Team:USTC/Notebook/8 1

Modeling

Everybody

Date: 8.1

Recorder: Chengle Zhang & Yu Xie Colony picking of Kozak

Pick 10 colonies of a plate containing Kozak in 10 test tubes (5 mL LB liquid medium, Cm+).
Cultivate the bacteria at 37°C and shock at 250 rpm/min.

Recorder：Yin Wu Double Digestion of sfGFP1-10 and KOZAK

Procedure: Add the materials into new EP tubes in the order of following table respectively.

sample	1	2	3	4
nuclease free water(μL)	14	14	14	14
10× Buffer(μL)	4	4	4	4
plasmid of KOZAK	0	0	20	20
sfGFP1-10	20	20	0	0
PstI(μL)	1	1	1	1
XbaI(μL)	1	1	0	0
BcuI(μL)	0	0	1	1
total(μL)	40	40	40	40

Mix gently and incubate at 37 degree Celsius for 15 mins .

Agarose gel electrophoresis Results: 图片名称 Agarose gel electrophoresis of digestion of sfGFP1-10 and KOZAK (from left to right:marker, sample 1-8)

Recorder: Kaiyue Ma Double Digestion of pET28a plasmid containing sup35

Procedure: Add the materials into new PCR tubes in the order of following table respectively.

sample	1	2
10× Green Buffer(μL)	2	2
plasmid	17	17
EcoRI(μL)	0.5	0.5
XbaI(μL)	0.5	0.5
total(μL)	20	20

Mix gently and incubate at 37 degree Celsius The thermal cycler. 30 minutes for tube 1, and 1 hour for tube 2.

Recorder: Yin Wu, Ya Jiang Gel Extraction of digestion product of sfGFP and KOZAK

Procedure: 1. Cut off the Gel part containing the target band. Get 8 tubes of digestion product. 2. Weigh the cut off Gel part. 3. Add Buffer B2 whose weight is 3 times of the Gel. 4. Add isopropanol whose volume is 1/3 of the Buffer B2. 5. Move the solution to adsorption column , 11000 rpm centifuge 30 s. 6.Put filtrate into the upper part of the same adsorption column,11000 rpm centifuge 30,discard filtrate. 7. Add 300 μL Buffer B2,11000 rpm centifuge 30 s, discard filtrate. 8. Add 500 μL Wash Solution, 12000 rpm centifuge 30 s, discard filtrate. Repeat once. 9. 12000 rpm centifuge 1 min. 10. Lying for 10 min. 11. Put the adsorption column in a new EP tube. Add 20 μL elution buffer which is pre-heated at 60 degree Celsius , 10 min's standing, 12000 rpm centifuge 1 min. Preserve in the EP tube with ddH₂O at 4 degree Celsius.

After extraction, we measured the OD A260/A280 and the concentration of digestion product.

sample	sfGFP1-10-1	sfGFP1-10-2	KOZAK-1	KOZAK-2
A260/A280	3.3	5.8	26.2	11.2
concentration(ng/μL)	2.30	1.76	1.93	2.28

Recorder: Yu Xie Colony picking of PR+GFP （13:10）

Pick 2 colonies of a plate containing PR+GFP in 2 test tubes (5 mL LB liquid medium, kana).
Cultivate the bacteria at 37°C and shock at 250 rpm/min.

Recorder: Chengle Zhang PCR of AD and BD

Experimental materials 1. Plasmid: pGAD-T7 and pGBK-T7(5 μL for each) from laboratory 436 of USTCSLS, Mr Liao; 2. Primer: AD 1, AD 2, BD 1, BD 2. Designed by ourselves, synthesized by Sheng Gong; 3. Sterilized ddH₂O, 2×HiFi-PCR Master, bought from Sheng Gong.

About primers

primer	AD 1	AD 2	BD 1	BD 2
sequence	5'-CCGGAATTCG CGGCCGCTTC TAGATGGCCAAT TTTAATCA AAGTGGGAA TATTGCTG-3'	5'-AACTGCAGCGG CCGCTACTAG >TACTACTCTTTTT TTGGGTTTGGT GGGGTATC-3'	5'-CCGGAATTCGC GGCCGCTTCT AGATGAAGCTACTG TCTTCTATCGAA C-3'	5'-AACTGCAGCGG CCGCTACTAGT ACTACGATACAGT CAACTGTCTTT GACC-3'

Procedure:

1.Prepare 2 PCR tubes and sequentially add：

sample	AD	BD
Sterilized ddH₂O	22 μL	22 μL
2×HiFi-PCR Master	25 μL	25 μL
pGAD-T7 (542.5 μg/mL)	1 μL	0
pGBK-T7 (506.7 μg/mL)	0	1 μL
AD Primer-1(10 μM)	2 μL	0
AD Primer-2(10 μM)	2 μL	0
BD Primer-1(10 μM)	0	2 μL
BD Primer-2(10 μM)	0	2 μL
total	50 μL	50 μL

2.PCR reaction Parameters setting：

stage	temperature	time
step 1	95	10 min
step 2	95	10 s
step 3	55	15 s
step 4	72	45 s
step 5	72	10 min
step 6	4	--

30 cycles(step 2 ~ step 4)

3.Agarose gel electrophoresis Result:

Recorder:Yinchenguang Lyu, Chengle Zhang Result of Gel Extraction of the product of last step |sample|AD|BD| |-| |concentration(ng/μL)|66.6|7.5| |260/280|1.97|3.48| |260/230|0.09|0.01|

Recorder: Yin Wu Ligation of sfGFP1-10 and KOZAK

Material: 1. double digestion product of sfGFP1-10 2. double digestion product of KOZAK 3. 10× T4 DNA ligase buffer,T4 DNA ligase(bought from Thermo Fisher Scientific) 4. nuclease-free water

Procedure: Add to the sample: 15 μL sfGFP 3 μL KOZAK 2 μL 10* T4 DNA Ligase Buffer 0.25 μL T4 DNA Ligase

Mix gently and incubate at 22 degree Celsius for 1 hour.

