iGEM 2019 — Team Shanghai_HS

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# Team:Shanghai HS/Notebook

Notebook

Solve the harm caused by cyanobacteria

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# Megamenu

## Cyanobarrier: Solve the harm caused by cyanobacteria

We anticipate our solution to aid in protecting the environment and avoiding people from getting sick because of drinking contaminated water.

![](https://igem.siqint.com/lanxijun/images/home/banner_down.jpg)

;

# Notebook

![](https://igem.siqint.com/lanxijun/images/home/bg.png)

* Project : DNA Cloning

  ![](https://igem.siqint.com/lanxijun/images/home/4-1.png)

  Saturday, 2019-7-20 ———————————————-
* PCR amplification of MlrA 1, Mlr2, and MlrA3.

  This experiment is to verify if the DNA sequence is suitable for protein expression.
  The process of creating the reaction system is as the following.
  - Add 18 ul of water
  - 25 ul of 2 x Phanta Buffer
  - 1 ul of dNTP
  - 2 ul for each of the two primers
  - 1 ul of the template
  - 1 ul of the DNA Polymerse
  Next, put reaction tube through the PCR machine.

  ![](https://igem.siqint.com/lanxijun/images/home/4-2.png)

  Add 0.5g Agarose with 50ml 1xTAE Buffer to get 1% gel. Microwave to melt the solution and add 1microliter of nucleic acid pigment. Then, add the matrix and wait till the gel concrete.
* Recycle the PCR product

  A. Add 5 microliter of 10 x loading buffer to the PCR system, perform agarose gel electrophoresis to factional DNA fragments. Conduct electrophoresis with 110 volts of electricity for 30 minutes, Image formation of gel on computer.
  B. Sever the target sectors from the gel
  C. Add 350 microliter of binding buffer, heat the product at 65 ℃ till the gel melts.
  D. Transfer the solution into the binding column.
  E. Revolve the solution in the centrifuge under 12000 rpm of revolution rate for 1 minute.
  F. Discard the yellowish waste solution.
  G. Add 500 microliter of wash buffer.
  H. Revolve the solution in the centrifuge under 12000 rpm of revolution rate for 1 minute.
  I. Discard the yellowish waste solution.
  J. Revolve the solution in the centrifuge under 12000 rpm of revolution rate for 2 minutes.
  K. Dry the binding column under room temperature.
  L. Add 30 microliter of 65 degrees celsius heated deionized water.
  M. Cap the solution, keeping the solution at room temperature, right until the solutes are uniformly dissolved.
  N. Revolve the solution in the centrifuge under 12000 rpm of revolution rate for 2 minutes, separating the DNA.
  Use nanodrop to measure the concentration of DNA.

  ![](https://igem.siqint.com/lanxijun/images/home/4-3.png)

  This is the formation of gel seen under an ultraviolet light.

  ![](https://igem.siqint.com/lanxijun/images/home/4-4.png)
* Conclusion

  The final concentration is 29.2 ng / 10^-6L
  Sunday, 2019-7-21 ————————————————

* LB Culture Medium

  A. Prepare 100ml solid culture medium & 600ml liquid culture medium
  Solid Medium - Tryptone 1 g + Yeast Extract 0.5 g + Sodium Chloride 1g
  Liquid Medium - Tryptone 6 g + Yeast Extract 3 g + Sodium Chloride 6 g + Agar 6-7.5 g.

  ![](https://igem.siqint.com/lanxijun/images/home/4-5.png)

  Use restriction enzymes to cut the plasmid.
* Linearizing the Plasmid using restriction enzymes.

  ![](https://igem.siqint.com/lanxijun/images/home/4-6.png)
* Electrophoresis

  A.1% gel: 0.25g Agarose + 25ml 1xTAE Buffer + heat, melt the solution, add 1microliter of nucleic acid pigment. Then, add the matrix and wait till the gel concretes.

