iGEM 2018 — Team GDSYZX

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# Team:GDSYZX/Notebook

Notebook

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### 7.23

#### Experiment Objectives

1、HMA2、 PCS1 PCR amplification
2、Detection of PCR product extraction results
3、Thermal transformation of plasmids

#### Experimental materials

Arabidopsis genomecDNA，Taq MIX（green），HMA2 gene primer 、PCS1 gene-specific primer 、agarose gel，TAE， goldview Stains
DNAsample（Receptive cells and plasmids）、LB

#### Experimental procedure

①22μl MIX(green) 、1μlHMA2 gene primer、1μl Arabidopsis genomecDNA
②2μl MIX（green）、1μlPCS1 gene primer、1μl Arabidopsis genomecDNA
③PCR
④Agarose gel electrophoresis

#### Experimental procedure

①Add 1.5ml1μl Connected DNA sample in the centrifuge tube，Gently mix
②Place in an ice box for 30min
③Insert the floating plate，place in 42 ℃water bath for 60s
④Placed in the ice for 5min
⑤In the super-clean bench, 1ml LB culture solution (for colony growth) was added to the receptor cells and gently mix
⑥37°C Oscillatory culture for 1h（180rpm）
⑦4000rpm/s Centrifugal for three times
⑧In the super-clean bench, the top 900 milliliters of l medium was removed
⑨Blow the rest of the bacteria liquid evenly, add the solid medium (for selecting the colony) and spread the bacteria liquid evenly with the applicator
⑩37°C overnight culture

#### Experimental results

PCR fail

### 7.24

#### Experiment Objectives

1、Thermal transformation of plasmids
2、PCR（pcs1&hma2）

#### Experimental materials

Igem plasmid、 competent cell

#### Experimental procedure

①The competent cells were divided into eight groups and put into the centrifuge tube
②Eight groups of plasmid 5 l were added to each centrifuge tube
③Placed in the ice for 30min
④Heat-shock 42°C 60s
⑤Placed in the ice for 5min
⑥Recovery：add 600μL LB 37°C 1h
⑦coated plates： Coated with chloramphenicol medium plate and incubated at 37 C overnight

#### Experimental results

PCR fail

### 7.25

#### Experiment Objectives

Extration Plasmid

#### Experimental materials

glucose、EDTA、Rnase，NaOH，SDS、KCl、 acetic acid、Guanidine hydrochloride，Tris-HCl、Potassium acetate、absolute ethyl alcohol
Reagent formula：
solutionⅠ：50 mL Tris-HCl(pH8.0)、10 mL 1 M EDTA、the volume is constant with distilled water to 1 L
solutionⅡ：0.2 mL 10 N NaOH、1 mL 10% SDS、8.8 mLddH2O
solutionⅢ： 195.4 g Guanidine hydrochloride、37.24 g potassium acetate、61 mL acetic acid 、the volume is constant with distilled water to 500mL

#### Experimental procedure

1. Add 250μl Buffer BL in the adsorption column AC, centrifuge(12000rmp) for 1 min to activated the silica gel film.
2. Take 2ml bacteria solution which was cultured overnight, centrifuge to collect bacteria.
3. Add 200μl solutionⅠ (with RNase A) to the suspended bacterial precipitation.
4. Add 200μl solutionⅡ , and turn it up and down gently for 4-7 times.
5. Add 200μl solutionⅢ, and turn it up and down gently for 4-7 times, mix it thoroughly (the solution is changed from fuchsia to pale yellow and white flocculation occurs at this time), and centrifuge(12000rmp) for 10-15 min.
6. Absorb the supernatant, transfer the supernatant into the adsorption column AC, and centrifugate (12000rpm) for 1 min. Then discard the waste liquid, and put the adsorption column AC back into the empty collecting tube.
7. Add 700μl Buffer W2 to adsorption column AC, centrifugate(12000rpm) for 1 min, and discard waste liquid.
8. Repeat step 7 once.
9. Put the adsorption cac back into the empty collecting tube and centrifuge(12000rpm) for 2min.
10. Remove the adsorption column AC and put it into a clean 1.5ml centrifuge tube. Let stand for 2min at 20℃.
Add 35-50μl Eluent and let stand for 2 min, and centrifuge(12000rpm) for 2 min.