Recorder: Yin Wu Transformation of recombinant plasmid of KOZAK and sfGFP1-10

Materials(all made by ourselves): 1. recombinant plasmid of KOZAK and sfGFP1-10 2. SOC media 3. competent cells

Procedure: 1. Put competent cells on ice. Then pre-chill by placing the tubes on ice. 2. Pipet the recombinant plasmid into competent cell tubes, 10 µL for each. 3. Flick the tube gently with your finger to mix. Incubate on ice for 30 minutes. Pre-heat waterbath now to 42°C. 4. Heat-shock the cells by placing into the waterbath for 90 seconds. Be careful to keep the lids of the tubes above the water level, and keep the ice close by. 5. Immediately transfer the tubes back to ice, and incubate on ice for 5 minutes. This helps the cells recover. 6. Add 200 µL of SOC media per tube, and incubate at 37°C for 1 hours. 7. Centrifuge the tube at 4000 rpm for 1 minutes, make the bacteria deposit at the bottom of the tube. 8. Discard 150 μL supernatant liquid and resuspend the bacteria. 9. Coat plate: add the solution to 2 plates (Cm+) and spread it, respectively, then cultivate it overnight.

Recorder: Ya Jiang Colony picking

Pick 4 colonies of a plate containing sfGFP in 4 test tubes (5 mL LB liquid medium, Cm+).
Cultivate the bacteria at 37°C and shock at 250 rpm/min overnight.

Recorder: Yinchenguang Lyu, Yonghao Liang Standardization of sup35

Experimental materials 1. PCR product of sup35, plasmid containing sup35. 2. Primer: standard-sup35-p, standard-sup35-r. Designed by ourselves, synthesized by Sangon Biotech; 3. Sterilized ddH₂O; 4.2×HiFi-PCR Master.

About primers

primer	1	2
sequence	5'-CCGGAATTC GCGGCCGC TTCTAGAT GTCGGATT CAAACCAG GGCAACA-3'	5'-TGCACTGCA GCGGCCGC TACTAGTAA TCATTCACC ACCTCATCGT CAACTTC-3'

Procedure:

1.Prepare 4 PCR tubes and two contianed sample 1, the other two contained sample 2：

sample	1 (plasmid 51.0ng/μL)	2 (plasmid 79.8ng/μL)
Sterilized ddH₂O	22.0 μL	22.0 μL
Template	1 μL	1 μL
standard-sup35-p	1 μL	1 μL
standard-sup35-r	1 μL	1 μL
2×HiFi-PCR Master	25 μL	25 μL
total	50 μL	50 μL

2.PCR reaction Parameters setting：

stage	temperature	time
step 1	95	5 min
step 2	95	1 min
step 3	57	1 min
step 4	72	1 min50 s
step 5	72	10 min
step 6	4	--

35 cycles(step 2 ~ step 4)

results failed. sample: from left to right: marker, sample 2,2(50 μL PCR product+10 μL loading buffer)

Plasmid Extraction for plasmids containing Kozak(BBa_K165002) Recorder: Yonghao Liang ,Chenguang Lyu and Yu Xie

Procedure: 1. Put bacteria solution into two EP tube respectively. Centrifuge bacteria solution at 11000 rpm, few sediment got. Remove the supernatant. Repeat once. 2. Add 250 μL Buffer P1, suspend cells. 3. Add 250 μL Buffer P2, mix well, 3 min's standing. 4. Add 350 μL Buffer P3, mix well. 5. 13400 rpm centrifuge 10 min, move all supernatant to adsorption column, 11000 rpm centrifuge 30 s, discard filtrate. Repeat until all the supernatant from the two tube is adsorped. 6. Add 500 μL Buffer DW1, 12000 rpm centifuge 30 s, discard filtrate. 7. Add 500 μL Wash Solution, 12000 rpm centifuge 30 s, discard filtrate. Repeat once. 8. 12000 rpm centifuge 1 min. 9. Lying for 10 min. 10. Put the adsorption column in a new EP tube. Add 50 μL ddH₂O, 10 min's standing, 12000 rpm centifuge 1 min. Preserve in the EP tube with ddH₂O at 4 degree Celsius.

After extraction, we measured the OD A260/A280 and the density of plasmids.

The OD A260/A280,OD A260/A280 and the concentration of plasmids are as follows:

sample	Kozak-1	Kozak-2	Kozak-3	Kozak-4	Kozak-5	Kozak-6	Kozak-7	Kozak-8	Kozak-9	Kozak-10
A260/A280	1.92	1.81	1.77	1.92	1.92	1.74	1.92	2.07	1.83	1.94
A260/A230	2.05	1.31	1.15	2.03	1.09	0.95	1.52	1.28	0.83	0.34
concentration(ng/L)	41.5	62.6	60.4	38.8	17.5	34.7	29.3	19.6	24.6	18.4

Then we check the plasmid with fastdigest enzyme EcoRI and PstI.

Agarose gel electrophoresis Result:

(from left to right: Kozak-1,Kozak-2,Kozak-3,Kozak-4,Kozak-5,Kozak-6,Kozak-7,Kozak-8,Kozak-9,Kozak-10)

Date: 8.2

Recorder: Kaiyue Ma PCR of sup35

Experimental materials PCR products of sup35, primer 1 & primer 2(10 μM), Sterilized ddH₂O, 2×HiFi-PCR Master

Procedure:

1.Prepare 2 PCR tubes and sequentially add：

sample	1（plasmid extraction; 78 ng/μL）	2（gel extraction;28 ng/μL）
Sterilized ddH₂O	22 μL	21 μL
2×HiFi-PCR Master	25 μL	25 μL
template	1 μL	2 μL
Primer 1	1 μL	1 μL
Primer 2	1 μL	1 μL
total	50 μL	50 μL

2.PCR reaction Parameters setting：

stage	temperature	time
step 1	95	7 min
step 2	95	1 min
step 3	60	1 min
step 4	72	1 min 50 s
step 5	72	10 min
step 6	4	--

30 cycles(step 2 ~ step 4)

