TRP-ADH-hAR-F PCR condition was set as follows:

NameTemperature Duration
Pre-denaturation94℃3 min
Denaturation94℃40s30x
Anealing60℃30s 
Extension72℃2min 
Final extension72℃7min

Gel electrophoresis of PCR products:

GelPCR product6 x Loading bufferVolume loadedDNA ladder
1% agarose20μl4μl10 μl5μl

T--SJTU-BioX-Shanghai--notebook-40.jpg

  1. re-inoculate yeast strain construct (10:15 pm)
yeast strainconstructmedia
9060-5ml YPD/ YPD agr
 ADH-NLc (9060-4)5ml YPD
 ADH-NLc (ALL)YPD agar
DJ03-5ml YPD/ YPD agr
 ADH-NLc (DJ03-4)5ml YPD
 ADH-NLc (ALL)YPD agar

20160811

  1. Sequencing

T--SJTU-BioX-Shanghai--notebook-41.jpg

20160812

  1. Endonuclease digestion

TRP-CU-NLc/ TRP-GAL-NLc digestion is aimed for transformation of yeast cells.

PlasmidEnzymeBufferddH2O
TRP-CU-NLc 14 μl0.5 μl NheI-HF
0.5 μl SacI-HF5 μl CutSmart9 μl
TRP-GAL-NLc 14 μl0.5 μl NheI-HF
0.5 μl SacI-HF5 μl CutSmart9 μl
TRP-ADH-hAR 112 μl1 μl HindIII-HF
1 μl ApaI5 μl CutSmart31 μl
LD-BS-HIS12 μl1 μl HindIII-HF
1 μl ApaI5 μl CutSmart31 μl


  1. KOD PCR for yeast transformation
TemplatePrimer
HIS-ADH-hAR 3BLKS-HIS F/R
HIS-GAL-hAR 3bBLKS-HIS F/R
KOD buf.5μl
dNTPs5 μl
Mg2+3 μl
Primer1x2 μl
Template1 μl
KOD pol1 μl
ddH2O34 μl
50 μl
  1. Put yeast colonies into liquid media
Namemedia
9060-110 ml YPD
9060-TRP-ADH-NLc-15 ml YPD
9060-TRP-ADH-NLc-25 ml YPD
9060-TRP-ADH-NLc-410 ml YPD
DJ03-TRP-ADH-NLc-410 ml YPD
  1. Make the construct HIS-ADH-hAR again, all procedures were as before.

Endonuclease digestion: DL15000 5μl T--SJTU-BioX-Shanghai--notebook-42.jpg Gel recovery elution volume: 30 μl T--SJTU-BioX-Shanghai--notebook-43.jpg Transformation of DH5a cells: 50 μl cells left after last time usage,and last time’s plate

20160813

  1. Transformation of yeast cells
NameConstructConstuct volumeplate
9060-1TRP-CU-NLc3/ TRP-GAL-NLc320 μlTRP-
9060-TRP-ADH-NLc-1HIS-GAL-hAR20 μlHIS-
9060-TRP-ADH-NLc-2HIS-ADH-hAR20 μlHIS-
9060-TRP-ADH-NLc-4HIS-GAL-hAR/ HIS-ADH-hAR20 μlHIS-
DJ03-TRP-ADH-NLc-4HIS-GAL-hAR/ HIS-ADH-hAR10 μlHIS-
  1. Pick up colonies of construct HIS-ADH-hAR as before

20160814

  1. Colony PCR for construct HIS-ADH-hAR

T--SJTU-BioX-Shanghai--notebook-44.jpg

  1. Plasmid extraction of construct HIS-ADH-hAR

T--SJTU-BioX-Shanghai--notebook-45.jpg

  1. Pick up anthor 10 colonies from plate of construct HIS-ADH-hAR

20160815

  1. Colony PCR of 10 colonies from yesterday
  2. Enzyme digestion of the plasmid extracted yesterday

HindIII-HF ApaI CutSmart T--SJTU-BioX-Shanghai--notebook-46.jpg

20160816

  1. The coelenterazine was put into the liquid nitrogen storage pot, MG group, position 6-1 (Red tube lid).
  2. Pick 8 colonies from TRP-CU-NLc and TRP-GAL-NLc plate and inoculate them on the TRP- plate.
  3. Taq PCR
PrimerTemplate
BLKS-HIS R/FHIS-ADH-hAR 1,2,3,4
HIS-ADH-hAR 3,2
LD-BS-HIS
HIS-GAL-Har 3b
TRP-ADH-hAR R/FHIS-ADH-hAR 1,2,3,4
HIS-ADH-hAR 3,2
TRP-ADH-hAR 1
TRP-GAL-hAR R/FHIS-GAL-Har 3b
TRP-GAL-Har 1
HIS-GAL-Har 2b
  1. Endonuclease digestion
TemplateEndonuclease Buffer
HIS-ADH-hAR 2,4EcoR IGreen FD