  ![](https://igem.siqint.com/lanxijun/images/home/4-7.png)

  Linearized pTOLO - Ex5
  B.Conduct electrophoresis with 110 volts of electricity for 30 minutes.
  C.Sever the target sectors from the gel.
  D.Collect the plasmids.
* Homologous Recombination

  ![](https://igem.siqint.com/lanxijun/images/home/4-8.png)

  Prepare LB Plate

  Melt the solid medium using the microwave.
  A. Add 50 ml 2000 x Kanamycin Monosulfate into 100 ml melted solid medium
  B.Pour the mixture into separate plates and wait for it to concrete.
* Transformation DH5αcell

  A.10 microliter homologous recombination product + 50 microliter competent cell, mix the solution
  B. Set the solution on ice and wait for 30 min
  C. 42 degrees celsius Heatshock for 45 sec
  D.Place it on ice for 2 min.
  E.Add 300 microliter LB liquid culture medium, place them inside the incubator at 37 degrees celsius at 220 RPM for an hour.
  F.Spread the medium evenly on the surface plate.
  G.Invert the plates and incubate at 37 degrees celsius overnight.
  Monday, 2019-7-22 ————————————————

* Find monoclonals on the petri dish and move them into small test tubes.

  a.Add 2.5 ul 2000x of Kanamycin Monosulfate to the 5 ml liquid LB medium.
  b.Use a sterilized toothpick to find the monoclonal.
  c. Shake for at least 10 hours at 220 rpm (37°)

  ![](https://igem.siqint.com/lanxijun/images/home/4-9.png)

  This is an image of the Petri dish.
* Use PCR to determine the positivity of the plasmid.

  System：
* Draw the plasmid

  a.Pour the bacterial fluid into a 2ml EP tube.
* Produce medium （9 Liters of LB medium）

  ![](https://igem.siqint.com/lanxijun/images/home/4_10.png)

  Prepare LB Plate

  Produce medium （9 Liters of LB medium）

  ![](https://igem.siqint.com/lanxijun/images/home/4_11.png)

  Thursday, 2019-7-25

* Preparation

  Fetch bacteria sample from a single bacteria colony.

  (1) Procedure

  A.Till the bacteria finished incubating, place them inside the large flask.

  ![](https://igem.siqint.com/lanxijun/images/home/4_12.png)

  (2) Protein Expression Testing

  A.Inject 3ml of lysis buffer into the cells

  ![](https://igem.siqint.com/lanxijun/images/home/4_13.png)

  This was the SDS - Page Gel that we dyed and decolorized.

  Friday, 2019-7-26

* Collect BL21 DE3 cells

  Fetch bacteria sample from a single bacteria colony.

  A.Add 15 mL bufferA, which contains 20 mM tris-chl and 100 mM NaCl, into 1 L E.coli cells pretreated by centrifugation. Then resuspend the cells
  B.Add DNA polymerase, MgCl2, PMSF containing 10 mg/ml of 2000x mother liquor of DNA polymerase, 2 M of 2000x mother liquor of MgCl2, 1M of 1000x mother liquor of PMSF.
  C.French press with pressure reaching 15,000 psi and lyse the cells twice.
  D.Centrifuge the cells with 15,000g centrifugal force for 10 minutes.
  E.Collect the supernatant of the cells, and then ultra-centrifuge the supernatant with 150,000g centrifugal force for an hour.
  F.Discard the supernatant after ultra-centrifugation. Add 6 mL buffer A per liter of cells into the sediment, and then resuspend. Then add DDM, a detergent, to a final concentration of 1 %. Incubate the solution under 4 degrees Celsius for 2 hours.
  G.Centrifuge the solution with 20,000g centrifugal force for 45 minutes.
  H.Collect the supernatant after centrifugation and bind the supernatant with Ni21-NTA. Add 1ml resin per liter of cells. Then incubate overnight.

  Sunday, 2019-7-28

* Further purification of MlrA protein using nickel column

  A.The protein that binds to Ni21-NTA is added to the affinity column. The liquid that flow through is collected.Using the method of gradient elution, while the amount of imidazole gradually increases, the amount of DDM simultaneously decreases.

   Ultimately, the final amount of imidazole reaches 200mM，and the percent of DDM within the affinity column reaches 0.018%

  ![](https://igem.siqint.com/lanxijun/images/home/4_14.png)
* URun SDS-PAGE Gel to identify the results of protein purification.

