Team:SJTU-BioX-Shanghai/Notebook

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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 1.6	NLc
human adrenergic receptor beta 2 （ADRB2)	hAR
yeast nucleotide binding alpha subunit (Gpa2) with 5 amino acids modification	Gpa2M
Yeast TRP section marker	TRP
Yeast HIS section marker	HIS
Yeast LEU section marker	LEU
6 x His-tag	his

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)

20160626

1. Plasmid extraction of pcDNA3-Nanolantern

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

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

20160629

1. Plasmid extraction of TRP-pADH1-NLc

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.-

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.

	Volume	OD600	Control/Experimetnal	Digested DNA
DJ03	3ml	0.9	1ml/2ml	0μl/10μl
9060	3ml	0.8	1ml/2ml	0μl/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.

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

20160714

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

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

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.

20160723

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

20160726

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

1. Gel extortion of ADRB2 gene

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

Reagents	Volume
KOD plus neo	1 μl
10x Buffer for KOD -Plus- Neo	5 μl
2mM dNTPs	5 μl
25mM MgSO4	3 μl
hAR	10 μl
10 pmol/µl Primer	1x2 μl
ddH2O	24 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

1. Gel extraction of endonuclease digested product

20160727

1. PCR amplification of ADRB2 gene.

450px

1. PCR cleaning

1. Gel electrophoresis of product in 3.

1. Gel extraction

20160728

1. ADRB2 gene fragment PCR

Name	Primer	Tm	Template(μl)	ddH2O (μl)	Total(μl)
hAR	hAR-R/hAR-F	48℃	(48℃) 2.4	11.2	20
ADH-hAR	TRP-ADH-hAR-R/ TRP-ADH-hAR-F	48℃	(48℃) 6	28	50
ADH-hAR		58℃	(58℃) 3	31	50
ADH-hAR		65℃	(48℃) 6	28	50

Cycle：

Name	Temperature	Duration
Pre-denaturation	95℃	3 min
Denaturation	95℃	40s	39x
Anealing	48/58/65℃	30s
Extension	68℃	1min 30s
Final extension	72℃	10min

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.

1. Gel recovery

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

Gel	DNA sample volume (after PCR cleaning)	6 x Loading buffer	DL2000
1% agarose	30 μl	6 μl	10 μl

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 volume	Competent cells	LB media	Spread on plate
10 μl	100 μl	600 μl	350 μl

20160729

1. Pick up positive colonies

Strains	Positive 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：

Reagents	Volume
Taq	1 μl
Buffer	2 μl
5μM dNTPs	1.76 μl
Culture	1 μl
10 pmol/µl Primer	0.4x2 μl
ddH2O	14.04 μl

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

Name	Temperature	Duration
Pre-denaturation	94℃	3 min
Denaturation	94℃	40s	35x
Anealing	60℃	30s
Extension	72℃	2min
Final extension	72℃	7min

1. Gel electrophoresis of PCR products in Step.1

Gel	PCR product	6 x Loading buffer	DNA ladder
1% agarose	20μl	4μl	5μl

1. Plasmid extraction

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

1. Endonuclease digestion

Plasmid	12μl
Endonuclease	1μl HindIII (HF)
1μl ApaI
Buffer	5μl Cutsmart
ddH2O	31μl

Gel electrophoresis

Gel	Digestion product	6 x Loading buffer	DL2000/15000
1% agarose	50μl	10μl	10μl

1. Gel recovery

Elution: 15μl in ddH2O

20160731

1. Ligation

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

Construct	Plasmid	DNA	Total volume
HIS2-GAL2	6.7 μl HIS2	9 μl GAL2	20 μl
HIS3-GAL3	7.4 μl HIS3	9.6 μl GAL3	20 μl

22℃ 1h

1. Transformation of DH5a competent cells

DNA volume	Competent cells	LB media	Spread on plate
10 μl	50 μl	300 μl	360 μl

20160801

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

20 μl system：

Reagents	Volume
Taq	1 μl
Buffer	2 μl
5μM dNTPs	1.76 μl
Culture	1 μl
10 pmol/µl Primer	0.4x2 μl
ddH2O	14.04 μl

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

Name	Temperature	Duration
Pre-denaturation	94℃	3 min
Denaturation	94℃	40s	30x
Anealing	60℃	30s
Extension	72℃	2min
Final extension	72℃	7min