### 7.27

#### Experiment Objectives

1、interlab coated plates
2、PCS1 PCR(The whole genome of arabidopsis cDNA was replaced as the template)
3、pPIC9K plasmid transformation

#### Experimental materials

bacterium solution、competent cell 、pPIC9K

#### Experimental procedure

①Take 100 milligrams of l fungus solution and add it to the medium
②Apply the medium evenly with glass rods that have been burned by an alcohol lamp until the solution is dried
③37°Covernight culture

#### Transformation

1.Thaw 50µL competent E. coli cells on ice for 10 minutes.
2.Add:5µl DNA（pPIC9K） from a ligation reaction mix.
3.Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
4.Place the mixture on ice for 30 minutes. Do not mix.
5.Heat shock at exactly 42°C for exactly 60 seconds. Do not mix.
6.Place on ice for 5 minutes. Do not mix.
7.Pipette 1m l of room temperature LB media into the mixture.
8.Incubate at 37°C and 4000 rpm for 3 minutes.
9.Mix the cells thoroughly by flicking the tube and inverting.
10.Incubate overnight at 37°C with plates upside down.

#### Experimental results

1.PCS1 PCR succeed
2.pPIC9Ktransformation succeed

### 7.28

#### Experiment Objectives

To isolate and purify the target gene and preserve it

#### Experimental materials

PCR product、Binding Buffer、Wash Buffer，ddH2O

#### Experimental procedure

①The PCR product was electrophoresed for 45min and put into the gel imager. The gel where the target strip was cut off, and the mass was weighed and put into the centrifuge tube，take such as volume Binding Buffer.

②Place the centrifuge tube in a metal bath to melt
③Transfer the liquid to the centrifugal adsorption column，standing for 1-2min.
④12000rpm centrifuge 30s，pour the liquid out of the collecting tube
⑤Add 600μl washing buffer，12000rpm centrifuge 30s，pour the liquid out of the collecting tube.
⑥Add 400μl washing buffer，12000rpm centrifuge 30s，pour the liquid out of the collecting tube.
⑦Place the adsorption column into the 60 C oven lid to dry for 5min(remove the alcohol from the rinse solution) until the alcohol does not smell.
⑧Take out the adsorption column ，add 50μl ddH2O，store it.

#### Experimental results

Success

### 7.30

#### Experiment Objectives

1.PCS1 PCR
2.colony PCR

#### Experimental materials

Mix（green），cDNA，PCS1 gene primer Sterile Water
0.1 µL Taq 、0.9µL dNTP、2µL 10×buffer 、1µL cell lysate、1 µL of 10 µM forward primer、1µL of 10 µM reverse primer、14µLddH2O

#### Experimental procedure

1.Add a single colony of cells to14 µL of water. Incubate at 95C for a minute to lyse the cells.
2.Combine 1 µL cell lysate, 0.1µL Taq,1 µL of 10 µM forward primer, 1µL of 10 µM reverse primer, and sterile water up to 20 µL.

#### Thermocycling

The PCR machine should be set to run the following steps:

### 7.31

#### Experiment Objectives

pPIC9K Extration Plasmid

#### Experimental procedure

1. Add 250μl Buffer BL in the adsorption column AC, centrifuge(12000rmp) for 1 min to activated the silica gel film.
2. Take 2ml bacteria solution which was cultured overnight, centrifuge to collect bacteria.
3. Add 200μl solutionⅠ (with RNase A) to the suspended bacterial precipitation.
4. Add 200μl solutionⅡ , and turn it up and down gently for 4-7 times.
5. Add 200μl solutionⅢ, and turn it up and down gently for 4-7 times, mix it thoroughly (the solution is changed from fuchsia to pale yellow and white flocculation occurs at this time), and centrifuge(12000rmp) for 10-15 min.
6. Absorb the supernatant, transfer the supernatant into the adsorption column AC, and centrifugate (12000rpm) for 1 min. Then discard the waste liquid, and put the adsorption column AC back into the empty collecting tube.
7. Add 700μl Buffer W2 to adsorption column AC, centrifugate(12000rpm) for 1 min, and discard waste liquid.
8. Repeat step 7 once.
9. Put the adsorption cac back into the empty collecting tube and centrifuge(12000rpm) for 2min.
10. Remove the adsorption column AC and put it into a clean 1.5ml centrifuge tube. Let stand for 2min at 20℃.

