Team:USTC/Notebook/7 7

Modeling

Everybody

Date: 7.7

Recorder：Kaiyue Ma We communicated with Dr. Dong Men from Wuhan Institute of Virology(WHIOV) through email, who promised to offer us the sup35 gene.

Recorder：Xuefeng Meng We get plasmids: pGAD-T7 and pGBK-T7(5 μL for each) from laboratory 436 of USTCSLS.

Date: 7.7

Recorder：Xingfu Qin TOP10 and BL21 competent cell preparation

Material Volume Top 10 competent cell 100 uL BL21 competent cell 100 uL LB plates（without any antibiotics） 2 LB fluid medium 200 mL*2 80 mM CaCl₂-20 mM MgCl₂ 150 mL 100 mM CaCl₂ -18% glycerin 10 mL 50 mL centrifuge tube 8 Using streakplate method to get single colony.

Procedure：

Place the Top 10 and BL21 competent cellin room temperature until it get melt.
Use inoculating loop to pick somebacteria solution and use streak plate method to inoculate it on the LB plate(without any antibiotics).
Cultivate the plates at 37℃ overnight.

Date: 7.8

Recorder：Xingfu Qin Colony picking

Pick the colonies in the test tubes (5mLLB liquid medium).
Cultivate the bacteria at 37°C andshockat 250 rpm/min for 6h

Amplify the bacteria

Transfer thebacteria medium into the 200mL LB fluidmedium and cultivate the at 37°C andshock at 250rpm/min Preparate competent cell

Measure the OD600 of the medium until itreach 0.4.

OD Top 10 0.384 BL21 0.634(failed) 1. Transfer the medium into the 50 mLcentrifuge tube(precool in ice) and then incubate on ice for 30 minutes.

Centrifuge the tube at 4000 rpm for 15min at 4℃.
Discard supernatant liquid and add 30 mL80 mM CaCl₂-20 mM MgCl₂ per tube.
Resuspend the bacteria and incubate onice for 30 minutes.
Centrifuge the tube at 2700 rpm for 15min at 4℃.
Discard supernatant liquid and add 2 mL100 mM CaCl₂ -18% glycerin per tube.
Resuspend the bacteria and subpackage100 uL per tube(1.5 mL).
Store the tube at -80°C.

Date: 7.8

Recorder: Kaiyue Ma Transformation of pGAD-T7 and pGBK-T7 Experimental materials: competent E·coil BL21 100 μL*2 (bought from Trangen Biotech) pGAD-T7 plasmid 1 μL pGBK-T7 plasmid 1 μL

Procedure: 1. Thaw competent cells on ice. Then pre-chill by placing the tubes on ice. 2. Pipet 1 µL of each plasmid separately into two competent cell tubes. 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 LB media per tube, and incubate at 37°C for 2 hours. 7. Centrifuge the tube at 4000 rpm for 2 minutes, make the bacteria deposit at the bottom of the tube. 8. Discard 800 μL supernatant liquid and resuspend the bacteria . 9. Coat plate: add the solution 200 μL in a plate and spread it , then cultivate it overnight.

Date: 7.9

Recorder: Kaiyue Ma

Results of Transformation 7.9-rot The results aren't well as expected, yet there still are enough colonies for us to work on.

Colony picking 1. Pick the colonies in the test tubes (5 mL LB liquid medium). 1. Pick the colonies of each plasmid into 3 tubes. 1. Cultivate the bacteria at 37°C and shock at 250 rpm/min overnight.

Recorder: Liu Haoyu, Jin Weijie, Wu Yin, Xi Dawei, Yan Dong

Study the publication Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein in order to acquire two fragments of GFP which is competent of self-associating
Searched the parts registry and found the DNA and amino acid sequence of BBa_E0040
Thought hard of primer designing and experiment designing.
Prepared experiment equipments.

Date: 7.10

Recorder: Kaiyue Ma Plasmid Extraction for pGAD-T7 and pGBK-T7

Procedure: 1. Centifuge 1.5 mL bacterium solution at 11000 rpm, few sediment getted. Remove the supernatant. Add another 1.5 mL bacterium solution to the EP tube and centifuge at 11000 rpm again. 2. Add 250 μL Buffer P1, resuspend cells. 3. Add 250 μL Buffer P2, mix well, 4 min's standing. 4. Add 350 μL Buffer P3, mix well. 5. 13400 rpm centifuge 10 min, move all supernatant to adsorption column, 11.6 rpm centifuge 30 s, discard filtrate. 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. Put the adsorption column in a new EP tube. Add 50 μL ddH₂O( heating in water bath at 55 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 density of plasmids.

	pGAD-T7-1	pGAD-T7-2	pGAD-T7-3	pGBK-T7-1	pGBK-T7-2	pGBK-T7-3
A260/A280	1.89	1.61	0.79	1.58	1.87	1.63
Density(ng/μl)	19.7	53.0	0.3	92.4	22.4	56.2

Date: 7.11

Recorder：Kaiyue Ma Transformation of pGAD-T7 and pGBK-T7 Experimental materials: competent E·coil top10 100 μL*2 (made by ourselves) pGAD-T7 plasmid 1 μL pGBK-T7 plasmid 1 μL

Procedure: 1. Thaw competent cells on ice. Then pre-chill by placing the tubes on ice. 2. Pipet 1 µL of each plasmid separately into two competent cell tubes. 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 LB media per tube, and incubate at 37°C for 2 hours. 7. Centrifuge the tube at 4000 rpm for 2 minutes, make the bacteria deposit at the bottom of the tube. 8. Discard 800 μL supernatant liquid and resuspend the bacteria . 9. Coat plate: add the solution 200 μL in a plate and spread it , then cultivate it overnight.(pGAD-T7 in 50 µg/mL Amp plate, pGBK-T7 in 10 µg/mL Kana plate)

Date: 7.11

Recorder：Xingfu Qin

Top10 competentcell transformation efficiency test

Material Volume Top 10 competent cell 100 uL PUC19 plasmid (0.1ng/uL) 1uL SOC fluid medium 900uL LB plates（AP 50ug/mL） 1 1. Absorb 1 uLplasmid on the surface of bacteria solution softly

Put the tubeson the ice about 30 mins.
Make a heatshock at 42 degree centigrade about 90sec
Put the tubeson the ice about 5 mins
Add 900ul SOCmedium into the tube and cultivate the bacteria at 37 degree centigrade andshock at 180rpm/min about 60 mins.
Add 100uL of thesolution in a plate and spread it evenly and cultivate it overnight

Date: 7.12

Recorder：Xingfu Qin

Results of Transformation

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There are about 200 Colonies on the plate.

So the transformation efficiency of the Top10 competent cell is about 2×10 7 cfu/ug DNA.

This transformation efficiency is enough for future experiments.

Date: 7.12

Recorder：Kaiyue Ma Colony picking

Pick 3 colonies of each plasmid in 6 test tubes (5 mL LB liquid medium).
Cultivate the bacteria overnight at 37°C and shock at 250 rpm/min.

Date: 7.12

Recorder：Yin Wu and Weijie Jin Transformation of GFP 1. Find the pSB1A2 plasmid(with Apr Ampicillin replication sequence in it) containing the gene of GFPmut3B(Part Name BBa_E0040) in 13L well of 2016 Kit Plate 4. 2. With a pipette tip, punch a hole through the foil cover into the corresponding well.(Do not remove the foil cover, as it could lead to cross contamination between the wells.) 3. Pipette 10 uL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended. The resuspension is red, as the dried DNA has cresol red dye. 4. Put competent cells on ice. Then pre-chill by placing the tubes on ice. 2. Pipet 1 µL of each plasmid separately into two competent cell tubes. 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 LB media per tube, and incubate at 37°C for 2 hours. 7. Centrifuge the tube at 4000 rpm for 2 minutes, make the bacteria deposit at the bottom of the tube. 8. Discard 800 μL supernatant liquid and resuspend the bacteria. 9. Coat plate: add two tubes of solution (200 μL each) in two 50 μL/mL Amp plates and spread it, respectively, then cultivate it overnight.

Date: 7.13

Recorder: Xuefeng Meng、Chengle Zhang Plasmid Extraction for pGAD-T7 and pGBK-T7

Procedure: 1. Centifuge 1.5 mL bacterium solution at 11000 rpm, few sediment getted. Remove the supernatant. Repeat twice. 2. Add 250 μL Buffer P1, resuspend cells. 3. Add 250 μL Buffer P2, mix well, 3 min's standing. 4. Add 350 μL Buffer P3, mix well. 5. 13400 rpm centifuge 10 min, move all supernatant to adsorption column, 11000 rpm centifuge 30 s, discard filtrate. 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.

	pGAD-T7-1	pGAD-T7-2	pGAD-T7-3	pGAD-T7-4	pGBK-T7-1	pGBK-T7-2	pGBK-T7-3	pGBK-T7-4
A260/A280	1.85	1.84	1.82	1.86	1.83	1.84	1.88	1.87
Density(ng/μl)	548.2	579.9	624.8	441.8	538.2	587.6	589.6	381.2

Recorder：Yin Wu Colony picking

Pick 2 colonies of each plate in 4 test tubes (5 mL LB liquid medium).
Cultivate the bacteria at 37°C and shock at 250 rpm/min.

Recorder:Weijie Jin, Yinan Chen,Dawei Xi Transformation of yeast GAL1 promoter

Procedure: 1. Find the BBa_J63010 plasmid(Ampicillin resistence) containing the gene of yeast GAL1 promoter(Part Name BBa_J63006) in 7D well of 2013 Kit Plate 5. 2. With a pipette tip, punch a hole through the foil cover into the corresponding well.(Do not remove the foil cover, as it could lead to cross contamination between the wells.) 3. Pipette 10uL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended. The resuspension is red, as the dried DNA has cresol red dye. 4. Put competent cells on ice. Then pre-chill by placing the tubes on ice. 5. Pipet 1 μL of each plasmid separately into two competent cell tubes. 6. Flick the tube gently with your finger to mix. Incubate on ice for 30 minutes. Pre-heat waterbath now to 42℃. 7. 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. 8. Immediately transfer the tubes back to ice, and incubate on ice for 5 minutes. This helps the cells recover. 9. Add 900 μL of LB media per tube, and incubate at 37℃ for 2 hours. 10. Centrifuge the tube at 4000 rpm for 2 minutes, make the bacteria deposit at the bottom of the tube. 11. Discard 800 μL supernatant liquid and resuspend the bacteria. 12. Coat plate: add the solution 200 μL in a plate which contains LB media and ampicilin and spead it , then cultivate it overnight.(cultivation starts on 17:45 at 7.13)

Date: 7.14

Recorder: Chengle Zhang, Shuai Shao Plasmid Extraction of GFP

Procedure: 1. Centrifuge 1.5 mL bacterium solution at 11000 rpm, few sediment got. Remove the supernatant. Repeat twice. 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 for 10 min, move all supernatant to adsorption column, 11000 rpm centrifuge for 30 s, discard filtrate. 6. Add 500 μL Buffer DW1, 12000 rpm centrifuge for 30 s, discard filtrate. 7. Add 500 μL Wash Solution, 12000 rpm centrifuge for 30 s, discard filtrate. Repeat once. 8. 12000 rpm centrifuge for 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 centrifuge 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.

	GFP-1	GFP-2	GFP-3	GFP-4
A260/A280	1.86	1.88	--	1.86
Density(ng/μl)	177.6	111.2	--	102.8

Note: There was a small hole at the bottom of the EP tube used for GFP-3. So all the sample went out of the tube while centrifuging, and GFP-3 failed.

Recorder:Yinchenguang Lyu,Yinan Chen,Dawei Xi Transformation of yeast GAL1 promoter

Procedure: 1. Find the BBa_J63010 plasmid(Ampicillin resistence) containing the gene of yeast GAL1 promoter(Part Name BBa_J63006) in 7D well of 2013 Kit Plate 5. 2. With a pipette tip, punch a hole through the foil cover into the corresponding well.(Do not remove the foil cover, as it could lead to cross contamination between the wells.) 3. Pipette 10uL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended. The resuspension is red, as the dried DNA has cresol red dye. 4. Put competent cells on ice. Then pre-chill by placing the tubes on ice. 5. Pipet 1 μL of each plasmid separately into two competent cell tubes. 6. Flick the tube gently with your finger to mix. Incubate on ice for 30 minutes. Pre-heat waterbath now to 42℃. 7. 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. 8. Immediately transfer the tubes back to ice, and incubate on ice for 5 minutes. This helps the cells recover. 9. Add 900 μL of LB media to one tube, 900 μL of SOC media to the other, and incubate both at 37℃ for 2 hours. 10. Centrifuge the tube at 4000 rpm for 2 minutes, make the bacteria deposit at the bottom of the tube. 11. Discard 800 μL supernatant liquid and resuspend the bacteria. 12. Coat plate: add the solution 200 μL in a plate which contains LB media and ampicilin and spead it , then cultivate it overnight.(cultivation starts at about 13:30 on 7.14)

Recorder: Xuefeng Meng, Yu Xie 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'-GAATTCGCGG CCGCTTCTAG ATGGCCAATT TTAATCAAAG TGGGAATATT GCTG-3'	5'-CTGCAGCGGC CGCTACTAGT ACTACTCTTT TTTTGGGTTT GGTGGGGTAT C-3'	5'-GAATTCGCGG CCGCTTCTAG ATGATGAAGC TACTGTCTTC TATCGAAC-3'	5'-CTGCAGCGGC CGCTACTAGT ACTACGATAC AGTCAACTGT CTTTGACC-3'
lenth (nt)	54	51	48	48
GC%	44.44	50.98	47.92	52.08
molecular weight (Da)	16690.78	15716.06	14733.50	14663.45
Tm (℃)	71.26	73.40	71.53	73.24

Procedure:

1.Prepare 6 PCR tubes and sequentially add：

sample	AD*3	BD*3
Sterilized ddH₂O	7 μL	7 μL
2×HiFi-PCR Master	10 μL	10 μL
pGAD-T7 (1.0μg/mL)	1 μL	0
pGBK-T7 (1.0μg/mL)	0	1 μL
AD Primer-1	1 μL	0
AD Primer-2	1 μL	0
BD Primer-1	0	1 μL
BD Primer-2	0	1 μL
total	20 μL	20 μL

2.PCR reaction Parameters setting：

stage	temperature	time
step 1	95	5 min
step 2	95	1 min
step 3	67	1 min
step 4	72	1 min
step 5	72	10 min
step 6	4	--

30 cycles(step 2 ~ step 4)

3.Agarose gel electrophoresis gel 1. Add 0.4 g agarose to 40 mL TAE buffer. 2. Dissolved by heating. 3. Cool down. 4. Pour into electrophoresis tank. 5. Mix 20 μL PCR product and 4 μL loading buffer. 6. Loading：DNA marker 6 μL, sample(*6) 24 μL. 7. Electrophoresis gel：110 V 30 min. 8. Dissolve about 2 μL Gelred to a container with TAE buffer and put the agarose gel in. 10. Autoradiography(UV).

Result Failed, only marker and Primer dimer are visible.

Recorder：Tianshu Liu Transformation of TEF2 1. Find the pSB1A3 plasmid containing the gene of TEF2 yeast constitutive promoter (Part Name BBa_K165037) in 21H well of 2016 Kit Plate 3. 2. Punch a hole through the foil cover into the corresponding well by a pipette tip. (Do not remove the foil cover, as it could lead to cross contamination between the wells.) 3. Pipette 10µL of ddH2O (distilled water) into the well. Pipette up and down a few times and lett for 5 minutes to make sure the dried DNA is fully resuspended. The resuspension is red, as the dried DNA has cresol red dye. 4. Put competent cells on ice. Then pre-chill by placing the tubes on ice. 5. Pipet 1 µL of each plasmid separately into two competent cell tubes. 6. Flick the tube gently with your finger to mix. Incubate on ice for 30 minutes. Pre-heat waterbath now to 42°C. 7. 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. 8. Immediately transfer the tubes back to ice, and incubate on ice for 2 minutes. This helps the cells recover. 9. Add 600 µL of LB media per tube, and incubate at 37°C for 2 hours. 10. Centrifuge the tube at 4000 rpm for 1 minutes, make the bacteria deposit at the bottom of the tube. 11. Discard 600 μL supernatant liquid and resuspend the bacteria. 12. Coat plate: add two tubes of solution (100 μL each) in two 1/1000 Cam plates and spread it, respectively, then cultivate it overnight.

result failed, there isn't any bacterial colony on the 1/1000 Cam plate.

Date: 7.13

Recorder：Xingfu Qin BL21 competent cell preparation

Material Volume LB plates 1 LB fluid medium 200 mL 80 mM CaCl₂-20 mM MgCl₂ 150 mL 100 mM CaCl₂ -18% glycerin 10 mL 50 mL centrifuge tube 4 Colony picking