iGEM 2021 — Team SCAU-China

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# Team:SCAU-China/Notebook

MESEG

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[SCAU China](https://2021.igem.org/Team%3ASCAU-China)

College of Life Sciences,

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Contact us:

020-85282180
[scau-china2021@stu.scau.edu.cn](scauchina2021%40163.com)

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# Team:SCAU-China/Engineering

MESEG

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Engineering

For MESEG project, we are planning to construct a fusion protein consisted of an ATG8 interacting motif (AIM), a MT that scavenge heavy metal, and a fluorescent protein for tracing. In this AIM and MT combination, the MT could be alternative according to its targeting heavy metal, which can be flexibly modified/substituted so that indeed expanding the target spectrum. We reasoned that the fusion protein, when expressed in *Chlamydomonas*, would specifically bind heavy metal via MT and, through the interaction between the AIM and ATG8, deliver the fusion protein–heavy metal complex to the vacuole, thus enhance its phytoremediation capability. So far, we went through an iteration of the engineering design cycle required by iGEM and demonstrated that our project was partially succussed.

[](https://static.igem.org/mediawiki/2021/2/26/T--SCAU-China--de1.mp4)

Video 1: The demonstration video of MESEG Project

As a proof of concept of MESEG, we first chose yeast as a model to test the phytoremediation function of our constructs.

## First Try of Wet Lab

We first used ATG19, a well-known yeast receptor as the starting material for MESEG construction. In yeast, the receptor ATG19 is required for the vacuolar deposition of Ape1, a vacuolar hydrolase. It binds Ape1 through its Ams1 binding domain (ABD) in the middle and binds ATG8 through ARR domain at its C-terminal (Yamasaki and Noda, 2017). It also can self-oligomerize through its coiled-coil (CC) region. Therefore, we kept the ARR domain and CC region, but replaced the ABD with SpMTL, a Cd binding protein derived from *Sedum plumbizincicola* and tagged a mCherry florescence protein at its N-terminal.

When the construct mCh-CC-SpMTL-ARR was expressed in the Cd-sensitive mutant *Δycf1*, it only slightly increased its Cd tolerance as compared with the construct with SpMTL (mCh-CC-ARR), but much weaker than the wild-type strain BY4741 (Figure 1). When the ARR domain is removed, the expression of mCh-CC-SpMTL conferred *Δycf1* mutant better Cd tolerance，indicating that the Cd binding of SpMTL might be interfered by the up and/or downstream domains.

![](https://static.igem.org/mediawiki/2021/1/1d/T--SCAU-China--engin1.png)

Figure 1. First generation of MESEG protein mCh-CC-SpMTL-ARR did not confer yeast Cd tolerance

The wild-type yeast strain BY4741 or Cd-sensitive mutant *Δycf1* were transformed with mCh-CC-ARR, mCh-CC-SpMTL-ARR or mCh-CC-SpMTL and grown on SD plates with indicated concentrations of CdCl2 for 3 d.

Moreover, using confocal laser scanning microscopy, we found that the protein mCh-CC-SpMTL-ARR is recruited to the pre-autophagosomal structure (PAS) regardless of nitrogen status (Figure 2), which is is similar to that of autophagic marker ATG8, implying that that this protein might might behave like cargo receptor ATG19 and is transported to the vacuole via autophagy route.

![](https://static.igem.org/mediawiki/2021/6/64/T--SCAU-China--engin2.png)

Figure 2. The subcellular localizations of fusion protein mCh-CC-SpMTL-ARR

The *Δycf1* mutant were transformed with pYES2 vectors expressing mCh-CC-SpMTL-ARR fusion proteins, and grown under nitrogen rich(+N) or nitrogen deficiency conditions (-N) for 12 hr before Confocal microscopy observation.