Recorder:Han Jinheng Plasmid Extraction for pGAL1-GFP

Materials: 1. 5 mL LB medium containing recombinant plasmid of pGal1 and GFP

Procedure: 1. Put bacteria solution into two EP tube respectively. Centrifuge bacteria solution at 11000 rpm, few sediment got. Remove the supernatant. Repeat once. 2. Add 250 μL Buffer P1, suspend cells. 3. Add 250 μL Buffer P2, mix well, 3 min's standing. 4. Add 350 μL Buffer P3, mix well. 5. 13400 rpm centrifuge 10 min, move all supernatant to adsorption column, 11000 rpm centrifuge 30 s, discard filtrate. Repeat until all the supernatant from the two tube is adsorped. 6. Add 500 μL Buffer DW1, 12000 rpm centifuge 30 s, discard filtrate. 7. Add 500 μL Wash Solution, 12000 rpm centifuge 30 s, discard filtrate. Repeat once. 8. 12000 rpm centifuge 1 min. 9. Lying for 10 min. 10. Put the adsorption column in a new EP tube. Add 50 μL ddH₂O, 10 min's standing, 12000 rpm centifuge 1 min. Preserve in the EP tube with ddH₂O at 4 degree Celsius.

After extraction, we measured the OD A260/A280 and the density of plasmids.

sample	sf-GFP-1	sf-GFP-2	sf-GFP-3	sf-GFP-4
A260/A280	1.87	1.86	1.87	1.86
concentration(ng/μL)	128.8	130.8	138.8	131.2

Recorder：Kaiyue Ma Standardization of sup35

1.Prepare 2 PCR tubes and sequentially add：

sample	1	2
Sterilized ddH₂O	32.5 μL	32.5 μL
5×PrimeSTAR Buffer (Mg²⁺Plus)	10 μL	10 μL
dNTP Mixture (2.5 mM each)	4 μL	4 μL
Template(PCR product)	0	1 μL
Template(plasmid )	1 μL	0
standard-sup35-p	1 μL	1 μL
standard-sup35-r	1 μL	1 μL
PrimeSTAR HS DNA Polymerase (2.5 U/μl)	0.5 μL	0.5 μL
total	50 μL	50 μL

2.PCR reaction Parameters setting：

for tube 1

stage	temperature	time
step 1	95	7 min
step 2	95	10 s
step 3	60	15 s
step 4	72	60 s
step 5	72	10 min
step 6	4	--

30 cycles(step 2 ~ step 4)

for tube 2

stage	temperature	time
step 1	95	7 min
step 2	95	10 s
step 3	57	15 s
step 4	72	50 s
step 5	72	10 min
step 6	4	--

35 cycles(step 2 ~ step 4)

3.result

failed

Recorder：Yonghao Liang **Colony picking of SUP35 **

Pick 1 colonies from 1 plate in 1 test tubes (5 mL LB liquid medium & 5μL kn+).
Cultivate the bacteria at 37°C and shock at 250 rpm/min.

Recorder: Yinchenguang Lyu & Yonghao Liang Agarose gel electrophoresis gel 1. Add 0.40 g agarose to 40 mL TAE buffer. 2. Dissolved by heating. 3. Cool down. 4. Pour into electrophoresis tank. 5. Mix 50 μL PCR product and 10 μL loading buffer. 6. Loading：PCR products: 27 μL : sample1,1,2,2,DNA marker 6 μL. 7. Electrophoresis gel：110 V 30 min. 8. Autoradiography(UV).

Result

Recorder:Yonghao Liang Plasmid Extraction for AP+ GFP and sup35-

Procedure: 1. Put bacteria solution into 3 EP tube respectively. Centrifuge bacteria solution at 11000 rpm, few sediment got. Remove the supernatant. Repeat once. 2. Add 250 μL Buffer P1, suspend cells. 3. Add 1 μL VisualLyse. 4. Add 250 μL Buffer P2, mix well, 3 min's standing. 5. Add 350 μL Buffer P3, mix well. 6. 13400 rpm centrifuge 10 min, move all supernatant to adsorption column, 11000 rpm centrifuge 30 s, discard filtrate. Repeat until all the supernatant from the two tube is adsorped. 7. Add 500 μL Buffer DW1, 12000 rpm centifuge 30 s, discard filtrate. 8. Add 500 μL Wash Solution, 12000 rpm centifuge 30 s, discard filtrate. Repeat once. 9. 12000 rpm centifuge 1 min. 10. Lying for 10 min. 11. Put the adsorption column in a new EP tube. Add 50 μL ddH₂O , 10 min's standing, 12000 rpm centifuge 1 min. Preserve in the EP tube with ddH₂O at 4 degree Celsius.

After extraction, we measured the OD A260/A280 and the concentration of plasmids.

sample	1	2
A260/A280	1.90	1.89
concentration(ng/μL)	41.6	21.5

Recorder: Chengle Zhang Double digestion of plasmid containing kozak sequence

Reaction system: 35 μL plasmid(about 50 ng/μL), 5 μL 10× Green Buffer,1 μL SpeI,1 μL PstI.

Agarose gel electrophoresis Result: 图片名称

Then we did gel extraction,the parameters of product are as follows:

sample	1	2
A260/A280	3.31	2.58
A260/A230	0.02	1.02
concentration(ng/μL)	4.2	13.1

Recorder: Chengle Zhang PCR of AD and BD

Experimental materials 1. Plasmid: pGAD-T7 and pGBK-T7 from laboratory 436 of USTCSLS, Mr Liao; 2. Primer: AD 1, AD 2, BD 1, BD 2. Designed by ourselves, synthesized by Sheng Gong; 3. Sterilized ddH₂O, 2×HiFi-PCR Master, bought from Sheng Gong.