1. Gel electrophoresis of PCR products in Step.1

Gel	PCR product	6 x Loading buffer	DNA ladder
1% agarose	20μl	4μl	5μl

10μl/well

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

	Name	volume（μl）
template	GAL-hAR	0.5
primer	TRP-ADH-Har	1

PCR conditions：

Name	Temperature	Duration
Pre-denaturation	95℃	3 min
Denaturation	95℃	40s	39x
Anealing	48℃	30s
Extension	68℃	1min 30s
Final extension	72℃	10min

electrophoresis：

Gel	PCR product	6 x Loading buffer	DL2000
1% agarose	5μl	1μl	5μl

1. PCR cleaning

Elution volume: 30μl；3 in 1 tube

1. Gel recovery

electrophoresis：

Gel	PCR cleaning product	6 x Loading buffer	DL2000
1% agarose	30μl	6μl	10μl

gel recovery：

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	Plasmid	DNA	Total volume
TRP-ADH-hAR	1.6 μl
TRP-ADH（切）	0.7 μl
ADH-hAR	20 μl

37℃ 0.5h

1. Transformation of DH5a competent cells

construct name	DNA volume	Competent cells	LB media	Spread on plate
TRP-ADH-hAR	10 μl	50 μl	600 μl	300 μl
GAL-3HA-UG27	6 μl	50 μl	600 μl	300 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：

Reagents	Volume
Taq	1 μl
Buffer	2 μl
5μM dNTPs	1.76 μl
Culture	1 μl
10 pmol/µl Primer	0.4x2 μl
ddH2O	14.04 μl

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

Name	Temperature	Duration
Pre-denaturation	94℃	3 min
Denaturation	94℃	40s	30x
Anealing	60℃	30s
Extension	72℃	2min
Final extension	72℃	7min

1. Gel electrophoresis of PCR products in Step.1

Gel	PCR product	6 x Loading buffer	Volume loaded	DNA ladder
1% agarose	20μl	4μl	10 μl	5μl

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
Endonuclease	1μl HindIII (HF)
1μl ApaI
Buffer	5μl Cutsmart
ddH2O	31μl

Gel electrophoresis

Gel	Digestion product	6 x Loading buffer	DL15000
1% agarose	50μl	10μl	10μl

1. Gel recovery

Elution: 40μl in ddH2O

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	Plasmid	DNA	Total volume
HIS-ADH-hAR	6.7 μl LD-BS-HIS3	13 μl TRP-ADH-hAR	20 μl

22℃ 1h

1. Transformation of DH5a competent cells

construct name	DNA volume	Competent cells	LB media	Spread on plate
HIS-ADH-hAR	10 μl	50 μl	600 μl	300 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：

Reagents	Volume
Taq	1 μl
Buffer	2 μl
5μM dNTPs	1.76 μl
Culture	1 μl
10 pmol/µl Primer	0.4x2 μl
ddH2O	14.04 μl

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

Name	Temperature	Duration
Pre-denaturation	94℃	3 min
Denaturation	94℃	40s	30x
Anealing	60℃	30s
Extension	72℃	2min
Final extension	72℃	7min

Gel electrophoresis of PCR products:

Gel	PCR product	6 x Loading buffer	Volume loaded	DNA ladder
1% agarose	20μl	4μl	10 μl	5μl

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：

Reagents	Volume
Taq	1 μl
Buffer	2 μl
5μM dNTPs	1.76 μl
Culture	1 μl
10 pmol/µl Primer	0.4x2 μl
ddH2O	14.04 μl

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

Name	Temperature	Duration
Pre-denaturation	94℃	3 min
Denaturation	94℃	40s	30x
Anealing	60℃	30s
Extension	72℃	2min
Final extension	72℃	7min

Gel electrophoresis of PCR products:

Gel	PCR product	6 x Loading buffer	Volume loaded	DNA ladder
1% agarose	20μl	4μl	10 μl	5μl

1. Plasmid extraction

HIS-ADH-hAR: 2,3 Elution: 40μl in ddH2O

1. Taq PCR of plasmid

20 μl system：

Reagents	Volume
Taq	1 μl
Buffer	2 μl
5μM dNTPs	1.76 μl
Extracted plasmid	1 μl
10 pmol/µl Primer	0.4x2 μl
ddH2O	14.04 μl

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

Name	Temperature	Duration
Pre-denaturation	94℃	3 min
Denaturation	94℃	40s	30x
Anealing	60℃	30s
Extension	72℃	2min
Final extension	72℃	7min

Gel electrophoresis of PCR products:

Gel	PCR product	6 x Loading buffer	Volume loaded	DNA ladder
1% agarose	20μl	4μl	10 μl	5μl

1. re-inoculate yeast strain construct (10:15 pm)

yeast strain	construct	media
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