Add 35-50μl Eluent and let stand for 2 min, and centrifuge(12000rpm) for 2 min.

#### Experimental results

Success

### 8.1

#### Experiment Objectives

Digest（pcs1&pPIC9k）

#### Experimental materials

Enzyme Master Mix for Plasmid Backbone (25ul total, for 5 rxns)
20ul Buffer 、20ul plasmid、1 ul BamHI、1 ul Not1、23 ul ddH20

#### Experimental procedure

Digest 37C/15 min, heat kill 80C/20 min

### 8.3

#### Experiment Objectives

1.making competent cells

#### Experimental materials

50ul competent cells 、1ng plasmids、LB

#### Experimental procedure

1、add 50ul competent cells、1ng plasmids，ice- bath for 30min
2、Put in 42 C water bath to keep warm for 1min
3、ice- bath for2 min
4、Add 1mlLB，culture at 37 degrees for 1 hour
5. Coated tablet to select

### 8.7

#### Experiment Objectives

1.colony PCR

#### Experimental materials

Mix（green），cDNA，PCS1 gene primer Sterile Water
0.1 µL Taq 、0.9µL dNTP、2µL 10×buffer 、1µL cell lysate、1 µL of 10 µM forward primer、1µL of 10 µM reverse primer、14µLddH2O

#### Experimental procedure

1.Add a single colony of cells to14 µL of water. Incubate at 95C for a minute to lyse the cells.
2.Combine 1 µL cell lysate, 0.1µL Taq,1 µL of 10 µM forward primer, 1µL of 10 µM reverse primer, and sterile water up to 20 µL.

#### Thermocycling

The PCR machine should be set to run the following steps:

### 8.9

#### Experiment Objectives

1.pPIC9K Extration Plasmid

#### Experimental procedure

1. Add 250μl Buffer BL in the adsorption column AC, centrifuge(12000rmp) for 1 min to activated the silica gel film.
2. Take 2ml bacteria solution which was cultured overnight, centrifuge to collect bacteria.
3. Add 200μl solutionⅠ (with RNase A) to the suspended bacterial precipitation.
4. Add 200μl solutionⅡ , and turn it up and down gently for 4-7 times.
5. Add 200μl solutionⅢ, and turn it up and down gently for 4-7 times, mix it thoroughly (the solution is changed from fuchsia to pale yellow and white flocculation occurs at this time), and centrifuge(12000rmp) for 10-15 min.
6. Absorb the supernatant, transfer the supernatant into the adsorption column AC, and centrifugate (12000rpm) for 1 min. Then discard the waste liquid, and put the adsorption column AC back into the empty collecting tube.
7. Add 700μl Buffer W2 to adsorption column AC, centrifugate(12000rpm) for 1 min, and discard waste liquid.
8. Repeat step 7 once.
9. Put the adsorption cac back into the empty collecting tube and centrifuge(12000rpm) for 2min.
10. Remove the adsorption column AC and put it into a clean 1.5ml centrifuge tube. Let stand for 2min at 20℃.
Add 35-50μl Eluent and let stand for 2 min, and centrifuge(12000rpm) for 2 min.

#### Experimental results

Fail

### 8.12~8.14

#### Experiment Objectives

1.colony PCR

#### Experimental materials

Mix（green），cDNA，PCS1 gene primer、 Sterile Water
0.1 µL Taq 、0.9µL dNTP、2µL 10×buffer 、1µL cell lysate、1 µL of 10 µM forward primer、1µL of 10 µM reverse primer、14µLddH2O
Note：use two kinds of primer（Species-specific primers/ Universal Primers）
Species-specific primers：（PCS1-F/PCS1-R）
Universal Primers：（AOX1-F/ AOX1-R）

#### Experimental procedure

Add a single colony of cells to14 µL of water. Incubate at 95C for a minute to lyse the cells.
1.Combine 1 µL cell lysate, 0.1µL Taq,1 µL of 10 µM forward primer, 1µL of 10 µM reverse primer, and sterile water up to 20 µL.