  The followings are the samples for Gel electrophoresis：
  1. The total sample of pTOLO-EX5-mlrA 2/3 in BL21(DE3) after the lysis of BL21 (DE3) cells
  2. The sample of precipitants after cell lysis and low-speed centrifugation
  3. The sample of supernatant after cell lysis and low-speed centrifugation
  4. The sample of supernatant after ultra-centrifugation
  5. The total sample after the membrane lysed
  6. The sample of precipitants after the membrane lysed
  7. The sample of supernatant after the membrane lysed
  8. The sample of impure proteins that first flow through the nickel column
  9. The sample after 5mM imidazole wash
  10. The sample after 10mM imidazole wash
  11. The sample after 20mM imidazole wash
  12. The sample after 200mM imidazole elution

  ![](https://igem.siqint.com/lanxijun/images/home/4_15.png)

  According to the results of Gel Electrophoresis, there is very few amount of proteins shown at the target strip. While on the contrary, we observed high concentrated protein in the impure proteins that first flowed through the nickel column and in the supernatant after the membrane lysed, though the size of the protein was slightly smaller than our target protein. We hypothesized that the protein with higher concentration was probably the MlrA without the sumo tag, otherwise it would just be another impure protein. Because of this uncertainty, we decided to react the impure proteins that first flowed through the nickel column and the supernatant after the membrane lysed with cyanotoxin separately. The results of the reaction will verify whether the two samples contains MlrA or not.

  ![](https://igem.siqint.com/lanxijun/images/home/4_16.png)

  ![](https://igem.siqint.com/lanxijun/images/home/4_17.png)

  Figure. 1 pTolo-EX5-MlrA3-detergent-Ni
  Figure. 2 pTolo-EX5-MlrA2-detergent-Ni

  ![](https://igem.siqint.com/lanxijun/images/home/4_18.png)

  ![](https://igem.siqint.com/lanxijun/images/home/4_19.png)

  Figure.3 pTolo-EX5-MlrA3-detergent
  Figure. 4 pTolo-EX5-MlrA2-detergent
* 24-hour reaction of MlrA with cynaotoxin

  aBesides using the supernatant after the membrane lysed and the impure proteins that first flowed through the nickel column to react with cyanotoxin, we also tested the purified MlrA 3 with 2mg/mL as its initial protein concentration. We tested 13 different reaction in total. As independent variables, the concentration of the target protein and the concentration of cyanotoxin were adjusted differently for each of the 13 reactions. The temperature was fixed at 27 degrees Celsius and the reaction time was fixed at 24 hours.

©2019 Microsoft Corporation. All rights reserved.

[![](https://igem.siqint.com/lanxijun/images/home/logo.jpg)](https://2019.igem.org/Team%3AShanghai_HS)

## About Us

We must explain to you how all seds this mistakens idea off denouncing pleasures and praising pain was born and I will give you a completed accounts off the system and expound.

*Consultation*

## Contact Info

* Rock St 12, Newyork City, USA
* (000) 000-000-0000
* Medizco@gmail.com
* Week Days: 09.00 to 18.00 Sunday: Closed


[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Shanghai HS/Experiments

Experiments

Solve the harm caused by cyanobacteria

[![](https://igem.siqint.com/lanxijun/images/home/logo.jpg)](https://2019.igem.org/Team%3AShanghai_HS)

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  [Team Members](https://2019.igem.org/Team%3AShanghai_HS/Team)

# Megamenu

## Cyanobarrier: Solve the harm caused by cyanobacteria

We anticipate our solution to aid in protecting the environment and avoiding people from getting sick because of drinking contaminated water.

![](https://igem.siqint.com/lanxijun/images/home/banner_down.jpg)

;

# Experiment Menu

![](https://igem.siqint.com/lanxijun/images/home/bg.png)

###### Documentation of the Development of Project

* PCR

  (1)PCR
  Polymerase Chain Reaction, PCR in short, is a biological technique that are able to produce many copies of a single piece of gene. In this case, our team’s goal is to make billions of copies of our target genes, MlrA2 and MlrA3, such that both MlrA genes would be available for later experimental processes.
  (2)Gel Electrophoresis
  Gel Electrophoresis is a technique carried out by biological researchers for separating and visualizing DNA fragments of different lengths based on size, electrical charge, and other physical properties of nucleic acids or proteins. Under 110 volts of electricity, the negatively-charged DNA fragments are able to travel from the negatively-charged anode to the positively-charged cathode, and the lengths they have traveled are determined by the number of base pairs each type of DNA fragment contained. Hence, by comparison with the distance marked by the restriction fragments, which is the known distance MlrA2 and MlrA3 would travel, we are able to identify the specific sector of gel containing the MlrA2 and MlrA3 fragments, and discard the materials for conducting PCR. Both types of MlrA genes are collected by severing the target sectors from the gel.