T--SJTU-BioX-Shanghai--notebook-47.jpg

20160820

  1. Test of the samples using KOD pcr

T--SJTU-BioX-Shanghai--notebook-48.jpg

20160827

  1. Transformation of ADH-NLc1/GAL-NLc1/CU-NLc1 yeast construct

His-GAL-Har 2b/2c/3b Endonuclease digestion

TemplateEndonuclease Buffer
His-GAL-Har 2b/2c/3bAflII/ NcoI3.1
  1. TRP-ADH plasmid
TemplateEndonuclease BufferTemplate V
TRP-ADH plasmidClaI/AscICutSmart20μl
  1. KOD PCR of hAR
TemplateprimerTm
hG/A549ADH-hAR-F/R58
  1. Ligation using recombinase

ADH-hAR (not cleaned or gel recovered) + TRP-ADH (previous)

  1. Transformation of DH5a

20160828

  1. Glucose sensing detection using plate reader

ADH GAL ADH/GAL/9060-1 9060-1 0 5 25 50 100 200

  1. Characterizing of GAL/CU/9060-1 under flo

20160829

  1. Enzyme digestion and gel recovery of TRP-ADH-hAR
  2. Ligation using T4 DNA ligase

TRP-ADH-hAR (gel recovered) + HIS (previous)

20160831

  1. Glucose sensing detection using plate reader

36h in YPGal ADH GAL CU 9060-1 0 5 25 50 100 200

  1. Colony PCR of HIS-ADH-hAR/TRP-ADH-hAR
  2. Freeze-dring of yest strain ADH-NLc/GAL-NLc/CU-Nlc/9060-1
  3. Revitalization of freeze-dried yeast strain CU-NLc

3h in culture + cryoprotectant -> 100μl in 5ml YPD

20160901

  1. Plasmid extraction of TRP-ADH-hAR/HIS-ADH-hAR
  2. Endonuclease digestion of HIS-ADH-hAR 1/2/3 and transformation of yeast strain TRP-ADH-NLc
  3. Freeze drying of yeast strain TRP-ADH-NLc and 9060-1

20160902

  1. Shake the yeast strain TRP-ADH-NLc1/TRP-GAL-NLc2/TRP-CU-NLc4/9060-1 for freeze drying and reactivation test

20160904

  1. KOD PCR of ADH-NLc 1 / GAL-NLc 2 / CU-NLc 4 / 9060 1
KOD buf.5μl
dNTPs5 μl
Mg2+3 μl
Primer1x2 μl
Template1 μl
KOD pol1 μl
ddH2O33 μl
Total50 μl
NameTemperature Duration
Pre-denaturation95℃5 min
Denaturation95℃40s39x
Anealing58℃30s 
Extension68℃2min 
Final extension72℃10min
Hold16℃infinite
NumberPrimerTemplate (each 3 tubes)
1CU-hAR-R/CU-hAR-F2TRP-ADH-hARTRP-ADH-hAR
2GAL-hAR-Rhis/GAL-hAR-FTRP-ADH-hARhG DNA
3GAL-hAR-R/GAL-hAR-FTRP-ADH-hARhG DNA
4ADH-hAR-Rhis/ADH-hAR-FhisTRP-ADH-hARhG DNA
  1. 2 displays no result
  2. Ligation using recombinase and transformation of DH5a cells
VectorDNA fragment
TRP-GAL (hAR cut)hAR (GAL)
TRP-ADH (hARcut)hAR-his (ADH)
  1. Colony PCR of transformed yeast cells (HIS-ADH-hAR)

primer: ADH-hAR-R/ADH-hAR-F

  1. Inoculate 4 colonies (ADH-NLc/ GAL-NLc/ CU-NLc/ 9060) in YPGal media

20160905

  1. Inoculate 4 colonies (ADH-hAR1/2/3/4) in YPD media
  2. Colony PCR of the construct TRP-GAL-hAR/TRP-ADH-hAR-his (failed, used DL15000 as ladder )

20160906

  1. Epinephrine response measurement

Culture process: 16 h in YPD -> 1 h in YPGal

EP (μM)ADH-hARaADH-hARbADH-hARcADH-hARdADH-NLc9060
0            
10            
25            
50            
75            
100            
500            
1000           

200 μl total volume, coelenterazine h in 10 μM final concentration

  1. Colony PCR of the construct TRP-GAL-hAR/TRP-ADH-hAR-his, extract the plasmids

20160907~20160911

  1. Construction of yeast genomic integration plasmid HIS-ADH-hAR-his, HIS-GAL-hAR-his and HIS-ADH-Gpa2M

20160912~20160918

  1. Transformation of yeast cells with HIS-ADH-hAR-his, HIS-GAL-hAR-his and HIS-ADH-Gpa2M and measure the response ability in glucose or epinephrine receptor or mimic pathological solutions.
  2. Test of the response ability after freeze drying process.

20160912~20160918

  1. Making our parts in pSB1C3 to finish the final step!

20160919~20161023

  1. Synthesized DNA from GENEWIZ to make further modification of ADRB2
  2. Dealing with the wiki work and presentation draft.
  3. Western blotting of His-tagged proteins and Immunocytochemistry analysis of membrane localization.

20161023~20161025

  1. Get good sleep to compensate for the coming jet-lag.

20161026

  1. Set off for Giant-Jamboree!