#### Thermocycling

The PCR machine should be set to run the following steps:

### 8.15

#### Experiment Objectives

1.Pcs1 PCR

#### Experimental materials

Mix（green），pcs1， coPCS1F,coPCS1R

#### Experimental procedure

1.Add a single colony of cells to14 µL of water. Incubate at 95C for a minute to lyse the cells.
2.Combine 1 µL cell lysate, 0.1µL Taq,1 µL of 10 µM forward primer, 1µL of 10 µM reverse primer, and sterile water up to 20 µL.

#### Thermocycling

3.Agarose gel electrophoresis
4.gum concentration:15ng
5.Separation and purification

#### materialsPCR product、Binding Buffer、Wash Buffer，ddH2O procedure ⑨The PCR product was electrophoresed for 45min and put into the gel imager. The gel where the target strip was cut off, and the mass was weighed and put into the centrifuge tube，take such as volume Binding Buffer ⑩Place the centrifuge tube in a metal bath to melt ⑪Transfer the liquid to the centrifugal adsorption column，standing for 1-2min ⑫12000rpm centrifuge 30s，pour the liquid out of the collecting tube ⑬Add 600μl washing buffer，12000rpm centrifuge 30s，pour the liquid out of the collecting tube ⑭Add 400μl washing buffer，12000rpm centrifuge 30s，pour the liquid out of the collecting tube ⑮Place the adsorption column into the 60 C oven lid to dry for 5min(remove the alcohol from the rinse solution) until the alcohol does not smell ⑯Take out the adsorption column ，add 50μl ddH2O，store it 8.16Experiment Objectives Digest（pcs1&pPIC9k） Experimental materials Enzyme Master Mix for Plasmid Backbone (25ul total, for 5 rxns) 20ul Buffer 、20ul plasmid、1 ul BamHI、1 ul Not1、23 ul ddH20 Experimental procedure Digest 37C/15 min, heat kill 80C/20 min Experiment Objectives ligation（pcs1&pPIC9k） Experimental materials pPIC9k、pcs1、 T4 DNA ligase buffer、T4 DNA ligase Experimental procedure 1.Add 9.5ul pPIC9k、7.5 ul pcs1、2 ul T4 DNA ligase buffer、0.8 ul T4 DNA ligase 2.Ligate 16~25℃/4h, heat kill 80C/20 min 3.Transform with 1-2 ul of product 8.17Experiment Objectives Transformed recombinant vector Experimental materials NEB competent cells Experimental procedure 1.Add 50ul competent cells，10ulNEB,，ice-bath for 30min，42℃ for 1min 2.Add 200ulSOC Medium，37℃ recover for1h 8.18Experiment Objectives ppic9k（colony pcr） Experimental materials Mix（green），cDNA，PCS1 gene primer、 Sterile Water 0.1 µL Taq 、0.9µL dNTP、2µL 10×buffer 、1µL cell lysate、1 µL of 10 µM forward primer、1µL of 10 µM reverse primer、14µLddH2O Note：use two kinds of primer（Species-specific primers/ Universal Primers） Species-specific primers：（PCS1-F/PCS1-R） Universal Primers：（AOX1-F/ AOX1-R） Experimental procedure Add a single colony of cells to14 µL of water. Incubate at 95C for a minute to lyse the cells. 1.Combine 1 µL cell lysate, 0.1µL Taq,1 µL of 10 µM forward primer, 1µL of 10 µM reverse primer, and sterile water up to 20 µL. Thermocycling The PCR machine should be set to run the following steps: 8.19Experiment Objectives Transformation Experimental materials LB broth、Ice、Selection plates Experimental procedure 1.Thaw 50µL competent E. coli cells on ice for 10 minutes. 2.Add:1µl DNA（pcs1&pPIC9K） from a ligation reaction mix. 3.Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex. 4.Place the mixture on ice for 30 minutes. Do not mix. 5.Heat shock at exactly 42°C for exactly 60 seconds. Do not mix. 6.Place on ice for 5 minutes. Do not mix. 7.Pipette 1m l of room temperature LB media into the mixture. 8.Incubate at 37°C and 4000 rpm for 3 minutes. 9.Mix the cells thoroughly by flicking the tube and inverting. Incubate overnight at 37°C with plates upside down. Experiment Objectives Extration Plasmid Experimental procedure 1. Add 250μl Buffer BL in the adsorption column AC, centrifuge(12000rmp) for 1 min to activated the silica gel film. 2. Take 2ml bacteria solution which was cultured overnight, centrifuge to collect bacteria. 3. Add 200μl solutionⅠ (with RNase A) to the suspended bacterial precipitation. 4. Add 200μl solutionⅡ , and turn it up and down gently for 4-7 times. 