  ![](https://igem.siqint.com/lanxijun/images/home/20.png)

  Figure1. Result of Gel Electophoresis after PCR

  (3)Extraction of MlrA
  We first add DNA binding buffer to the gel and melt the gel at 65℃. We then extract the MlrA genes from the gel with a centrifuge, a machine with a part that spins mixtures of different substances around so that substances with different masses may separate due to centrifugal force. After four rounds of revolutions, DNA then will be extracted out of the gel mixture.
  (4)Concentration Testing
  Finally, we measure the absorbance of the resultant DNA of MlrA2 and MlrA3 with a nanodrop, the final result is 29.2nanogr am per microliter
  This piece of data helps us estimate the volume of materials required in the later process of Homologous Recombination.
* Molecular Cloning with DH5α Cells.

  (1)Preparation of the Cultural Medium
  Preparation of cultural medium for the growth of DH5α competent cells, so that the MlrA genes can be successfully duplicated by the cells.
  (2)Cleaving of Restriction Site
  Restriction endonucleases, or restriction enzymes, are enzymes that cut DNA molecules at a limited number of specific locations. As we cleave the empty plasmid with restriction enzymes, the plasmid is getting linearized such that the gene of interest, the gene containing the MlrA gene sequence, will be able to bind to the plasmid at restriction sites during homologous recombination.
  (3)Gel Electrophoresis
  In this case, gel electrophoresis helps us identify and extract the linearized plasmid out of the plasmid-restriction enzyme mixture.

  ![](https://igem.siqint.com/lanxijun/images/home/21.png)

  Figure 2. Gel Electrophoresis during Molecular Cloning

  (4)Homologous Recombination
  Homologous recombination is the recombination of two fragments of DNA with similar nucleotide sequences at the corresponding fronts and ends by the homologous recombination enzyme. In this process, the homologous recombination enzyme catalyzed the attachment of the MlrA2 and MlrA3 genes to the linearized plasmids, and the formation of a complete circular plasmid.
  (5)Transformation
  Transformation is the phenomenon defined as a change of genotype due to assimilation of external DNA by a cell. Since DH5α is a competent cell, cells with cell membranes etched with tiny holes, plasmids with MlrA genes can easily enter the DH5α cell and become a part of DH5α cell’s genotype, such that the plasmid can utilize the materials in the DH5α cell to replicate it self during culturing.
  (6)Culturing
  Pick from monoclonal DH5α cell culture and incubate them in fluid until they have replicated enough amount of plasmids for 10 hours.
  (7)Plasmid-Target Gene Testing: PCR and Electrophoresis
  In this case, PCR is applied to determine the presence or absence of MlrA genes on the plasmid. Due to the fact that the primer in this PCR process is paired with promoter on the gene of interest, the gene containing MlrA gene sequence, if the MlrA gene is successfully transplanted to the plasmid, the plasmids would be proliferated, and vice versa.

  Conduct electrophoresis again to test whether the plasmids have been proliferated or not. As long as the distance the plasmid gene traveled matches with the appropriate gene mark, the plasmids are then proliferated and contain the MlrA genes. Since our plasmids do match with the mark, the MlrA genes are present on our plasmids.

  ![](https://igem.siqint.com/lanxijun/images/home/22.png)

  Figure 3. Electrophoresis for Gene Testing
  Line 1 to 5 contains plasmids inserted with MlrA 2 genes, Line 6 to 10 contains plasmids inserted with MlrA 3 genes.
  The results showed that MlrA is inserted into the plasmids.

  (8)Plasmid Extraction
  We then extract the plasmids from the bacterial fluid with a centrifuge after six rounds of revolutions.
  (9)DNA Sequencing
  Decipher the sequence of the plasmids to see whether any mutations have happened to the gene. The results suggest that no mutation has happened, and the plasmids can be used for later experimental processes.
* Molecular Cloning with BL21(DE3) Cells

  (1)Transformation
  Transfer the plasmids into BL21(DE3) competent cells so that the protein can be expressed from the MlrA genes on the plasmids by the materials in BL21(DE3).