About primers

primer	AD 1	AD 2	BD 1	BD 2
sequence	5'-CCGGAAT TCGCGGCCGCTT CTAGATGG CCAATTTT AATCAAA GTGGGAAT ATTGCTG-3'	5'-AACTGCAGC GGCCGCTAC TAGTACTAC TCTTTTTTT GGGTTTGGT GGGGTATC-3'	5'-CCGGAATTC GCGGCCGCTT CTAGATGA AGCTACTG TCTTCTA TCGAAC-3'	5'-AACTGCAGC GGCCGCTAC TAGTACTAC GATACAGT CAACTGTC TTTGACC-3'

Procedure:

1.Prepare 4 PCR tubes and sequentially add：

sample	AD-1	AD-2	BD-1	BD-2
Sterilized ddH₂O	22 μL	22 μL	22 μL	22 μL
2×HiFi-PCR Master	25 μL	25 μL	25 μL	25 μL
pGAD-T7	1 μL	1 μL	0	0
pGBK-T7	0	0	1 μL	1 μL
AD Primer-1(10 μM)	2 μL	2 μL	0	0
AD Primer-2(10 μM)	2 μL	2 μL	0	0
BD Primer-1(10 μM)	0	0	2 μL	2 μL
BD Primer-2(10 μM)	0	0	2 μL	2 μL
total	50 μL	50 μL	50 μL	50 μL

2.PCR reaction Parameters setting：

stage	temperature	time
step 1	95	10 min
step 2	95	1 min
step 3	59	1 min
step 4	72	54 s
step 5	72	10 min
step 6	4	--

30 cycles(step 2 ~ step 4)

3.Agarose gel electrophoresis Result:

(from left to right:AD-1,AD-1,AD-2,AD-2,BD-1,BD-1,AD-2,AD-2)

Then we did gel extraction only to BD,the parameters of product are as follows:

sample	BD-1	BD-2
A260/A280	1.83	1.85
A260/A230	0.91	0.26
concentration(ng/μL)	75.2	16.6

Recorder: Ya Jiang PCR of bacteria containing recombinant plasmid of Kozak and sfGFP1-10

About primers

primer	1(VF2)	2(VR)
sequence	5'- TGCCACC TGACGTC TAAGAA-3'	5'-ATTACCGC CTTTGAGT GAGC-3'

Procedure:

1.Prepare 7 PCR tubes and sequentially add：

sample	1,2,3,4,5,6,7
Sterilized ddH₂O	32 μL
5×PrimeSTAR Buffer (Mg²⁺Plus)	10 μL
dNTP Mixture (2.5 mM each)	42 μL
Template(bacteria)	1.5 μL
Primer 1	1 μL
Primer 2	1 μL
PrimeSTAR HS DNA Polymerase (2.5 U/μl)	0.5 μL
total	50 μL

2.PCR reaction Parameters setting：

stage	temperature	time
step 1	95	5 min
step 2	95	10 s
step 3	55	15 s
step 4	72	70 s
step 5	72	10 min
step 6	4	--

30 cycles(step 2 ~ step 4)

Agarose gel electrophoresis Results: 图片名称 Agarose gel electrophoresis of PCR product (from left to right: marker,sample 1,2,3,4,5,6,7)

Recorder: Chengle Zhang PCR of AD

Experimental materials 1. Plasmid: pGAD-T7 from laboratory 436 of USTCSLS, Mr Liao; 2. Primer: AD 1, AD 2. Designed by ourselves, synthesized by Sheng Gong; 3. Sterilized ddH₂O, 2×HiFi-PCR Master, bought from Sheng Gong.

About primers

primer	AD 1	AD 2
sequence	5'-CCGGAAT TCGCGGCCG CTTCTAGAT GGCCAATTT TAATCAAAGTG GGAATATTGCTG-3'	5'-AACTGCA GCGGCCGCT ACTAGTACT ACTCTTTTT TTGGGTTTGGT GGGGTATC-3'	5'-CCGGAAT TCGCGGCCG CTTCTAGAT GAAGCTACT GTCTTCTATCG AAC-3'	5'-AACTGCA GCGGCCGCT ACTAGTACT ACGATACAG TCAACTGTCTT TGACC-3'

Procedure:

1.Prepare 2 PCR tubes and sequentially add：

sample	AD-1	AD-2
Sterilized ddH₂O	22 μL	22 μL
2×HiFi-PCR Master	25 μL	25 μL
pGAD-T7	1 μL	1 μL
AD Primer-1(10 μM)	2 μL	2 μL
AD Primer-2(10 μM)	2 μL	2 μL
total	50 μL	50 μL

2.PCR reaction Parameters setting：

stage	temperature	time
step 1	95	10 min
step 2	95	1 min
step 3	55	1 min
step 4	72	54 s
step 5	72	10 min
step 6	4	--

30 cycles(step 2 ~ step 4)

3.Agarose gel electrophoresis Result:

(from left to right:AD-1,AD-1,AD-2,AD-2)

Then we did gel extraction,the parameters of product are as follows:

sample	AD-1	AD-2
A260/A280	1.87	1.83
A260/A230	0.31	0.16
concentration(ng/μL)	61.8	28.1

Recorder: Ya Jiang Transformation of plasmids pUC57 containing sfGFP11

Materials(all made by ourselves): 1. plasmids pUC57 containing sfGFP11 2. SOC media 3. competent cells

Procedure: 1. Put competent cells on ice for 3-5 min. 2. Pipet the recombinant plasmid into competent cell tubes, 1 µL for each. 3. Flick the tube gently with your finger to mix. Incubate on ice for 30 minutes. Pre-heat waterbath now to 42°C. 4. Heat-shock the cells by placing into the waterbath for 90 seconds. 5. Immediately transfer the tubes back to ice, and incubate on ice for 5 minutes. This helps the cells recover. 6. Add 200 µL of SOC media per tube, and incubate at 37°C for 45 min. 7. Add bacteria to LB liquid media (5mL). Incubate at 37°C over night.

Recorder: Kaiyue Ma Gel extraction of standard sup35NM

sample	1
A260/A280	1.91
concentration(ng/μL)	77.6

I AM SOOOO AWESOME!

Recorder: Yin Wu and Chengle Zhang PCR of AD and BD

Experimental materials 1. Plasmid: pGAD-T7 and pGBK-T7 from laboratory 436 of USTCSLS, Mr Liao; 2. Primer: AD 1, AD 2, BD 1, BD 2. Designed by ourselves, synthesized by Sheng Gong; 3. Sterilized ddH₂O, 2×HiFi-PCR Master, bought from Sheng Gong.