20160812

1. Endonuclease digestion

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

	Plasmid	Enzyme	Buffer	ddH2O
TRP-CU-NLc 1	4 μl	0.5 μl NheI-HF
0.5 μl SacI-HF	5 μl CutSmart	9 μl
TRP-GAL-NLc 1	4 μl	0.5 μl NheI-HF
0.5 μl SacI-HF	5 μl CutSmart	9 μl
TRP-ADH-hAR 1	12 μl	1 μl HindIII-HF
1 μl ApaI	5 μl CutSmart	31 μl
LD-BS-HIS	12 μl	1 μl HindIII-HF
1 μl ApaI	5 μl CutSmart	31 μl

1. KOD PCR for yeast transformation

Template	Primer
HIS-ADH-hAR 3	BLKS-HIS F/R
HIS-GAL-hAR 3b	BLKS-HIS F/R

KOD buf.	5μl
dNTPs	5 μl
Mg2+	3 μl
Primer	1x2 μl
Template	1 μl
KOD pol	1 μl
ddH2O	34 μl
总	50 μl

1. Put yeast colonies into liquid media

Name	media
9060-1	10 ml YPD
9060-TRP-ADH-NLc-1	5 ml YPD
9060-TRP-ADH-NLc-2	5 ml YPD
9060-TRP-ADH-NLc-4	10 ml YPD
DJ03-TRP-ADH-NLc-4	10 ml YPD

1. Make the construct HIS-ADH-hAR again, all procedures were as before.

Endonuclease digestion: DL15000 5μl Gel recovery elution volume: 30 μl Transformation of DH5a cells: 50 μl cells left after last time usage，and last time’s plate

20160813

1. Transformation of yeast cells

Name	Construct	Constuct volume	plate
9060-1	TRP-CU-NLc3/ TRP-GAL-NLc3	20 μl	TRP-
9060-TRP-ADH-NLc-1	HIS-GAL-hAR	20 μl	HIS-
9060-TRP-ADH-NLc-2	HIS-ADH-hAR	20 μl	HIS-
9060-TRP-ADH-NLc-4	HIS-GAL-hAR/ HIS-ADH-hAR	20 μl	HIS-
DJ03-TRP-ADH-NLc-4	HIS-GAL-hAR/ HIS-ADH-hAR	10 μl	HIS-

1. Pick up colonies of construct HIS-ADH-hAR as before

20160814

1. Colony PCR for construct HIS-ADH-hAR

1. Plasmid extraction of construct HIS-ADH-hAR

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

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

Primer	Template
BLKS-HIS R/F	HIS-ADH-hAR 1,2,3,4
HIS-ADH-hAR 3,2
LD-BS-HIS
HIS-GAL-Har 3b
TRP-ADH-hAR R/F	HIS-ADH-hAR 1,2,3,4
HIS-ADH-hAR 3,2
TRP-ADH-hAR 1
TRP-GAL-hAR R/F	HIS-GAL-Har 3b
TRP-GAL-Har 1
HIS-GAL-Har 2b

1. Endonuclease digestion

Template	Endonuclease	Buffer
HIS-ADH-hAR 2,4	EcoR I	Green FD

20160820

1. Test of the samples using KOD pcr

20160827

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

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

Template	Endonuclease	Buffer
His-GAL-Har 2b/2c/3b	AflII/ NcoI	3.1

1. TRP-ADH plasmid

Template	Endonuclease	Buffer	Template V
TRP-ADH plasmid	ClaI/AscI	CutSmart	20μl

1. KOD PCR of hAR

Template	primer	Tm
hG/A549	ADH-hAR-F/R	58

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
dNTPs	5 μl
Mg2+	3 μl
Primer	1x2 μl
Template	1 μl
KOD pol	1 μl
ddH2O	33 μl
Total	50 μl

Name	Temperature	Duration
Pre-denaturation	95℃	5 min
Denaturation	95℃	40s	39x
Anealing	58℃	30s
Extension	68℃	2min
Final extension	72℃	10min
Hold	16℃	infinite

Number	Primer	Template (each 3 tubes)
1	CU-hAR-R/CU-hAR-F2	TRP-ADH-hAR	TRP-ADH-hAR
2	GAL-hAR-Rhis/GAL-hAR-F	TRP-ADH-hAR	hG DNA
3	GAL-hAR-R/GAL-hAR-F	TRP-ADH-hAR	hG DNA
4	ADH-hAR-Rhis/ADH-hAR-Fhis	TRP-ADH-hAR	hG DNA

1. 2 displays no result
2. Ligation using recombinase and transformation of DH5a cells

Vector	DNA 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-hARa	ADH-hARb	ADH-hARc	ADH-hARd	ADH-NLc	9060
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!

Bio-X Institutes

Shanghai Jiao Tong University

800, Dongchuan Road 200240, Shanghai, China

sherlockdoylecaoty@sjtu.edu.cn