* Enzyme function test

  1.Transfer E.coli from the small bottles to large flasks. Then shake the flasks until the OD (Optical Density) is between 0.6 and 0.8. The purpose of these steps is to make E.coli grow to the logarithmic phase. The cells at the logarithmic phase are good materials for metabolism and laboratory research, and also the best materials for strains, because they have the same physiological characteristics, balanced growth of the components and constant growth rate.

  ![](https://igem.siqint.com/lanxijun/images/home/23.png)

  Figure 4. E.coli in large flasks.

  2. Add IPTG to the flasks. IPTG is an extremely inducing agent widely used in the laboratory. Additionally, we also need to take a sample without adding IPTG, in order to control the variables in the following experiments.
  3. Place the flasks in the shaker and induce protein expression overnight.
  4. In order to test whether mlrA is expressed, we take 4 different samples. These 4 samples correspond to the supernatant solution and precipitate before and after induction, respectively. Then let these samples run the protein gel(SDS-Page gel) together for verification.

  ![](https://igem.siqint.com/lanxijun/images/home/24.png)

  Figure 5. The result of SDS-Page gel electrophoresis

  Our experimental results show that mlrA is mainly expressed in the sediment. This shows that our prediction of mlrA is a membrane protein is correct.
* Purification of membrane protein mlrA

  1. French press E.coli for 2 times. When this step is complete, all the cell’s membrane is broken and the cell contents are discharged. This makes it easy for us to carry out the following centrifugal.
  2. 15,000g centrifuge for 10 minutes, collect the supernatant solution, and then centrifugate the solution for another hour at the force of 15,0000g. So we can make sure that the mlrA is ultimately in the precipitation after the centrifuation. The next thing we need to do is purify the mlrA from the impure protein.
  3. Add buffer to the precipitation and let it re-hang, then add a certain concentration of DDM(N-Dodecyl-β-D-maltoside, this substance is able to isolate the membrane protein and maintain the normal structure of the membrane protein). Extract for 2 hours at 4 degrees Celsius.
  4. The solution after extract is re-engaged for 20,000g centrifuge for 45 minutes. Because DDM is added, mlrA is present in the supernatant solution after centrifugal. Collect the supernatant solution and incubate overnight with Ni21-NTA affinity column (Qiagen) binding.

  ![](https://igem.siqint.com/lanxijun/images/home/25.png)

  Figure 6. Ni21-NTA affinity column (Qiagen) binding

  5. Purify mlrA protein with nickel column. Add the protein incubated overnight to the purification column and collect flow through. Since most of the mlrA has been combined with the binding sites on the nickel column, most of the flow through collected at this time is impure protein.
  6. Gradient elute mlrA with imidazole competition agent. Imidazole will compete with mlrA on the binding sites of the nickel column. When the concentration of imidazole increases, the mlrA is gradually eluded.

  ![](https://igem.siqint.com/lanxijun/images/home/26.png)
* Identify purification results

  In order to determine whether the protein we finally purified is MlrA, we take 12 different samples from different periods of the experiment and let these samples run SDS-Page gel.

  ![](https://igem.siqint.com/lanxijun/images/home/27.png)

  ![](https://igem.siqint.com/lanxijun/images/home/28.png)

  ![](https://igem.siqint.com/lanxijun/images/home/29.png)

  ![](https://igem.siqint.com/lanxijun/images/home/30.png)

  Figure 7. The result of the SDS-Page gel electrophoresis
  Figure 7A
  Line 1: MlrA2 total sample
  Line 2: MlrA2 supernatant sample
  Line 3: MlrA2 precipitate sample
  Line 4: MlrA2 precipitate sample after low speed centrifugation
  Line 5: MlrA2 supernatant sample before high speed centrifugation
  Line 6: MlrA2 supernatant sample after high speed centrifugation
  Line 7: MlrA2 precipitate sample after membrane lysed
  Line 8: MlrA2 supernatant sample after membrane lysed
  Figure 7B
  Line 9: MlrA3 total sample
  Line 10: MlrA3 supernatant sample
  Line 11: MlrA3 precipitate sample
  Line 12: MlrA3 precipitate sample after low speed centrifugation
  Line 13: MlrA3 supernatant sample before high speed centrifugation
  Line 14: MlrA3 supernatant sample after high speed centrifugation
  Line 15: MlrA3 precipitate sample after membrane lysed
  Line 16: MlrA3 supernatant sample after membrane lysed
  Figure 7C
  Line 17: MlrA2 supernatant sample after membrane lysed (same as sample 8)
  Line 18: Impure MlrA2 sample that first flow through the nickel column
  Line 19: The sample after 5mM imidazole wash
  Line 20: The sample after 10mM imidazole wash
  Line 21: The sample after 20mM imidazole wash
  Line 22: The sample after 200mM imidazole wash
  Figure 7D
  Line 23: MlrA3 supernatant sample after membrane lysed (same as sample 16)
  Line 24: Impure MlrA3 sample that first flw through the nickel column
  Line 25: The sample after 5mM imidazole wash
  Line 26: The sample after 10mM imidazole wash
  Line 27: The sample after 20mM imidazole wash
  Line 28: The sample after 200mM imidazole wash

  The result of the SDS-Page gel electrophoresis is unexpected. We can only observe very small amounts of protein at the target strip of the elution samples. However, the supernatant solution and flow through do exist higher concentration of protein at the upper position than the target strip. We theoretically speculate that this higher concentration of protein may be the mlrA without the SUMO tag; but it could also be just a kind of impure protein. Because of this uncertainty, we decide to try it use both the supernatant solution and the flow through to react with microcystin to verify that if these two samples contain the MlrA we really need.
  In addition to using samples we obtained in our experiments, we also used previously purified mlrA3(one specific kind of mlrA gene which is extracted from Sphingomonas sp. ACM-3962) with an initial concentration of 2 mg/ml to react with microcystin. We conducted a controlled variable experiment and tried a total of 13 reactions. In these reactions, we adjusted the MlrA concentration and the microcystin concentration respectively; the reaction temperature is 27 degrees Celsius, and the reaction time is 24 hours.
  Using HPLC to detect the effect of MlrA on the decomposition of microcystin

  ![](https://igem.siqint.com/lanxijun/images/home/31.png)

  Figure 8. HPLC (High Performance Liquid Chromatography)
  HPLC (High Performance Liquid Chromatography),is a technique used to separate, identify, and quantify each component in a mixture. It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column fulled with a solid adsorbent material. Each component in the sample interacts slightly differently with the absorbent material, causing different flow rates for the different components and leading to the separation of the components as they flow out the column.

  ![](https://igem.siqint.com/lanxijun/images/home/32.png)

  Figure 9: 13 samples we used to run the SDS-Page Gel
  Sample 1 - The total sample of pTOLO-EX5-mlrA 2/3 in BL21(DE3) after the lysis of BL21 (DE3) cells.
  Sample 2 - precipitants after cell lysis and low-speed centrifugation.
  Sample 3 - supernatant after cell lysis and low-speed centrifugation.
  Sample 4 - supernatant after ultra-centrifugation.
  Sample 5 - The total sample after the membrane lysed.
  Sample 6 - precipitants after the membrane lysed.
  Sample 7 - supernatant after the membrane lysed.
  Sample 8 - impure proteins that first flow through the nickel column.
  Sample 9 - The sample after 5mM imidazole wash.
  Sample 10 - The sample after 10mM imidazole wash.
  Sample 11 - The sample after 20mM imidazole wash.
  Sample 12 - The sample after 200mM imidazole elution.
  Sample 13 - Blank or Buffer.

  We added 13 samples after 24 hours of reaction to the HPLC instrument, and the computer will generate the data and peak map separately after more than 8 hours.

  ##### To view the result of HPLC, please visit Result

©2019 Microsoft Corporation. All rights reserved.

[![](https://igem.siqint.com/lanxijun/images/home/logo.jpg)](https://2019.igem.org/Team%3AShanghai_HS)

## About Us

We must explain to you how all seds this mistakens idea off denouncing pleasures and praising pain was born and I will give you a completed accounts off the system and expound.