About primers

primer	AD-1	AD-1	BD-1	BD-1
sequence	5'-CCGGAATT CGCGGCCGC TTCTAGATG GCCAATTT TAATCAAAGTG GGAATATTGCTG-3'	5'-AACTGCAG CGGCCGCTA CTAGTACTA TTTTTTTG GGTTTGGTGGG GTATC-3'	5'-CCGGAATT CGCGGCCGC TTCTAGATG AAGCTACT GTCTTCTATCG AAC-3'	5'-AACTGCAG CGGCCGCTA CTAGTACTA CGATACAG TCAACTGTCTTTGAC C-3'

Procedure:

1.Prepare 4 PCR tubes and sequentially add：

sample	AD-1	AD-1	BD-1	BD-1
Sterilized ddH₂O	22 μL	22 μL	22 μL	22 μL
2×HiFi-PCR Master	25 μL	25 μL	25 μL	25 μL
pGAD-T7	1 μL	1 μL	0	0
pGBK-T7	0	0	1 μL	1 μL
AD Primer-1(10 μM)	2 μL	2 μL	0	0
AD Primer-2(10 μM)	2 μL	2 μL	0	0
BD Primer-1(10 μM)	0	0	2 μL	2 μL
BD Primer-2(10 μM)	0	0	2 μL	2 μL
total	50 μL	50 μL	50 μL	50 μL

2.PCR reaction Parameters setting：

stage	temperature	time
step 1	95	10 min
step 2	95	1 min
step 3	57	1 min
step 4	72	54 s
step 5	72	10 min
step 6	4	--

30 cycles(step 2 ~ step 4)

3.Agarose gel electrophoresis Result:

(from left to right:AD-1,AD-1,AD-1,AD-1,BD-1,BD-1,BD-1,BD-1)

Date: 8.3

Recorder: Kaiyue Ma Results of IPTG induction of sup35NM+GFP

The result shows the induction succeeded. We are ready to do the characterization experiments.

Recorder: Yin Wu Plasmid Extraction for pUC57-sfGFP11

Materials: 1. 5 mL LB medium containing plasmid pUC57-sfGFP11

Procedure: 1. Put bacteria solution into two EP tube respectively. Centrifuge bacteria solution at 11000 rpm, few sediment got. Remove the supernatant. Repeat once. 2. Add 250 μL Buffer P1, suspend cells. 3. Add 250 μL Buffer P2, mix well, 3 min's standing. 4. Add 350 μL Buffer P3, mix well. 5. 13400 rpm centrifuge 10 min, move all supernatant to adsorption column, 11000 rpm centrifuge 30 s, discard filtrate. Repeat until all the supernatant from the two tube is adsorped. 6. Add 500 μL Buffer DW1, 12000 rpm centifuge 30 s, discard filtrate. 7. Add 500 μL Wash Solution, 12000 rpm centifuge 30 s, discard filtrate. Repeat once. 8. 12000 rpm centifuge 1 min. 9. Lying for 10 min. 10. Put the adsorption column in a new EP tube. Add 50 μL ddH₂O, 10 min's standing, 12000 rpm centifuge 1 min. Preserve in the EP tube with ddH₂O at 4 degree Celsius.

After extraction, we measured the OD A260/A280 and the density of plasmids.

sample	sfGFP11 -1	sfGFP11 -2
A260/A280	1.87	1.88
concentration(ng/μL)	187.1	208.1

Recorder: Yu Xie Double digestion of sup35

Reaction system:

sample	10 μL Sup35(77 ng/μL)
nuclease free water(μL)	6
10×Buffer(μL)	2
PstI(μL)	1
XbaI(μL)	1
total(μL)	20

Agarose gel electrophoresis Result: 图片名称

Then we did gel extraction,the parameters of product are as follows:

sample	sup35-1
A260/A280	2.10
A260/A230	0.02
concentration(ng/μL)	13.1

Recorder: Yinchenguang Lyu Gel Extraction of digestion product of PCR of AD and BD

Result |sample|AD|BD| |-| |A260/A280|1.87|1.73| |A260/A230|0.25|0.05| |concentration(ng/μL)|107.3|37.9|

Recorder: Yonghao Liang Plasmid Extraction for pr+sup35

Materials:5 mL LB medium containing recombinant plasmid of pr+gfp

Procedure: 1. Put bacteria solution into two EP tube respectively. Centrifuge bacteria solution at 11000 rpm, few sediment got. Remove the supernatant. Repeat once. 2. Add 250 μL Buffer P1, suspend cells. 3. Add 250 μL Buffer P2, mix well, 3 min's standing. 4. Add 350 μL Buffer P3, mix well. 5. 13400 rpm centrifuge 10 min, move all supernatant to adsorption column, 11000 rpm centrifuge 30 s, discard filtrate. Repeat until all the supernatant from the two tube is adsorped. 6. Add 500 μL Buffer DW1, 12000 rpm centifuge 30 s, discard filtrate. 7. Add 500 μL Wash Solution, 12000 rpm centifuge 30 s, discard filtrate. Repeat once. 8. 12000 rpm centifuge 1 min. 9. Lying for 10 min. 10. Put the adsorption column in a new EP tube. Add 50 μL ddH₂O, 10 min's standing, 12000 rpm centifuge 1 min. Preserve in the EP tube with ddH₂O at 4 degree Celsius.

After extraction, we measured the OD A260/A280 and the density of plasmids.

sample	pr+gfp
A260/A280	1.93
concentration(ng/μL)	37.5

Recorder:Kaiyue Ma & Yonghao Liang Characterization Experiments of Sup35+GFP Procedure: 1. Sample 1 is the control. 2. Sample 2 has been heat at 42 degree for 10 mins. 3. Observe under fluorescence microscope.

Results: Control: 图片名称 Sup35+GFP: Conclusion: 1. Size of E.coli is too small that we can not tell whether the GFP have aggregate or not. 2. The induction is too strong that it expresses too much GFP . 3. Maybe GFP will not aggregate in E.coli.