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[SCAU China](https://2021.igem.org/Team%3ASCAU-China)

College of Life Sciences,

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Contact us:

020-85282180
[scau-china2021@stu.scau.edu.cn](scauchina2021%40163.com)

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# Team:SCAU-China/Experiments

MESEG

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TAP Medium Protocol for *Chlamydomonas reinhardtii*

##### From Gorman, D.S., and R.P. Levine (1965) Proc. Natl. Acad. Sci. USA 54, 1665-1669.

* Make the following stock solutions:

  ###### 1. TAP salts

  |  |  |
  | --- | --- |
  | NH4Cl | 15.0 g |
  | MgSO4·7H2O | 4.0 g |
  | CaCl4·2H4O | 2.0 g |

  Water to 1 liter

  ###### 2. Phosphate solution

  |  |  |
  | --- | --- |
  | K2HPO4 | 28.8 g |
  | KH2PO4 | 14.4 g |

  Water to 100 mL

  ###### 3. Hutner’s trace elements

  Hutner et al. (1950) Proc. Am. Philos. Soc. 94, 152-170

  For 1-liter final mix, dissolve each compound in the volume of water.
  The EDTA should be dissolved in boiling water, and the FeSO4 should be prepared last to avoid oxidation.

  | compound | amount | water |
  | --- | --- | --- |
  | EDTA disodium salt | 50 g | 250 mL |
  | ZnSO4·7H2O | 22 g | 100 mL |
  | H3BO3 | 11.4 g | 200 mL |
  | MnCl2·4H2O | 5.06 g | 50 mL |
  | CoCl2·6H2O | 1.61 g |
  | CuSO4·5H2O | 1.57 g |
  | (NH4)6Mo7O24·4H2O | 1.10 g |
  | FeSO4·7H2O | 4.99 g |

  Mix all solutions except EDTA. Bring to boil, then add EDTA solution. The mixture should turn green. When everything is dissolved, cool to 70 degrees C. Keeping temperature at 70℃, add 85 mL hot 20% KOH solution (20 grams/100 mL final volume). Do not use NaOH to adjust the pH.

  Bring the final solution to 1-liter total volume. It should be clear green initially. Stopper the flask with a cotton plug and let it stand for 1-2 weeks, shaking it once a day. The solution should eventually turn purple and leave a rust-brown precipitate, which can be removed by filtering through two layers of Whatman#1 filter paper. If necessary, repeat the filtration until the solution is clear. Store refrigerated or frozen convenient aliquots. Some people shorten the time for the precipitate to form by bubbling the solution with filtered air.

  If no precipitate forms, the solution is still usable. However, you might want to check the pH in this case and adjust it to around 7.0 using either KOH or HCl as needed.

##### To make the final medium, mix the following:

1. 2.42 g Tris
2. 25 mL solution #1 (salts)
3. 0.375 mL solution #2 (phosphate)
4. 1.0 mL solution #3 (trace elements)
5. Water to 1 liter.
6. For solid medium, add 15 g agar per liter.
7. Autoclave.
8. For Tris-minimal medium omit the acetic acid and titrate the final solution to pH 7.0 with HCl.

Yeast (*Saccharomyces cerevisiae*) Transformation Protocol

##### Prepare cells

1. Take the strain stored in 20% glycerol, and culture it at YPDA plate, 28℃ for 3 days.
2. Pick a fresh S. cerevisiae monoclonal from a YPDA plate and culture it in 3 mL liquid YPDA medium, 28℃, 200 rpm overnight.
3. Transfer the yeast solution in step 2 to 10 mL YPDA liquid at 1:10, 28℃, 200 rpm overnight. OD600 after step 3 should be 4.0~6.0.
4. Pack 1 mL yeast solution reached OD600 value into 1.5 mL tube and centrifuge for 5000 rcf/1 min and remove supernatant.

##### Solution A:

1. Before preparation, please boil DTT at 100℃ for 5 mins, then put it on ice.
2. 400 μL 50% PEG4000 (in TE) Repeated freezing at -80℃ and thawing before use.
3. 50 μL 1 M DTT (DL-Dithiothreitol)
4. 50 μL 2 M LiAC (in ddH2O)

##### Transformation:

Before preparation, please boil carrier DNA (Salmon sperm ssDNA) at 100℃ for 5 mins, then put it on ice.

1. Add the following solutions to cells after cell preparation:
   125 mL solution A
   5 mL carrier DNA
   0.1-1 mg plasmids
2. Vortex it fully and incubated at 42℃ for 1 hour. Vibrate every ten minutes.
3. Vortex for 10 mins, centrifuge for 5000 rcf/1 min and remove supernatant.
4. Resuspend the yeast in 200 μL of 2% glucose.
5. Apply the corresponding screening plate.
6. Inverted and cultured the plate at 28℃ for 2~3 d.

Yeast (*Saccharomyces cerevisiae*) Cell Immobilization

##### Preparation of *Chlamydomonas reinhardtii*:

1. Collect *Chlamydomonas* in logarithmic phase, centrifuge at 2000 rpm for 10 minutes, and remove the culture medium.
2. Wash it with ddH2O for three times, and use it immediately. Adjust the algae concentration to 1.6 g/L with ddH2O.

##### Embedding:

1. Take the adjusted concentration of algal solution and mix it with 2% sodium alginate and 7% PVA solution according to the volume ratio of 1:1.
2. Place the beaker containing a certain volume of 0.1 M CaCl2 solution on the magnetic stirrer with a built-in rotor to keep it in a slow stirring state.
3. Drip the mixed solution of algae into CaCl2 solution at a uniform speed with No. 4.5 injection needle.
4. Allow to stand at 4℃ for 12 hours. Then, pour out the solution and wash it with ddH2O three times (at this time, there are small balls with a diameter of about 1.5 mm).
5. Transfer the pellet into 4% (W/V) boric acid solution (pH 6.7 by HCl) and let it stand in a refrigerator at 4℃ for 12 hours.

PVA Dissolution Protocol

Stainless steel is generally recommended as the material of dissolution tank. The speed of the mixer is 100-200 rpm. If the stirring is weak, PVA particles will settle and tend to form blocks at the bottom of the dissolution tank, so pay attention. If the stirring is too strong, it is easy to bubble.

1. Put PVA slowly into water and stir constantly.
2. After fully dispersed in water, stir and raise the temperature to 95℃, keep the temperature for about 60 minutes, PVA will be completely dissolved. Stir and cool to room temperature.

However, partial alcoholysis of PVA is easy to produce fast agglomerates during dissolution, so please pay attention to the following:

1. The water temperature during feeding shall be kept below 25℃.
2. The feeding speed of PVA is slightly slow.
3. After feeding, fully stir according to the original temperature for 10~15 minutes, then start heating up, and continue stirring during heating up.

##### reference

<http://www.kuraray.com.cn/Public/Uploads/image/product/20171116/20171116093132_47178.pdf>

Electric Transformation for *Chlamydomonas reinhardtii*

### Method one

##### Agents and materials:

1. Tap liquid medium with corresponding antibiotics (Zeocin, Paromomycin, Hygromycin B) and TAP plates with corresponding antibiotics (10 μg/mL Zeocin, or 20 μg/mL Paromomycin, or 10 μg/mL Hygromycin B).
2. Clean 0.4 cm electric shock cup (soaked in absolute ethanol and dried in the sterile environment of hood). Before electric conversion, pay attention to setting a 16℃ water bath, and advance the electric shock cup to 16℃ (at least 15 minutes).
3. Sterile 10% Tween-20 (filtration sterilization)
4. Sucrose: Prepare 1 M concentration, filter sterilization or high temperature and high-pressure sterilization. When in use, dilute it into tap, and the final concentration is 60 mM.
5. Starch (for cells without cell wall): Weigh 10 g corn starch into a 50 mL centrifuge tube, add 95% or 100% ethanol and completely resuspend corn starch and soak for at least 24 hours. 1000 g, centrifuge it for 1 min, remove ethanol, add 60 mM sucrose for resuspension to an appropriate amount of screening medium, repeated this step 5 times, and finally resuspended it in screening medium with 60mM sucrose at 25% (W/V)
6. Boiled salmon sperm DNA (10 mg/mL) (optional)
7. Sterile 50 mL centrifuge tubes

##### Protocol:

1. Culture *Chlamydomonas* cells in 25 mL TAP liquid medium until the cell density is about 1~5×106 cells/mL (22 ℃, 3~4 days, 120 rpm), then transfer an appropriate amount of *Chlamydomonas* cells to 100 mL TAP liquid medium and continue to culture until the cell density is about 1~5×106 cells/mL.
2. Place *Chlamydomonas* cells on ice for 10 mins, transfer them to a 50 mL sterile centrifuge tube, and add sterile 10% Tween-20 in the ratio of 1:2000. For example, 50 μL 10% Tween-20 is added to 100 mL of cells (optional: Tween-20 needs to be added when fewer cells are obtained, which is conducive to cell redundancy).
3. Collect cells by centrifugation: 4℃, 2000 g, 5 mins (if tween-20 is added in step 2, centrifugation at 4℃, 5000 g, 5 mins can be selected and centrifuge it at 3000 rpm, 5 mins this time).
4. Operate in the hood: remove the supernatant as much as possible, resuspend the cells with precooled TAP liquid medium (added 60 mM sucrose), the cell density after resuspension is about 4×108 cells/mL, and place it on ice for 15 minutes. At the same time, precool the electric shock cup.
5. Operation in the hood: add 250 μL of resuspended cells, 5 μL of 10 mg/mL boiled salmon sperm DNA, and 7 μL of DNA to be transformed in a 0.4 cm electric shock cup, mix evenly, and cover;
6. Take a water bath at 16℃ for 5 mins, place it in the electromotor, and set the parameters (Bio Rad gene pulser II: 0.6-0.65 kV, 25 μF and no resistance; BTX Gemini X2: voltage 800 V, resistance 1575 Ω, capacitance 50 μF).
7. Wipe off the water in the shell of the electric shock cup with absorbent paper, put it flat into the electric shock instrument, clamp the electric shock cup on both sides, and put down the safety cover. Press pulse, that is, conduct electric shock and record the time. 10-14 ms is the normal time range (the electric conversion is completed when the prompt tone is heard). After electric shock, transfer it to ice and place for 10 mins immediately .
8. If resuscitation is not required, the transformed cells shall be coated directly on the screening plate (for *Chlamydomonas* cells with cell wall defected, 0.6~0.8 mL 25% corn starch solution shall be added and coated on the surface of the plate). After the plate is dried in a sterile environment, it shall be cultured under the set culture conditions (resuscitation is required this time, see article 9 of this section).
9. If resuscitation is needed, transfer the transformed cells to 40 mL TAP liquid medium (added 60 mM sucrose) for culture for 24-48 h, and recover overnight in slow and weak light on the shaking table. The cells were collected by centrifugation, resuspended in 1 mL TAP liquid medium, and coated on the plate. The method is the same as that in step 6. Treatment of electric shock Cup: clean it after use, add absolute ethanol into the electrode cup, cover it with a hat and store it for a long time. Take it out before electric conversion and dry it in hood (under sterile conditions).

### Method two

##### Preparation

1. Early logarithmic *Chlamydomonas* cc4533 (cmj030) (OD650 between 0.6-1.0)
2. TOS (containing 40 mM sucrose (filtration sterilization) TAP solution)
3. Plate containing 10 μg /mL of zeocin

##### Protocol

1. Well cultured *Chlamydomonas*, 160 mL, 3500 rpm (1000 g), 10 mins
2. Pour out the supernatant and add 10 mL TOS for suspension then centrifuge at 3500 rpm (1000 g) for 5 mins
3. Pour out the supernatant and add 10 mL TOS for suspension then centrifuge at 3500 rpm (1000 g) for 5 mins
4. Pour out the supernatant and adjust cell concentration to 2×108 cells / ml
5. Then add the following agents into 1.5 mL centrifuge tube.

| Transformation group | Control group |
| --- | --- |
| 2 μg plasmids (linearized) | the same volume of water |
| --- | --- |
| 250 μL *Chlamydomonas* | 250 μL *Chlamydomonas* |
| Mix well, 16 degrees for at least 30 mins (or more than 80 mins). | |

6. Transfer to an electric shock cup placed on ice. Use gene pulser II and a pulse controller plus apparatus (Bio-Rad), 4 mm shock cup, 25 UF, 800 V
7. Transfer immediately into a 15 mL centrifuge tube containing 13 mL TOS and shake it gently for 6-8 h under weak light (5 mmol photons m-2 s-1)
8. Centrifuge it at 3500 rpm (1000 g) for 5 mins, 500 μL TOS, then coat on a plate of 10 μg/mL zeocin, weak light 5 mmol photons m-2 s-1, screen after 7-14 days.

The Protocol of *E. coli* (DH-5α) Transformation

##### The protocol of preparing competent *E. coli* cells by CaCl2

1. Take *E. coli* (DH-5α) cell tube from -70℃ and inoculate on the SOB plate without resistance at 37℃, 18~28 h to get single colony.
2. Culture *E. coli* in 1 mL liquid SOB medium each tube at 200 rpm under 37℃ overnight about 3~4 h.
3. Drop 500 μL cultured *E. coli* solution in to 50 mL new liquid SOB medium (1:100 dilution) to culture 2~3 h until the solution reaches about OD600=0.3~0.6. Then put it on ice.
4. Centrifuge the *E. coli* solution in a 2 mL tube (1.5 mL/tube) at 5000 rpm, 4℃ for 2 mins to collect the cells and drop out supernatant.
5. Add 300~500 μL cold 0.1 M CaCl2 solution to resuspend the *E. coli* cells and put the tube on the ice quietly for about 25 mins.
6. Centrifuge the preparing cell tube at 5000 rpm, 4℃ for 2 mins to collect the cells and drop out supernatant.
7. Add 50~100 μL cold 0.1 M CaCl2 and resuspend the cells on the ice. Then store the cell at 4℃.

##### *E. coli* (DH-5α) transformation protocol

1. Take competent cell tube from 4℃ and put on the ice.
2. Add 10 μL plasmids solution into each tube of 100 μL competent cells and put it back on the ice quietly for 30 mins.
3. Use metal bath at 42℃ to hot shock for 90 s, then put it on ice for 5 mins.
4. Add 500~800 μL LB to culture at 220 rpm under 37℃ for 1 h to recover resistant.
5. Centrifuge the tube at 12000 rpm for 1 min, discard the upper liquid and leave 100 microliters for heavy suspension.
6. Put the transformed *E. coli* solution on the surface of the exact resistant LB media plate and place under 37℃ for about 10~12 h (overnight).

SD-ura Medium Protocol

|  |  |  |
| --- | --- | --- |
|  | 100 mL | 200 mL |
| YNB(0.67%) | 0.67 g | 1.34 g |
| -Ura DO Supplement(0.2%) | 0.077 g | 0.154 g |
| Galactose (2%)/glucose (2%) | 2 g | 4 g |
| Adjust pH = 5.8 and add ddH2O to make the corresponding volume | | |
| Agar(2%) | 2 g | 4 g |
| Sterilization at 121℃ for 15 minutes | | |

YPDA Medium Protocol

|  |  |  |
| --- | --- | --- |
|  | 100 mL | 200 mL |
| YPD | 5 g | 10 g |
| Ade (Yeast specific) | 0.012 g | 0.024 g |
| Agar | 2 g | 4 g |
| Add ddH2O to make the corresponding volume. | | |

Agarose Gel Recover Protocol

* A suitable concentration of agarose gel was prepared, and DNA fragments were separated by electrophoresis. After the DNA fragment was separated, the gel was placed under the ultraviolet lamp, and the gel containing DNA fragments was quickly cut off and the excess gel was removed as far as possible.
* Weigh the gel block and transfer it to 1.5 or 2 mL centrifuge tube. According to 100 mg gel block is equivalent to 100 μL. For volume calculation, add equal volume buffer GDP. 50~55 degrees C water bath for 7~10 minutes, let the gel block completely dissolve. During the water bath, mix upside down twice to accelerate the sol. If the weight of the gel block is 200 mg, 200 μL Buffer GDP is added. When the gel concentration exceeds 2%, 2 times the volume of Buffer GDP was added. When processing more than 5 KB fragments, after adding 3 times volume (gel volume), add 1 time volume (gel volume) isopropanol and mix it evenly. Then follow step 3 to operate.
* The droplets on the tube wall are collected by brief centrifugation. The HiPure DNA Mini column was sheathed in a 2 mL centrifuge tube. Put 700 μL transfer the solution to the column. 12000 g centrifuged for 30 ~ 60 seconds.
* (Optional: Sol more than 700 μL) Pour the waste filtrate and put the column back into a 2 mL centrifuge tube. Transfer the remaining solution to the column, 12000 g centrifugation for 30 ~ 60 seconds.
* Discard the filtrate and put the column back into a 2 mL centrifuge tube. Add 300 μL buffer GDP into the column. Let it stand for 1 minute. 12000 g centrifuged for 30~60 seconds.
* Discard the filtrate and put the column back into a 2 mL centrifuge tube. Add 600 μL buffer DW2 (diluted with absolute ethanol) into the column, 12000 g centrifugation 30~60 seconds.
* (Optional) Discard the filtrate and put the column back into a 2 mL centrifuge tube. Add 600 μL buffer DW2 (diluted with absolute ethanol) into the column. 12000 g centrifugation for 30~60 seconds.
* Discard the filtrate and put the column back into a 2 mL centrifuge tube, 12000 g centrifuge for 2 minutes. For some sensitive applications (when most of the elution buffer needs to be added to the connecting reaction solution): open the cover of the column and dry by air for 5 minutes to remove ethanol completely.
* The column is sleeved in a 1.5 mL centrifuge tube and 15-30 μL elution buffer is added to the center of column membrane. Let it stand for 2 mins, then, 12000 g centrifuged for 1 minute. Drop the post and save the DNA at -20℃.

Gel recover kit is from Guangzhou Magen Biotechnology.

Colony PCR Protocol

* Reaction system preparation:

  |  |  |
  | --- | --- |
  |  | 10 μL |
  | 2 × Taq Master Mix(Vazyme,Dye plus) | 5 μL |
  | Primer F | 0.5 μL |
  | Primer R | 0.5 μL |
  | ddH2O | 3 μL |
  | Bacterial solution to be tested | 1 μL |
* PCR program

  Pre denaturation 95℃ for 3 mins
  Denaturation: 95℃ 15 s
  Renaturation 55℃ 15 s
  Extension 72℃ 30 s
  Number of repetitions × 34
  Extend again at 72℃ for 5 mins

Agarose Gel Electrophoresis Protocol

* 1.5% agarose (1.5 g / 100 mL) + 0.5 × TBE 100 mL.
* Put it in the microwave for melting.
* Accession 6 μL/100 mL EB.
* Pour the mixture into the mold and wait for freezing.
* Point (DL2000 DNA marker) 5 μL, 10 × DNA Loading buffer(Vazyme) 1 μL mixed with DNA sample.
* Electrophoresis (3~5 V/cm)

Single Restriction Enzyme Digestion Protocol

* The reaction system is prepared according to the following table.

  |  |  |  |
  | --- | --- | --- |
  |  | 10 μL | 50 μL |
  | ScaI enzyme | 0.2 μL | 1 μL |
  | 10 × Buffer | 1 μL | 5 μL |
  | plasmid DNA | 2 μL | 10 μL |
  | Nuclease free ddH2O | To 6.8 μL | To 50 μL |
* Incubate at 37℃ for one hour.

The enzyme was bought from NEB company.

Western Blot Protocol

##### Agents:

* Sealing solution: 5% non-fat milk powder, prepared by 1 × PBS).
* Transfer buffer (1 L): Tris 5.8 g, Glycine 2.9 g, Methanol 200 mL(add before use).
* 10 × PBS: 0.2 mol/L Tris-HCl (pH 7.5), 1.5 mol/L NaCl.
* 1 × PBST: 1 × PBS, 0.1% TritonX-100 (1000 μL)

##### Protocol:

* Electrophoresis and membrane transfer
  1. Add x μL sample and n μL protein extract to 2 × protein sample buffer.
  2. Constant temperature at 100℃ and denaturation for 10 mins.
  3. Loading, then electrophoresis at 8 mA, 120 mins(A piece of gel).
  4. Transfer to polyvinylidene difluoride membranes: Activate membranes with ethanol for 1 min and rinse with transfer buffer before preparing the stack. And the filter paper needs to be soaked in the transfer buffer. Cut off the excess gel, assemble the stack according to the gel on the negative electrode (black) and the membrane on the positive electrode (white), and no bubbles are allowed. There are two layers of filter paper (soaked in transfer buffer) between the sponge pad and gel or membrane.
  5. Constant current 80 mA, overnight.
* Seal off and crossbreeding
  1. Sealing: transfer the membrane into the sealing solution and incubate the membrane at room temperature rinsing, 70 rpm for 1 h then discard sealing solution.
  2. Add hybrid solution I (the first antibody), 70 rpm, more than 1 h.
  3. 1 × PBST wash three times, 10 mins each time.
  4. Add hybrid solution II (the second antibody), room temperature, 70 rpm, 1 h.
  5. 1 × PBST wash three times, 10 mins each time.
* Development
  1. Transfer the washed membrane to a clean container then add luminous solution A and B, 500 μL each.
  2. Install the film in the X-ray cassette (watch out for bubbles) then use X-ray film to develop in a certain time (dark room).
  3. Develop in developing solution then wash in water. Finally, fix it in fixing solution (dark room).

SDS-PAGE Electrophoresis Protocol

##### Agents:

* 30% gel storage solution: Acrylamide 30 g, Bis-acrylamide 0.8 g, 100 mL ddH2O.
* 10% ammonium persulfate (AP) solution: AP 2 g, 20 mL ddH2O.
* 1 mol/L HCl
* Separation gel buffer, 1.5 mol/L Tris-HCl: Tris 18.15 g, adjust pH value to 8.8 by HCl. Constant volume to 100 mL.
* Concentration gel buffer, 0.5 mol/L Tris-HCl: Tris 6 g, adjust pH value to 6.8 by HCl. Constant volume to 100 mL.
* 10% SDS: SDS 5 g, dissolved in 50 mL hot water.
* 10% TEMED: prepare 20 mL when use.
* 3 × electrode buffer: Tris 6 g, Glycine 28.8 g, SDS 1 g. Constant volume to 1000 mL.

##### Protocol:

* Clean and dry the glass plate with a blower.
* Install electrophoresis tank and glass plate.
* Prepare four 100 mL small beakers for water, sealing solution, concentrated glue and separating glue respectively. Prepare 1 straw and 1 glass rod respectively.
* Use a small beaker to prepare the sealing glue solution according to the table below, mix it evenly, pour it into the sealing groove of the electrophoresis tank immediately, and let it stand for 5~10 mins until it solidifies.

  | Agents | 15% Separation gel/μL | 5% Concentration gel/μL |
  | --- | --- | --- |
  | H2O | 2664 | 1332 |
  | Upper Tris | - | 528 |
  | Lower Tris | 1056 | - |
  | 40% Acrylamide | 420 | 210 |
  | 10% SDS | - | 100 | 50 |
  | 7% APS | 63.6 | 31.8 |
  | TEMED | 5.64 | 2.82 |
* Fill a small beaker with water, fill the two layers of glass with water, and check whether there is water leakage at the bottom and both sides of the glass. If there is water leakage, reinstall it.
* Use a small beaker to prepare the lower layer of gel (separation gel) according to the above table. The gel concentration is 15%, mix evenly, fill the gel, its height is 1~1.5 cm away from the comb, and then cover a layer of water with a straw. The setting time is 15~30 mins. When the gel and water are layered, pour out the water.
* Use a small beaker to prepare the upper gel (concentrated gel) according to the above table. The gel concentration is 5%. Mix it evenly, fill it with glue and insert it into the comb. The setting time is 10~20 mins. Pull out the comb after solidification.
* Add diluted electrode buffer into the upper tank of the electrophoresis tank and soak it to the low glass surface, but it shall not exceed the high glass surface. Pay attention not to water leakage.
* Add diluted electrode buffer into the lower tank of the electrophoresis tank, and the volume is basically the same as that of the upper tank.
* Loading and then setting electrophoresis program according to your plan.

Yeast Metal Tolerance Measurement Protocol

* Select a single colony and inoculate it in YPDA liquid medium until OD600 =1.
* The cell suspension was diluted to OD600=0.5 and OD600=0.01, respectively.
* Then use a pipette to suck 1-5 μL (depending on the size of the plate) and drop it vertically. Repeat 3 times for each concentration.

Plasmid Extraction Protocol

* Centrifuge at 10000 g for 1 minute. Collect 1-5 mL cells.
* Discard the medium. Gently tap on the absorbent paper to absorb the residual liquid. Add 250 μL buffer P1/RNase A mixture to resuspend the cells in a high-speed vortex.
* Add 250 μL buffer NP2 into the heavy suspension and mix it upside down gently for 8-10 times.
* Add 350 μL buffer NP3 and immediately reverse it 8-10 times to completely neutralize the solution.
* Centrifuge at 13000–16000 g for 2 minutes.
* Install HiPure DNA Mini column II in the collection tube. Carefully transfer the supernatant to the column and centrifuge at 13000 g for 30-60 minutes.
* Discard the filtrate. Put the column into the collection tube. Add 500 μL buffer PW1 to the column and centrifuge at 13000 g for 30-60 seconds.
* Discard the filtrate. Put the column into the collection tube. Add 600 μL buffer PW2 (diluted with absolute ethanol) to the column and centrifuge at 13000 g for 30-60 seconds.
* (Optional) Repeat step 8 one time.
* Discard the filtrate. Put the column into the collection tube. Centrifuge at 13000 g for 3 mins.
* Put the column in a sterilized 1.5 ml centrifuge tube. Add 30 - 100 μL elusion buffer or sterilized ddH2O to the center of the membrane of the column, stand for 1 minute, 13000 g centrifuge for 1 minute to elute DNA.
* Store at -20℃.

Yeast Total Protein Extraction Protocol

* Take 1~5 × 106 liquid yeast cells into 1.5 mL microtube, 8000 g, and centrifuge at 4℃ for 2 minutes.
* Carefully remove the supernatant, slowly add 1 mL ice precooled sterile water to the precipitation, and blow gently and repeatedly with a pipette to resuspend the precipitation.
* 8000 g, centrifuged at 4℃ for 2 minutes. Remove the supernatant carefully and remove the liquid as much as possible.
* Add 25 μL of Yeast Protein Extraction Reagent to the precipitation, and blow gently and repeatedly with a pipette to resuspend the precipitation.
* Put it into a 30℃ water bath and warm it for more than 30 minutes, during which gently shake the centrifugal tube for 1~2 times. The following operations can be carried out according to step 6.
* Take out the centrifuge tube from the water bath at 30℃, and centrifuge 12000 g at 4℃ for 5 minutes. Carefully remove the supernatant, add 25 μL PBS solution, 25 μL 2 × Protein SDS PAGE Loading Buffer (code no.9172) to the precipitation, and vortex oscillate for 2 minutes to resuspend the precipitation.
* Take 12.5 μL of the suspension of step 6 into a new 1.5 mL microtube, and take 10 μL for electrophoretic detection after 10 minutes of warm bath at 100℃.

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[SCAU China](https://2021.igem.org/Team%3ASCAU-China)

College of Life Sciences,

![](https://static.igem.org/mediawiki/2021/5/5b/T--SCAU-China--CollegeLogo.jpg)

Contact us:

020-85282180
[scau-china2021@stu.scau.edu.cn](scauchina2021%40163.com)

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# Team:SCAU-China/Results

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)