*Consultation*

## Contact Info

* Rock St 12, Newyork City, USA
* (000) 000-000-0000
* Medizco@gmail.com
* Week Days: 09.00 to 18.00 Sunday: Closed

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Shanghai HS/Results

Experiments

Solve the harm caused by cyanobacteria

[![](https://igem.siqint.com/lanxijun/images/home/logo.jpg)](https://2019.igem.org/Team%3AShanghai_HS)

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  [Description](https://2019.igem.org/Team%3AShanghai_HS/Description)
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  [Attributions](https://2019.igem.org/Team%3AShanghai_HS/Attributions)
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# Megamenu

## Cyanobarrier: Solve the harm caused by cyanobacteria

We anticipate our solution to aid in protecting the environment and avoiding people from getting sick because of drinking contaminated water.

![](https://igem.siqint.com/lanxijun/images/home/banner_down.jpg)

;

After we have conceived our project, we went to the lab and tried to accomplish our expectations. Both successes and failures were taken place and were used to improve our project by iterating our practical experimental steps. On this page, we want to convince you that what exactly we managed to realize and what achievements we are able to show in our project.
Our research was essentially divided into three main sections. We will share the results we obtained from each individual step.

* DNA Cloning and the Construction of Plasmid

  Key Achievements
  -Successful construction of the pTOLO-EX5-mlrA plasmid
  -Transformation of DH5-alpha cell and BL21(DE3)

  ![](https://igem.siqint.com/lanxijun/images/home/33.png)

  ![](https://igem.siqint.com/lanxijun/images/home/34.png)

  Using the restriction enzymes Ncol and Xcol, we linearized the pTOLO-EX5 and were able to reconstruct the plasmid we want with MlrA gene we got using the homologous recombination method. Then, the homologous recombinant products were added into DH5-alpha competent E.coli for transformation.

  ![](https://igem.siqint.com/lanxijun/images/home/35.png)

  The gel electrophoresis result of the plasmid we got
* Plasmid Extraction.

  Key Achievements
  - Removal of impurities such as DNA, RNA and impure proteins by centrifugation to obtain a relatively pure plasmid
  - Transformation of the plasmid into BL21(DE3) E.coli competent cells
* Protein Purification

  (As you can see from the picture, we use N-Dodecyl-β-D-maltoside, which is also known as DDM, to transfer the target membrane protein mlrA from the supernatant to the membrane of DDM. Since DDM provides the membrane which mlrA depends on, with the DDM concentration increases, the concentration of the mlrA in the supernatant decreases to zero. That is the last step of protein purification.

  ![](https://igem.siqint.com/lanxijun/images/home/36.png)

  After centrifuge the solution, to further purify the protein, we put Ni21-NTA with the supernatant and incubate overnight. The mlrA will be combined with the specific binding sites on Ni21-NTA. Then, using the method of gradient elution, while the amount of imidazole, which is a competitive agent in this step, increases, it will compete with mlrA on the binding sites of the Ni column. Therefore, the mlrA is gradually elated.

  ##### For further and detailed explain action, please view our Notebook and Experiment.

* Enzyme Function Test

  Key Achievements
  -Purification of the MlrA protein using nickel column
  -Degradation of MC-LR (Microcystin-LR) and test the results.

  To examine whether or not our purification result is correct, we ran SDS-PAGE gel to identify the results.

  Surprisingly, according to the results we got, very few amounts of protein showed on the target strip of the elution samples. However, the supernatant solution and flow through did exist higher concentration of protein at the upper position than the target strip. Theoretically, we speculated that this higher concentration of protein maybe the mlrA without the SUMO tag (pTolo-EX5 is the carrier of the mlrA we designed): without the tag, it becomes lighter than usual, thus at a upper position; but it could also be just a kind of impure protein. Because of this uncertainty, we decide to let both the supernatant solution and the flow through react with microcystin to verify that if these two samples contain the MlrA we really need.

  In addition to using samples we obtained in our experiments, we also used previously purified mlrA3(one specific kind of mlrA gene which is extracted from Sphingomonas sp. ACM-3962) with an initial concentration of 2 mg/ml to react with microcystin. We conducted a controlled variable experiment and tried a total of 13 reactions. In these reactions, we adjusted the MlrA concentration and the microcystin concentration respectively; the reaction temperature is 27 degrees Celsius, and the reaction time is 24 hours.

  By adding the protein that binds to the Ni21-NTA to the affinity column, we were able to collect the liquid that went through. We ran the SDS-Page Gel with thirteen chosen samples to verify the results of the protein purification. It turns out, we successfully purified MlrA2&3, indiciating that it can be devoted to degrading MC-LR.

  ![](https://igem.siqint.com/lanxijun/images/home/37.png)

  Sample 1 - The total sample of pTOLO-EX5-mlrA 2/3 in BL21(DE3) after the lysis of BL21 (DE3) cells.

  Sample 2 - precipitants after cell lysis and low-speed centrifugation.
  Sample 3 - supernatant after cell lysis and low-speed centrifugation.
  Sample 4 - supernatant after ultra-centrifugation.
  Sample 5 - The total sample after the membrane lysed.
  Sample 6 - precipitants after the membrane lysed.
  Sample 7 - supernatant after the membrane lysed.
  Sample 8 - impure proteins that first flow through the nickel column.
  Sample 9 - The sample after 5mM imidazole wash.
  Sample 10 - The sample after 10mM imidazole wash.
  Sample 11 - The sample after 20mM imidazole wash.
  Sample 12 - The sample after 200mM imidazole elution.
  Sample 13 - Blank or Buffer.

  ##### For further explanations, please visit Experiment.

  High Performance Liquid Chromatography (HPLC)
  To quantify whether MC-LR can be degraded from the samples, we conducted a control variable experiments,
  and the following are the data we got:

  ![](https://igem.siqint.com/lanxijun/images/home/38.png)

  The original HPLC peak map of MC-LR at the concentration of 0.5mg/ml

  ![](https://igem.siqint.com/lanxijun/images/home/39.png)

  The HPLC peak map we get when the concentration between MlrA and MC-LR is 9:1

  ![](https://igem.siqint.com/lanxijun/images/home/40.png)

  1.By adding the protein that we have designed, and letting it react with the MC-LR, we find that they successfully reacted just as we expected.
  2.From the HPLC results, we find that as the ratio of the mlrA we have designed increases, the percentage of the reaction product increases and the amount of MC-LR decreases consequencely. This result successful indicates that the protein mlrA we have designed is able to react with MC-LR and release the product which is 160 times less toxic than MC-LR, as we mentioned in the Background.
  3.Although the result of the HPLC did show that our experiment is successful basically, there are still some remained issues of the protein we have designed:
  a)The efficiency of the protein we designed to degrade MC-LR still needs to be improved. We control the change in samples’ concentration by adding different volumes of the same concentration of mlrA and MCLR to the sample. As it is shown in the figure above, only when the concentration ratio of mlrA is extremely high—— for example, 9:1 or 8:2 —— can most of the MC-LR be reacted effectively. In fact, according to the data we got, the mlrA we got is able to transfer about 39% of the MC-LR to the relatively non-toxic product when the concentration ratio is 9:1. When it is applied to the real world environment, however, the efficiency needs to be boosted undoubtedly, since it is unrealistic for waterworks to reach such a high concentration ratio.
  b)The future goal of our project is to invent a device to purify the MC-LR in the water using the protein mlrA we have designed. Since the protein mlrA is an enzyme, it can be reused by enzyme immobilization technology. Nevertheless, the application of the device is still not practical, due to time constraints. We hope that in the future iGEM competition, there will be teams to solve the issues we remained.
* Unsuccessful moments

  Throughout our project, we have encountered minor obstacles and failures along the way. Although these roadblocks had little impact on our end result, we did have to devote more time to recover from these unfortunate moments. One of the main failures would be the explosion of the SDS-PAGE Gel ran to identify the results of protein purification. This gel exploded in the process of heating and displayed an unclear image for examination. To solve this issue, we tried to wait for the color of the gel to dissimilate but the result was the same. We had no choice but to run the SDS-PAGE Gel again and was finally able to observe the purification result precisely.

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## About Us

We must explain to you how all seds this mistakens idea off denouncing pleasures and praising pain was born and I will give you a completed accounts off the system and expound.

*Consultation*

## Contact Info

* Rock St 12, Newyork City, USA
* (000) 000-000-0000
* Medizco@gmail.com
* Week Days: 09.00 to 18.00 Sunday: Closed