Recorder: Kaiyue Ma Characterization of sup35NM in E. coli 1. Add 5ul bacteria to 5 mL LB liquid medium (kn+) 2. Cultivate at 37 °C for 2.5 hrs, shock at 250 rpm 3. Add 1ul 23.83 mg/mL IPTG 4. Add 1.5 mL of bacteria to an EP tube, cultivate at 37 °C for 1 hrs, shock at 250 rpm. Get another 1.5 mL, cultivate at 42 °C for 1 hrs

Recorder: Kaiyue Ma&Yonghao Liang Transformation of plasmid pSB1A3

Materials(all made by ourselves): 1. plasmid pSB1A3 2. SOC media 3. competent cells

Procedure: 1. Put competent cells on ice. Then pre-chill by placing the tubes on ice. 2. Pipet the recombinant plasmid into competent cell tubes, 2 µL for each. 3. Flick the tube gently with your finger to mix. Incubate on ice for 30 minutes. Pre-heat waterbath now to 42°C. 4. Heat-shock the cells by placing into the waterbath for 90 seconds. Be careful to keep the lids of the tubes above the water level, and keep the ice close by. 5. Immediately transfer the tubes back to ice, and incubate on ice for 5 minutes. This helps the cells recover. 6. Add 900 µL of SOC media per tube, and incubate at 37°C for 1 hours. 7. Centrifuge the tube at 4000 rpm for 1 minutes, make the bacteria deposit at the bottom of the tube. 8. Discard 950 μL supernatant liquid and resuspend the bacteria. 9. Add 5 μL solution to 3 tubes (Amp) and spread it, respectively(6 tubes in total), then cultivate it overnight.

Recorder: Guanchao Ding & Yu Xie Double digestion of AD and BD

Reaction system:

sample	10 μL AD(107.3 ng/μL)	15 μL BD(77 ng/μL)
nuclease free water(μL)	6	1
10×Buffer(μL)	2	2
PstI(μL)	1	1
XbaI(μL)	1	1
total(μL)	20	20

Failed.

colony picking for PYescGAP Chengle Zhang

Pick 6 colonies of a plate containing PYescGAP in test tubes (5 mL LB liquid medium, Ap+).
Cultivate the bacteria at 37°C and shock at 250 rpm/min overnight.

Recorder: Wenkai Han, Yin Wu PCR of sfGFP1-10

Procedure:

1.Prepare 4 PCR tubes and sequentially add：

sample	1-4
Sterilized ddH₂O	32.5 μL
5×PrimeSTAR Buffer (Mg²⁺Plus)	10 μL
dNTP Mixture (2.5 mM each)	4 μL
Template (sfGFP)	1 μL
standard-sfGFP1-10-p	1 μL
standard-sfGFP1-10-r	1 μL
PrimeSTAR HS DNA Polymerase (2.5 U/μl)	0.5 μL
total	50 μL

2.PCR reaction

Parameters setting：

stage	temperature	time
step 1	95	5 min
step 2	95	10 s
step 3	55	15 s
step 4	72	43 s
step 5	72	10 min
step 6	4	--

30 cycles(step 2 ~ step 4)

Agarose gel electrophoresis Results: 图片名称

Recorder: Yu Xie Ligation of kozak and sup35

Material: 1. double digestion product of kozak 2. double digestion product of sup35 3. 10× T4 DNA ligase buffer,T4 DNA ligase(bought from Thermo Fisher Scientific) 4. nuclease-free water

Procedure: 1.Add to either of samples:

Sample	volume
kozak cut	4μL
sup35	10.6μL
nuclease free water	3.2μL
10* T4 DNA Ligase Buffer	2μL
T4 DNA Ligase	0.2μL

2.Mix gently and incubate at 22 degree Celsius for 1 hour.

Recorder: Yu Xie Transformation of recombinant plasmid of kozak and sup35

Materials: 1. recombinant plasmid of kozak and sup35 2. SOC media 3. competent cells

Procedure: 1. Put competent cells on ice. Then pre-chill by placing the tubes on ice. 2. Pipet the recombinant plasmid into competent cell tubes, 10 µL for each. 3. Flick the tube gently with your finger to mix. Incubate on ice for 30 minutes. Pre-heat waterbath now to 42°C. 4. Heat-shock the cells by placing into the waterbath for 90 seconds. Be careful to keep the lids of the tubes above the water level, and keep the ice close by. 5. Immediately transfer the tubes back to ice, and incubate on ice for 5 minutes. This helps the cells recover. 6. Add 200 µL of SOC media per tube, and incubate at 37°C for 1 hours. 7. Centrifuge the tube at 5500 rpm for 1 minutes, make the bacteria deposit at the bottom of the tube. 8. Discard 150 μL supernatant liquid and resuspend the bacteria. 9. Coat plate: add the solution to 2 plates (Cm+) and spread it, respectively, then cultivate it overnight.

Recorder: Yin Wu, Wenkai Han **Gel Extraction of PCR of sfGFP1-10 **

Procedure: 1. Cut off the Gel part containing the target band. Get 4 tubes of digestion product. 2. Weigh the cut off Gel part. 3. Add Buffer B2 whose weight is 3 times of the Gel. 4. Add isopropanol whose volume is 1/3 of the Buffer B2. 5. Move the solution to adsorption column , 11000 rpm centifuge 30 s. 6.Put filtrate into the upper part of the same adsorption column,11000 rpm centifuge 30,discard filtrate. 7. Add 300 μL Buffer B2,11000 rpm centifuge 30 s, discard filtrate. 8. Add 500 μL Wash Solution, 12000 rpm centifuge 30 s, discard filtrate. Repeat once. 9. 12000 rpm centifuge 1 min. 10. Lying for 10 min. 11. Put the adsorption column in a new EP tube. Add 20 μL elution buffer which is pre-heated at 60 degree Celsius , 10 min's standing, 12000 rpm centifuge 1 min.