5. Add 200μl solutionⅢ, and turn it up and down gently for 4-7 times, mix it thoroughly (the solution is changed from fuchsia to pale yellow and white flocculation occurs at this time), and centrifuge(12000rmp) for 10-15 min. 6. Absorb the supernatant, transfer the supernatant into the adsorption column AC, and centrifugate (12000rpm) for 1 min. Then discard the waste liquid, and put the adsorption column AC back into the empty collecting tube. 7. Add 700μl Buffer W2 to adsorption column AC, centrifugate(12000rpm) for 1 min, and discard waste liquid. 8. Repeat step 7 once. 9. Put the adsorption cac back into the empty collecting tube and centrifuge(12000rpm) for 2min. 10. Remove the adsorption column AC and put it into a clean 1.5ml centrifuge tube. Let stand for 2min at 20℃. Add 35-50μl Eluent and let stand for 2 min, and centrifuge(12000rpm) for 2 min. 8.20Experiment Objectives ppic9k-copcs1（colony pcr） Experimental materials Mix（green），cDNA，PCS1 gene primer、 Sterile Water 0.1 µL Taq 、0.9µL dNTP、2µL 10×buffer 、1µL cell lysate、1 µL of 10 µM forward primer、1µL of 10 µM reverse primer、14µLddH2O Note：use two kinds of primer（Species-specific primers/ Universal Primers） Species-specific primers：（PCS1-F/PCS1-R） Universal Primers：（AOX1-F/ AOX1-R） Experimental procedure 1.Add a single colony of cells to14 µL of water. Incubate at 95C for a minute to lyse the cells. 2.Combine 1 µL cell lysate, 0.1µL Taq,1 µL of 10 µM forward primer, 1µL of 10 µM reverse primer, and sterile water up to 20 µL. Thermocycling The PCR machine should be set to run the following steps: 8.21Experiment Objectives 1.Pcs1 pcr 2.pPIC9K-copcs1 (colony pcr) 3.competent yeast Experimental materials (making competent yeast) PEG(50%)、Licl、linear TtDNA、DNA,FishSperm Experimental procedure 1.Add 1ml connection product into 1.5ml centrifugal tube 2.2000rpm centrifuge 5min 3.Throw away the supernatant 4.Add 1ml ddH20 5.2000rpm centrifuge 5min 6.Throw away the supernatant 7.Add 300μl ddH20 8.2000rpm centrifuge 5min 9.500μl 0.1M Licl ,4000rpm centrifuge 3min 10.Throw away the supernatant 11.Add 200μl PEG(50%)、30μl 1M Licl 、20μl DNA,FishSperm、50μl linear TtDNA 12.Standing 30℃ 30min，42℃ 25min 13.7000rpm centrifuge 3min 14.Throw away the supernatant 15.Add 500μl ddH20 Experimental materials(Transform into the yeast) Competent yeast 、PEG、TE 、MD Experimental procedure 1.Add 50μl competent yeast、2μl plasmids(pPIC9K-copcs1)MIX 2.Add 500μl (PEG/LiAc,DMSO）MIX 3.Ice-bath at 30℃ for 1h，mix every 15min 4.Add 1ml YPD，cultivate in shaker at 30℃ for 1h 5.3500rpm centrifuge 5min，throw away the supernatant 6.Add 150μl TE，coated plates in MD 7.30℃ cultivate 8.22Experiment Objectives Yeast resistance in cdcl2 test（3 methods） Experimental materials Cdcl2、yeast、YPD Experimental procedure 1.Add 100μl 0.5mM Cdcl2 in YPD，use ss-spreader to spread evenly×3 2.Add 100μl 0.6mM Cdcl2 in YPD，use ss-spreader to spread evenly×3 3.Add 100μl 0.7mM Cdcl2 in YPD，use ss-spreader to spread evenly×3 4.Add 100μl 0.8mM Cdcl2 in YPD，use ss-spreader to spread evenly×3 5.Add 100μl 0.9mM Cdcl2 in YPD，use ss-spreader to spread evenly×3 6.Add 100μl 1mM Cdcl2 in YPD，use ss-spreader to spread evenly×3 Experimental procedure 1.Mix Cdcl2 、yeast first(0.5mM Cdcl2、0.6mM Cdcl2、0.7mM Cdcl2、0.8mM Cdcl2 、0.9mM Cdcl2 、1mM Cdcl2) 2.Use ss-spreader to spread evenly Experimental procedure 1.Add 100μl yeast（with different concentration） in YPD, cultivate for 8h 2.Dropwise added 2μl Cdcl2 on the yeast Note Different concentration：0.5mM Cdcl2、0.6mM Cdcl2、0.7mM Cdcl2、0.8mM Cdcl2 、0.9mM Cdcl2 、1mM Cdcl2 8.23Experiment Objectives Making yeast growth curve Experimental materials Yeast（mother liquid）、YPD（Solid） Experimental procedure 1.Dilute the yeast（mother liquid）with water to different concentration. Concentration: 10-2 、10-3、10-4 、10-5、10-6 2.Coat the yeast（with different concentration）on YPD medium，cultivate it. 3. Count the number of the colony 4. Make the yeast growth curve 8.24~8.25Experiment Objectives 1.PAGE（proteins electrophoresis, ） Experimental materials 30%Acr、10%SDS、1.5mol/L pH8.8 Tris-Hcl Buffer、1.0mol/L pH6.8 Tris-Hcl Buffer、0.05mol/L pH8.0 Tris-Hcl Buffer、10%AP、TEMED、sample solution、staining solution、destaining solution、 coagulant Experimental procedure 1.Cleaning the glass plate 2.Filling the glass plate with buffer and coagulant 3.Loading the sample 4. Electrophoresis 5.Staining&decolorize 8.25Experiment Objectives 1.Protein purification 2.Induce protein’s expression


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# Team:GDSYZX/Experiments

Experiment

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  + [Human practices](https://2018.igem.org/Team%3AGDSYZX/Human_Practices)
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* AWARDS
  + [Entrepreneurship](https://2018.igem.org/Team%3AGDSYZX/Entrepreneurship)

# Experiment

![](https://static.igem.org/mediawiki/2018/e/ee/T--GDSYZX--experiment--首页.jpg)

During 7.24~8.26, our GDSYZXigem 2018 team have spent so much time together and we have done lots of achievements and experiments. We nearly made our progress every day and we are really happy about them. The following are some experiments processes photos which might interest you. These are all the success we have made.

![](https://static.igem.org/mediawiki/2018/4/48/T--GDSYZX--discription--pic1.jpg)
![](https://static.igem.org/mediawiki/2018/c/c0/T--GDSYZX--discription--pic2.jpg)
![](https://static.igem.org/mediawiki/2018/3/30/T--GDSYZX--discription--pic3.jpg)
![](https://static.igem.org/mediawiki/2018/f/f4/T--GDSYZX--discription--pic4.jpg)
![](https://static.igem.org/mediawiki/2018/4/4f/T--GDSYZX--discription--pic5.jpg)
![](https://static.igem.org/mediawiki/2018/9/94/T--GDSYZX--discription--pic6.jpg)

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# Team:GDSYZX/Results

result

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  + [Description](https://2018.igem.org/Team%3AGDSYZX/Description)
  + [Design](https://2018.igem.org/Team%3AGDSYZX/Design)
  + [Experiments](https://2018.igem.org/Team%3AGDSYZX/Experiments)
  + [Notebook](https://2018.igem.org/Team%3AGDSYZX/Notebook)
  + [InterLab](https://2018.igem.org/Team%3AGDSYZX/InterLab)
  + [Model](https://2018.igem.org/Team%3AGDSYZX/Model)
  + [Results](https://2018.igem.org/Team%3AGDSYZX/Results)
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* PARTS
  + [Parts](https://2018.igem.org/Team%3AGDSYZX/Parts)
* SAFETY
  + [Safety](https://2018.igem.org/Team%3AGDSYZX/Safety)
* HUMAN PRACTICE
  + [Human practices](https://2018.igem.org/Team%3AGDSYZX/Human_Practices)
  + [Education & Engagement](https://2018.igem.org/Team%3AGDSYZX/Public_Engagement)
* AWARDS
  + [Entrepreneurship](https://2018.igem.org/Team%3AGDSYZX/Entrepreneurship)

# Result

First, we got the gene wild type pcs1 from the arabidopsis cDNA. Then we use ‘BamH 1’ and ‘Not 1’as the restriction enzyme to cut the wild type pcs1 and pEASY-Blunt, a kind of cloning vector. And then we linked them and transformed them to DH5α. Here is the colony PCR with it. Then, we connected wtpcs1 with pPIC9K and transformed them into BL21. The next picture is the plasmid of the pPIC9K-wtpcs1.The band is the location of pPIC9K-wtpcs1.

![](https://static.igem.org/mediawiki/2018/0/0c/T--GDSYZX--result--pic1.jpg)

We also did the same things with copcs1. The first picture is the PCR of copcs1. The next picture is the colony PCR of pEASY-copcs1 in DH5α. The last picture is the plasmid of pPIC9K-copcs1.

![](https://static.igem.org/mediawiki/2018/d/de/T--GDSYZX--result--pic2.jpg)

Furthermore, we transformed the pPIC9K-wtpcs1 and pPIC9K-copcs1 into the GS115. And then we cultured them in MD culture medium without histidine. Here is the picture.

![](https://static.igem.org/mediawiki/2018/a/a2/T--GDSYZX--result--pic3.jpg)

And we extracted the protein of prokaryotic expression, including the wtpcs1 and copcs1. We used the IPTG to induce BL21 to express protein. We divided them into 4 groups. And then we used IPTG to induce them for 4 hours, 6 hours, 8 hours, 12 hours，respectively. There is the result. If we obtained the protein, it would locate in 54 kD. However, the protein can’t be express, since pcs1 is a eukaryotic gene.

![](https://static.igem.org/mediawiki/2018/1/1c/T--GDSYZX--result--pic4.jpg)

In addition, we have done the experiment about yeast’s tolerance to cadmium chloride. We divided them into six groups. There were added 100 microliters from 500 micro mole per liter to 1000 micro mol per liter. There is a gap of 100 between each group. We made three copies for each group in order to make the data be more convictive. The picture shows that yeast can’t survive in 900 micro mole per liter.

![](https://static.igem.org/mediawiki/2018/1/1d/T--GDSYZX--result--pic5.jpg)

Furthermore, we have done some modeling. First of all, we attempt to accomplish the yeast growth curve. Look at the graph. We diluted the yeast solution at seven different levels and make three copies for each one. And then, we calculate the mean.

![](https://static.igem.org/mediawiki/2018/0/08/T--GDSYZX--result--pic6.jpg)

Then, we calculate the regression curve. The R-square is equal to 0.9954.

![](https://static.igem.org/mediawiki/2018/a/aa/T--GDSYZX--result--pic7.jpg)

Secondly, we try to find out the relation between cadmium concentration and yeast concentration. We cultured them in different concentration in 1 micro liter.
We measure the od value and compute the yeast concentration from the yeast growth curve.

![](https://static.igem.org/mediawiki/2018/4/41/T--GDSYZX--result--pic8.jpg)

Here is the scatter diagram. It seems that the second point is outlier.

![](https://static.igem.org/mediawiki/2018/3/32/T--GDSYZX--result--pic9.jpg)

Deleting the outlier, we obtained the regression curve.

![](https://static.igem.org/mediawiki/2018/f/fb/T--GDSYZX--result--pic10.jpg)

Eventually, we make another group to find out the relation in 5 micro liters. We change the cadmium concentration and draw a new graph. The second point is a outlier. Canceling the outlier, we gain the regression curve.

![](https://static.igem.org/mediawiki/2018/3/34/T--GDSYZX--result--pic11.jpg)

![](https://static.igem.org/mediawiki/2018/5/56/T--GDSYZX--result--pic12.jpg)