After extraction, we measured the OD A260/A280 and the concentration of digestion product.

sample	1	2	3	4
A260/A280	3.06	2.12	1.83	1.65
concentration(ng/μL)	7.8	13.6	33.2	28.8

Recorder: Wenkai Han, Yin Wu PCR of sfGFP1-10

Procedure:

1.Prepare 4 PCR tubes and sequentially add：

sample	1-4
Sterilized ddH₂O	32.5 μL
5×PrimeSTAR Buffer (Mg²⁺Plus)	10 μL
dNTP Mixture (2.5 mM each)	4 μL
Template (sfGFP)	1 μL
standard-sfGFP1-10-p	1 μL
standard-sfGFP1-10-r	1 μL
PrimeSTAR HS DNA Polymerase (2.5 U/μl)	0.5 μL
total	50 μL

2.PCR reaction

Parameters setting：

stage	temperature	time
step 1	95	5 min
step 2	95	10 s
step 3	55	15 s
step 4	72	43 s
step 5	72	10 min
step 6	4	--

30 cycles(step 2 ~ step 4)

Agarose gel electrophoresis Results: 图片名称

Recorder: Yin Wu, Wenkai Han **Gel Extraction of PCR of sfGFP1-10 **

Procedure: 1. Cut off the Gel part containing the target band. Get 4 tubes of digestion product. 2. Weigh the cut off Gel part. 3. Add Buffer B2 whose weight is 3 times of the Gel. 4. Add isopropanol whose volume is 1/3 of the Buffer B2. 5. Move the solution to adsorption column , 11000 rpm centifuge 30 s. 6.Put filtrate into the upper part of the same adsorption column,11000 rpm centifuge 30,discard filtrate. 7. Add 300 μL Buffer B2,11000 rpm centifuge 30 s, discard filtrate. 8. Add 500 μL Wash Solution, 12000 rpm centifuge 30 s, discard filtrate. Repeat once. 9. 12000 rpm centifuge 1 min. 10. Lying for 10 min. 11. Put the adsorption column in a new EP tube. Add 20 μL elution buffer which is pre-heated at 60 degree Celsius , 10 min's standing, 12000 rpm centifuge 1 min.

After extraction, we measured the OD A260/A280 and the concentration of digestion product.

sample	1	2
A260/A280	1.83	1.83
concentration(ng/μL)	46.9	60.8

Recorder: Kaiyue Ma&Yonghao Liang Transformation of plasmid YeLGAP

Materials(all made by ourselves): 1. plasmid YeLGAP 2. SOC media 3. competent cells

Procedure: 1. Put competent cells on ice. Then pre-chill by placing the tubes on ice. 2. Pipet the recombinant plasmid into competent cell tubes, 2 µL for each. 3. Flick the tube gently with your finger to mix. Incubate on ice for 30 minutes. Pre-heat waterbath now to 42°C. 4. Heat-shock the cells by placing into the waterbath for 90 seconds. Be careful to keep the lids of the tubes above the water level, and keep the ice close by. 5. Immediately transfer the tubes back to ice, and incubate on ice for 5 minutes. This helps the cells recover. 6. Add 900 µL of SOC media per tube, and incubate at 37°C for 1 hours. 7. Centrifuge the tube at 4000 rpm for 1 minutes, make the bacteria deposit at the bottom of the tube. 8. Discard 950 μL supernatant liquid and resuspend the bacteria. 9. Add 5 μL solution to 3 tubes (Amp) and spread it, respectively(6 tubes in total), then cultivate it overnight.

Recorder:Kaiyue Ma & Yonghao Liang Characterization Experiments of Sup35+GFP Procedure: 1. Sample 1 is the control(37 degree). 2. Sample 2 has been heat at 42 degree for 10 mins. 3. Observe under fluorescence microscope.

Results: 37 degree: 图片名称 42 degree:

Conclusion: In the picture of group 42 degree, we can see that there are some brightened dot in the cell, which means the GFP may aggregate a little after being heat-shocked. But in the picture of group 37 degree, GFP spread more even in the cell.

Date: 8.4 Recorder：Ya Jiang Double Digestion of BBa_K081005 (constitutive promoter family member and RBS) and pUC57(containing sfGFP11)

Procedure: In either of the samples of BBa_K081005, add nuclease-free water 19 μL 10*Green Buffer 4 μL plasmid 15 μL BcuI 1 μL PstI 1 μL Mix gently and incubate at 37 degree Celsius for 15 min.

In either of the samples of pUC57(containing sfGFP11), add nuclease-free water 14 μL 10*Green Buffer 4 μL plasmid 20 μL PstI 1 μL XbaI 1 μL Mix gently and incubate at 37 degree Celsius for 15 min. Agarose gel electrophoresis Results: 图片名称

Recorder: Ya Jiang *Gel Extraction of digestion product of BBa_K081005 (constitutive promoter family member and RBS) and pUC57(containing sfGFP11)

sample	1	2	3
A260/A280	failed	1.81	1.91
concentration(ng/μL)	failed	31.8	29.3

I discard sample 1 (sfGFP11 digested from pUC57).

Plasmid Extraction for PYescGAP Recorder: Yu Xie and Chengle Zhang

Special notes:using visualize material.

sample	1	2	3	4	5	6
A260/A280	1.84	1.86	1.86	1.85	1.85	1.85
A260/A230	1.21	2.36	2.32	2.29	2.20	2.32
concentration(ng/μL)	329.9	738.6	469.3	458.5	383.1	325.6

(note: Sample2 may not be used because when experimenting mistake of pipetting deposition did occur.)

Then we use fastdigest enzyme PstI and EcoRI to check the plasmid. The results are as follows: It shows all the plasmid is OK.

Recorder：Yin Wu Double Digestion of PYescGAP and sfGFP1-10

Procedure: In either of the samples of PYescGAP, add nuclease-free water 19 μL 10*Green Buffer 4 μL plasmid 15 μL EcoRI 1 μL PstI 1 μL Mix gently and incubate at 37 degree Celsius for 15 min.

In either of the samples of sfGFP1-10, add nuclease-free water 14 μL 10*Green Buffer 4 μL plasmid 20 μL EcoRI 1 μL PstI 1 μL Mix gently and incubate at 37 degree Celsius for 15 min. Agarose gel electrophoresis Results: 图片名称

Recorder: Yinchenguang Lyu PCR of AD and BD

Experimental materials 1. Plasmid: pGAD-T7 and pGBK-T7(5 μL for each) from laboratory 436 of USTCSLS, Mr Liao; 2. Primer: AD 1, AD 2, BD 1, BD 2. Designed by ourselves, synthesized by Sangon Biotech; 3. Sterilized ddH₂O, 2×HiFi-PCR Master, bought from Sangon Biotech.

About primers

primer	AD 1	AD 2	BD 1	BD 2
sequence	5'-CCGGAATT CGCGGCCGC TTCTAGATGGC CAATTTTAA TCAAAGTGGGA ATATTGCTG-3'	5'-AACTGCAG CGGCCGCTA CTAGTACTACT CTTTTTTTG GGTTTGGTGGG GTATC-3'	5'-CCGGAATT CGCGGCCGC TTCTAGATGAA GCTACTGTC TTCTATCGA AC-3'	5'-AACTGCAG CGGCCGCTA CTAGTACTACG ATACAGTCA ACTGTCTTT GACC-3'

Procedure:

1.Prepare 2 PCR tubes and sequentially add：

sample	AD	BD
Sterilized ddH₂O	22 μL	22 μL
2×HiFi-PCR Master	25 μL	25 μL
pGAD-T7 (542.5 μg/mL)	1 μL	0
pGBK-T7 (506.7 μg/mL)	0	1 μL
AD Primer-1(10 μM)	2 μL	0
AD Primer-2(10 μM)	2 μL	0
BD Primer-1(10 μM)	0	2 μL
BD Primer-2(10 μM)	0	2 μL
total	50 μL	50 μL

2.PCR reaction Parameters setting：

stage	temperature	time
step 1	95	10 min
step 2	95	1 min
step 3	59	1 min
step 4	72	45 s
step 5	72	10 min
step 6	4	--

30 cycles(step 2 ~ step 4)

3.Agarose gel electrophoresis Result:

Then we did gel extraction,the parameters of product are as follows:

sample	AD	BD
A260/A280	1.90	2.55
A260/A230	0.11	0.02
concentration(ng/μL)	81.4	11.3

Recorder: Yin Wu Gel Extraction of digestion product of PYescGAP and sfGFP1-10

sample	PYescGAP-1	PYescGAP-2	sfGFP1-10-1	sfGFP1-10-2
A260/A280	1.89	1.87	2.20	1.94
concentration(ng/μL)	65.3	67.3	9.7	12.1

Recorder: Jiang Ya, Wenkai Han Ligation of sfGFP1-10 and PYescGAP

Material: 1. double digestion product of sfGFP1-10 2. double digestion product of PYescGAP 3. 10× T4 DNA ligase buffer,T4 DNA ligase(bought from Thermo Fisher Scientific) 4. nuclease-free water

Procedure: Add to the sample: 5 μL sfGFP 1.5 μL PYescGAP 11.5 μL nuclease-free water 2 μL T4 DNA Ligase Buffer 0.2 μL T4 DNA Ligase

Mix gently and incubate at 22 degree Celsius for 1 hour.

**Recorder: Shao Shuai & Kaiyue Ma & Yonghao Liang ** Characterization Experiments of Sup35+GFP Procedure: 1. Add 2.383 mg/ml and 0.283 mg/ml IPTG into 2 tubes(sample 1) respectively, then cultivate at 37 degree for 4 hours. 2. Add 2.383 mg/ml and 0.283 mg/ml IPTG into 2 tubes(sample 2) respectively, then cultivate at 42 degree for 4 hours. 3. Observe under fluorescence microscope.

Results: 37 degree,2.383 mg/ml IPTG: 图片名称

42 degree,2.383 mg/ml IPTG: 图片名称

37 degree,0.2383 mg/ml IPTG: 图片名称

42 degree,0.2383 mg/ml IPTG: 图片名称

Conclusion: 1.When we add 2.383 mg/ml IPTG into the solution, the fluorescence becomes much weaker than the last time when we add 23.83 mg/ml IPTG into the solution. But as the bacteria is too small, we still can't tell whether the GFP have aggregated or not. 2.When we add 0.2383 mg/ml IPTG into the solution, the IPTG is too dilute to induce the E.coli to express GFP. 3.In a word, the characterization experiment of SUP35+GFP on E.coli is failed. So next time, we will do it on yeast, which has a much bigger cell body.

Recorder: Ya Jiang, Yin Wu Transformation of recombinant plasmid of sfGFP1-10 and PYescGAP

Recorder: Chengle Zhang PCR of AD and BD with enzyme pfu

Agarose gel electrophoresis Result: 图片名称

Then we did gel extraction.The information about the product is as follows:

sample	AD	BD
A260/A280	1.89	1.89
A260/A230	0.48	0.89
concentration(ng/μL)	111.2	97.9

Recorder: Yu Xie Double digestion of AD, BD and PYescGAP

Reaction system:

sample(μL)	13 μL AD	10 μL BD	16 μL PYescGAP-1	16 μL PYescGAP-2
nuclease free water(μL)	3	6	0	0
10×Buffer(μL)	2	2	2	2
PstI(μL)	1	1	1	1
EcoRI(μL)	1	1	1	1
total(μL)	20	20	20	20

Agarose gel electrophoresis Result: 图片名称

Then we did gel extraction,the parameters of product are as follows:

sample	AD	BD	PYescGAP-1	PYescGAP-2
A260/A280	10.34	5.43	1.91	1.90
A260/A230	0.01	0.01	0.91	0.21
concentration(ng/μL)	7.5	9.6	79.4	60.8

Recorder: Kaiyue Ma PCR of AD and BD with enzyme Primerstar

After PCR,we directly digest the production with enzyme PstI and EcoRI.

Agarose gel electrophoresis Result: 图片名称

It shows that our experiment failed.

Recorder: Kaiyue Ma and Chenguang Lvyi PCR of AD and BD with enzyme Primerstar

Agarose gel electrophoresis Result: 图片名称 (For the picture,the left four are AD,the right four are BD.) Then we did gel extraction.The result is as follows: