iGEM 2022 — Team nanjing-nfls

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{% block title %}Notebook{% endblock %}
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{% block pageTitle %}notebook{% endblock %}
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- Construction of Bacteria
- Bacterial Transformation
- Liquid Culture & Glycerol Stocks
- Restrictive Endonuclease Digestion
- Recovering Plasmid DNA
- Diagnostic Endonuclease Digest & PCR
- Agarose Gel Electrophoresis
- Protein Expression Analysis
- Cell Fractionation
- SDS-PAGE & Coomassie
- Degradation Assay
- Co-Culture with Microcystis Aeruginosa
- Culture Setup
- Population of Microcystis Aeruginosa
- RNA Extraction and Reverse Transcription
- Quantitative PCR

#### **Construction of Bacteria**

##### **Bacterial Transformation**

Performed and Documented by **Chenni Wang**

###### **Materials**

Plasmid vector DNA (pET23b)

Insert DNA

DNA ligase and buffer

Competent state E. coli (strain Bl21 (DE3))

LB media

LB plate with appropriate antibiotic for the LB agar

###### **Equipment:**

Eppendorf tube

Shaking incubator at 37 °C

Stationary incubator at 37 °C

Water bath at 42 °C

Bucket of ice

Microcentrifuge tubes

Sterile spreading device (i.e. pipette tip)

###### **Procedures:**

1. Combine the following in a PCR or Eppendorf tube:

   * Plasmid Vector DNA
   * Insert DNA
   * Ligase Buffer (1μL/10μL reaction for 10X buffer)
   * 1μL DNA Ligase
   * H2O to a total of 10μL
   * For a control group, add H2O instead of insert DNA.
2. Incubate at room temperature (approximately 25°C) for 2 hours before proceeding to bacterial transformation.
3. Incubate at room temperature (approximately 25°C) for 2 hours before proceeding to bacterial transformation.
4. Retrieve competent cells from -80°C refrigerator, and defrost on ice for approximately 30 minutes.
5. Retrieve agar plates containing the appropriate antibiotic from storage at 4°C, and let it warm up to room temperature. Leave in 37°C incubator for later use.
6. Mix 5μL of plasmid DNA into 50 μL of competent cells in a microcentrifuge tube. Gently mix the components. For a control group, add 1μL of water to verify that the LB agar’s antibiotic functions properly.
7. Incubate the competent cell/DNA mixture on ice for approximately 30 minutes.
8. Heat shock each transformation tube by placing the bottom half of the tube into a 42°C water bath, leaving it for approximately 45 seconds.
9. Put the tubes back on ice for 2 minutes.
10. Add 250 μL LB media (without antibiotic) to the bacteria, and grow in 37°C shaking incubator for 45 min.
11. Plate an appropriate amount of the transformation onto the 10 cm LB agar plate containing the corresponding antibiotic.
12. Incubate plates at 37°C overnight.

##### **Liquid Culture & Glycerol Stocks**

###### **Materials:**

LB media

Appropriate antibiotic for LB agar

Previously cultured bacteria

50% filter sterilized glycerol

###### **Equipment:**

Sterile 1.5 mL Eppendorf Tubes

Sterile loop for bacteria retrieval

###### **Procedures:**

1. Add liquid LB to a tube or flask and add the appropriate concentration of antibiotic.
2. Using a sterile loop, select a single colony from the LB agar plate.
3. Drop the loop into the liquid LB mixture and swirl.
4. Loosely cover the culture with a sterile cap that is not air tight. Incubate bacterial culture at 37°C for 12-18 hours in a shaking incubator.
5. After observed bacterial growth, add 500 μL of the overnight culture to 500 μL of 50% glycerol in a 2 mL screw top tube, and gently mix.
6. Freeze the glycerol stock tube at -80°C.

#### **Restrictive Endonuclease Digestion**

Documented by **Yunxian Ding** and **Yunzhu Chen**

##### **Recovering Plasmid DNA:**

###### **Materials:**

Overnight culture of bacteria transformed with plasmid

PBS buffer

Denaturing solution

Renaturing solution

100% and 70% ethanol

###### **Equipment:**

Desktop microcentrifuge

Desktop vortexer

###### **Procedures:**

1. Grow an overnight culture of bacteria.
2. Centrifuge the culture to pellet the bacteria before proceeding with DNA preparation.
3. Remove the supernatant and resuspend the bacteria in buffer.
4. Add a denaturing solution from the DNA extraction kit to the resuspended bacteria.
5. Add a renaturing solution from the DNA extraction kit to the denatured bacteria.
6. Pellet the proteins and genomic DNA by centrifugation, and remove the plasmid-containing supernatant.
7. Add ethanol (or isopropanol) to precipitate the plasmid DNA.
8. Centrifuge at 12000 rpm for 10 minutes to pellet the DNA.
9. Wash the pellet with 70% ethanol to remove excess salt.
10. Resuspend the DNA pellet with PBS buffer.

##### **Diagnostic Endonuclease Digest & PCR**

###### **Materials\*:**

Plasmid DNA

Restriction enzyme

Restriction digest buffer

Forward Primer

Reverse Primer

5 μL 10X Taq buffer with MgCl2

1 μL dNTP mix

Taq DNA Polymerase

\*A TaKaRa PCR amplification kit was used in our experiments. Its details can be found here: <https://www.takara.co.kr/file/manual/pdf/R011_e.v1906Da.pdf>

###### **Equipment:**

Thin-walled PCR tube

Ice Bucket

PCR Machine

###### **Procedures:**

1. In a 1.5mL tube combine the following:

   * 1 μg of plasmid DNA sample
   * 1 μL of each restriction enzyme
   * 3 μL restriction digest buffer
   * H2O up to total volume of 30 μL

   Mix gently by pipetting.
2. Incubate tube at 37 °C for 1 hour.
3. Place thin-walled PCR tubes on ice. Set up a 50 μL reaction:

   * 2 μL template DNA
   * 5 μL 10X Taq buffer with MgCl2
   * 1 μL dNTP mix
   * 2.5 μL of 10 μM Forward Primer
   * 2.5 μL of 10 μM Reverse Primer
   * 0.2 μL 5 units/μL Taq DNA Polymerase
   * H2O up to total volume of 50 μL
4. Place reaction tubes in PCR machine. Set the standard time and temperature for each step in a PCR cycle:

   Initial Denaturation for 2 minutes at 94°C.

   Denature for 30 seconds at 94°C.

   Anneal primers for 30 seconds at 5°C below Tm.\*

   Extend DNA for 2 minutes at 72°C.
5. Repeat for 20-30 cycles. Final Extension for 5 minutes at 72°C.

\*We amplified 3 different sequences with different primer Tm, so they were not performed simultaneously.

##### **Agarose Gel Electrophoresis:**

###### **Materials:**

DNA samples

Gel loading dye

Electrophoresis buffer

TAE

Agarose

Gelred

###### **Equipment:**

Well combs

Gel box

Microwave

Electrophoresis chamber

Voltage source

UV light source

Casting tray

![](https://static.igem.wiki/teams/4125/wiki/notebook/a-pic-1.jpg)

FIG.1 Agarose Gel and Reagents

###### **Procedures:**

* Mix 1g of agarose powder and 8μL of GelRed with 100 mL 1xTAE in a microwavable flask.
* Microwave for 1-3 min until the agarose is completely dissolved.
* Let agarose solution cool down to about 50 °C in about 5 mins.
* Pour the agarose into a gel tray with the well comb in place.
* Place the gel at room temperature for 20-30 mins, until it has completely solidified.
* Place the gel into the gel box of electrophoresis unit. Fill gel box with 1xTAE until the gel is covered.
* Carefully load a molecular weight ladder.
* Add loading buffer to each DNA sample. Carefully load samples into the additional wells of the gel.
* Run the gel at 120 V for approximately 90 minutes.
* Turn off power, disconnect the electrodes, and remove the gel from the gel box.
* Visualize DNA fragments using UV light source.

![](https://static.igem.wiki/teams/4125/wiki/notebook/a-pic-2.png)

FIG.2 Restrictive Endonuclease Digestion Results

#### **Protein Expression Analysis**

Performed and Documented by **Qinyi Wang** and **Zhiwen Ji**

##### **Cell Fractionation**

###### **Materials:**

Bacteria Samples

0.1mM IPTG inductor

PBS buffer

0.1mM EDTA

10 μg/mL lysozyme

0.01mM MgCl2

2% Triton X-100

###### **Equipment:**

Microcentrifuge tubes

Ultracentrifuge tubes

Desktop centrifuge

Ultracentrifuge

Ultrasound sonicator

Ice bucket and beaker

###### **Procedures:**

* The bacteria were grown at 37°C for 12h after 0.1mM IPTG induction.
* Cells were collected by centrifuge of 6000 rpm for 10 min.
* After 2 washes with PBS buffer, cells were resuspended in PBS containing 0.1mM EDTA and 10 μg/mL lysozyme.
* After incubation of 2 hours on ice, the cell suspensions were treated with ultrasound sonication on ice (5 minutes, 1 cycle).
* Intact cells were separated from the lysates with a slow centrifuge of 6000 rpm for 10 min.
* At this point, the supernatant contained the whole cell lysates. We retrieved it with ultracentrifugation at 50,000 rpm for 1h to pellet the total membrane fraction.
* The supernatant containing soluble cytoplasmic and periplasm fractions was collected and stored at -4°C for later assays.
* The pellet of total membrane fraction was resuspended into PBS buffer containing 0.01 mM MgCl2 and 2% Triton X-100 for solubilizing the inner membrane, and incubated at room temperature for 30 minutes.
* The suspension was ultracentrifuged again at 50,000 rpm for 1h. The supernatant contained the inner membrane proteins, and the pellet contained the outer membrane proteins.
* All samples were collected and stored at -4°C for later assays.

##### **SDS-PAGE & Coomassie**

###### **Materials:**

1. 1X PBS
2. Protein loading buffer
3. Precast SDS-PAGE gel
4. SDS-PAGE running buffer
5. Prestained protein ladder
6. Distilled deionized (DDI) water
7. Coomassie Stain Solution:

   * Ethanol 75 mL
   * Glacial Acetic Acid 25 mL
   * DDI H2O 150 mL
   * Coomassie Brilliant Blue R-250 0.5 g
8. Destain Solution:

   * Ethanol 60 mL
   * Glacial Acetic Acid 20 mL
   * DDI H2O 120 mL

###### **Equipment:**

Heat block

SDS-PAGE gel tank & chamber

Power supply

Spatula

Platform shaker

Gel dryer

###### **Procedures:**

* Prepare the precast gel as follows:

  a)Remove the gel from the plastic packaging.

  b)Remove tape and plastic comb.

  c)Rinse the wells with DDI water 3 times, Shake the gel gently between washes to remove residual water.

  d)Load the gel into the chamber of the SDS-PAGE gel tank, fill the chamber with the 1X running buffer.
* Add 16 µL of loading buffer to 20 µL of each sample. The mixture was heated at 100°C for 5 minutes.
* Load 20 µL of each samples into the gel.
* Load 16 µL of the prestained protein ladder.
* Place the lid on the tank and plug the electrode cords into the power supply.
* Run the gel at 100 V for approximately 15 minutes, until the dye has migrated into the running gel.
* Increase the voltage to 150 V and continue for approximately 40 minutes, until the dye front has reached the bottom of the gel.
* Remove the run gel from the apparatus and remove the spacers and glass plates. Place the gel into a small tray.
* Rinse the gel 3 times for 5 minutes with 100 ml deionized water to remove SDS and buffer salts. Discard each rinse.
* Add 50 mL Coomassie Bright Blue R-250 staining solution and stain for 30 minutes with gentle shaking at 100 rpm. Discard the stain.
* Add 30 ml destain solution and destain for 1 minute with gentle shaking at 100 rpm.
* Pour off and discard the destain solution. Add 50 mL of destain solution.
* Destain with gentle shaking until the gel is visibly destained, for approximately 2.5 hours. Discard the destain solution.
* Add 50 ml DDI H2O and rinse for 5 minutes with gentle shaking at 100 rpm.
* Dry the gel on the gel dryer at 60°C for 1 hour with a sheet of filter paper below the gel.

![](https://static.igem.wiki/teams/4125/wiki/notebook/a-pic-3.png)

FIG.3 SDS-PAGE Gel Stained with Coomassie

#### **Degradation Assay**

Performed and Documented by **Qinyi Wang** and **Zhiwen Ji**

###### **Materials:**

MC-LR (This study used MC-LR synthesized by Taiwan Algal Science Inc.)

Bacteria from glycerol stock

Methanol and trifluoroacetic acid as the mobile phase of HPLC

PBS buffer

LB agar plate and media

###### **Equipment:**

Desktop microcentrifuge

Microcentrifuge tubes

Shaking incubator at 37 °C

HPLC vials

High-performance liquid chromatography machine

Mass spectrograph machine

###### **Procedures:**

* 1.Samples with MC-LR at concentrations 0.1 mg/L, 0.25 mg/L, 0.5 mg/L, 1 mg/L, 2.5 mg/L, 5 mg/L, 10 mg/L in PBS buffer were analyzed using HPLC to derive the standard curve.

  ![](https://static.igem.wiki/teams/4125/wiki/notebook/a-pic-4.png)
* Recombinant strain of pET23b-inaK+mlrA was incubated overnight on a 120 rpm shaker at 37 °C.
* After cell growth, retrieved cell colony were centrifuged at 8000 rpm for 5 min and wash with PBS buffer twice. The cells were resuspended in PBS reaction system of 20mL containing MC-LR and cultivated on a 120 rpm shaker at 37 °C for 10 hours. The same culture medium without any bacteria was used as a control. Both the treatment group and the control group were triplicated.
* Samples were collected from the cultures every hour, and centrifuged at 10,000 rpm for 1 min to separate the bacteria from the medium.
* Supernatant from the sample were placed into HPLC vials for concentration assay. The concentration of cyclic MC-LR was measured by the area in HPLC graph and compared to the standard curve.

  ![](https://static.igem.wiki/teams/4125/wiki/notebook/a-pic-5.png)

  FIG.5 Degradation Assay Results
  (error = standard deviation)
* Samples from the 10th hour were analyzed with mass spectrograph to determine the final product of linearized microcystin.

  ![](https://static.igem.wiki/teams/4125/wiki/notebook/a-pic-6.png)

  FIG.6 Mass Spectrometry of Degradation Product

#### **Co-Culture with microcystis aeruginosa**

Performed and Documented by **Qinyi Wang, Yunxian Ding,** and **Chenni Wang**

##### **Culture Setup**

###### **Materials:**

Microcystis aeruginosa

Engineered E. coli

PBS buffer

###### **Equipment:**

Spectrophotometer

250 mL Erlenmeyer flask

###### **Procedures:**

* 1.Grow an overnight culture of bacteria and microcystis aeruginosa.
* 2.Add the two types of cells into an 250 mL Erlenmeyer flask. Adjust the culture so that all experimental groups had cyanobacteria density of 8.96±0.32×106 CFU/mL and E. coli density of 1.52±0.24×109 CFU/mL.
* Control group was cultured with only microcystis aeruginosa. Both the treatment group and the control group were triplicated.
* 3.The flasks were kept at 25 °C for 7 consecutive days.

##### **Population of microcystis aeruginosa**

###### **Materials:**

Co-culture of microcystis aeruginosa and engineered E. coli

Distilled water

###### **Equipment:**

Hemocytometer

Microscope

###### **Procedures:**

1. Gently swirl the flask to ensure the cells are evenly distributed.Before the cells have a chance to settle, take out 0.5 mL of cell suspension using a sterile pipette and place in an Eppendorf tube. Add distilled water to total volume 2.5 mL.
2. Pipette 100 µL of diluted cell suspension and apply to the hemocytometer.
3. Using a microscope, focus on the grid lines of the hemocytometer with a 10X objective. Count the live cells in one set of 16 squares. If necessary, dilute again until all cells are distinct from each other.

   Note: although the E. coli and cyanobacteria were not separated, only cyanobacteria are identifiable under optical microscope precision. Therefore, the counting is not interfered.

   ![](https://static.igem.wiki/teams/4125/wiki/notebook/a-pic-7.png)

   FIG.7 microcystis aeruginosa Population Density
   (error = standard deviation)

##### **RNA extraction and reverse transcription**

###### **Materials:**

Co-culture of microcystis aeruginosa and engineered E. coli

PBS buffer

TRIzol

Chloroform

Isopropanol

Ethanol

RNAse-free water

Reverse Transcription Buffer

MgCl2

dNTP mix

DTT

RNase Inhibitor

MultiScribe Reverse Transcriptase

DEPT-treated water

RNA-specific primer

###### **Equipment:**

Desktop microcentrifuge

Desktop vortexer

Ice bucket

RNAse-free tube

Thermal cycler

###### **Procedures:**

1. 1 mL of cell culture was retrieved and centrifuged at 8000 rpm for 10 minutes.
2. The pellet was resuspended in PBS and TRIzol to be lysed, and sat at room temperature for 5 minutes.
3. Extract RNA from the samples: add 0.2 mL of Chloroform per 1 mL of TRIzol used. Shake by hand for 10 seconds.
4. Incubate the samples for 3 minutes on ice and centrifuge for 15 minutes at 10,000 rpm at 4°C to separate RNA from the rest of the cell lysate.
5. Transfer the aqueous layer to a new RNAse-free tube. Add 1 volume of Isopropanol to the extracted aqueous layer. Incubate at -20°C for 2 hours.
6. Centrifuge for 20 minutes at 10,000 rpm at 4°C and discard the supernatant.
7. Wash the pelleted RNA by resuspending the pellet in 0.5 ml of 75% ethanol and vortex for a few seconds. Centrifuge for 5 minutes at 10,000 rpm at 4 °C.
8. Remove the ethanol wash and air-dry the pellet for 5 minutes.
9. Resuspend RNA pellet in RNase-free water.
10. Prepare reverse transcription master mix: (listed are final concentrations)

    * 1X Reverse Transcription Buffer
    * 1.75 mM MgCl2
    * 0.5mM (each) dNTP mix
    * 5.0 mM DTT
    * 1.0 U/µL RNase Inhibitor
    * MultiScribe Reverse Transcriptase 2.5 U/µL
    * DEPT-treated water to total volume
11. 11.Add 1 µL of template RNA and 1 µL of 10 µM primer into 20 µL reaction system containing the above master mix.
12. 12.Incubate at:

    * 65 °C for 5 minutes
    * 4 °C for 2 minutes
    * 37 °C for 30 minutes
    * 95 °C for 5 minutes
    * 4 °C until retrieved.

##### **Quantitative PCR**

###### **Materials:**

Reverse transcribed cDNA

SyBr Green PCR Mix

qPCR primers

Distilled water

###### **Equipment:**

Desktop microcentrifuge

Desktop vortexer

qPCR machine

96 well plate

###### **Procedures:**

1. Prepare reaction mix for each well as follows:

   * 2 µL 1:10 diluted cDNA (or water, for control groups)
   * 5 µL 2x SyBr Green PCR Mix
   * 0.05 µL 10uM forward primer
   * 0.05 µL 10uM reverse primer
   * 2.9 µL water
2. Run qPCR at the following temperatures:

   * 50°C,2 minutes
   * 95 °C,10 minutes
   * 95°C, 15 seconds; 60°C,1 minute for 40 cycles.

Note: although the E. coli and cyanobacteria were not separated, only genes unique to microcystis aeruginosa were examined in qPCR. Moreover, the 16S gene not present in E. coli was used as reference gene, so results were not interfered.

![](https://static.igem.wiki/teams/4125/wiki/notebook/a-pic-8.png)

FIG.8 Metabolic Activity: Relative Gene Expression Levels on Day 7
(error = standard deviation)

![](https://static.igem.wiki/teams/4125/wiki/notebook/a-pic-9.png)

FIG. 9 Microcystin-LR Synthesis: Relative Gene Expression Levels on Day 7
(error = standard deviation)

##### References:

[1].Li, L., Gyun Kang, D. and Joon Cha, H., 2004. Functional display of foreign protein on surface of Escherichia coli using N‐terminal domain of ice nucleation protein. Biotechnology and bioengineering, 85(2), pp.214-221.

[2].Shi, H. and Su, W.W., 2001. Display of green fluorescent protein on Escherichia coli cell surface. Enzyme and microbial technology, 28(1), pp.25-34.

[3].Liu, M., Ni, H., Yang, L., Chen, G., Yan, X., Leng, X., Liu, P. and Li, X., 2019. Pretreatment of swine manure containing β-lactam antibiotics with whole-cell biocatalyst to improve biogas production. Journal of Cleaner Production, 240, p.118070.

[4].Qin, L., Zhang, X., Chen, X., Wang, K., Shen, Y. and Li, D., 2019. Isolation of a novel microcystin-degrading bacterium and the evolutionary origin of mlr gene cluster. Toxins, 11(5), p.269.

[5].Nishizawa, T., Asayama, M., Fujii, K., Harada, K.I. and Shirai, M., 1999. Genetic analysis of the peptide synthetase genes for a cyclic heptapeptide microcystin in Microcystis spp. The journal of biochemistry, 126(3), pp.520-529.

[6].Addgene.org. 2022. Addgene: Protocols for Molecular Biology, Plasmid Cloning, and Viral Preps. [online] Available at:  [Accessed 1 October 2022].

[7].Assets.fishersci.com. 2022. TaqMan Reverse Transcription Reagents. [online] Available at:  [Accessed 1 October 2022].

Hosting.med.upenn.edu. 2022. Berger Lab Protocol: Quantitative PCR from Human Cell RNA. [online] Available at:  [Accessed 1 October 2022].

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{% block title %}engineering{% endblock %}
{% block pageTitle %}engineering{% endblock %}
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- Overview
- Stage 1 Design
- mlr Gene Cluster
- Ice Nucleation Protein (InaK)
- Stage 2 Construction
- Stage 3 Verification
- Stage 4 Further Analysis

#### **Overview**

This year, team Nanjing\_NFLS designed and characterized 2 new parts. By going through the engineering cycle of Design – Construction – Verification – Further Analysis, we succeeded in creating a microcystin degrading bacteria.

| Part Number | Type | Description | Length |
| --- | --- | --- | --- |
| [BBa\_K4125000](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3964000) | Basic | mlrA from Sphingopyxis sp.m6 | 1010 bp |
| [BBa\_K4125001](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3964000) | Composite | mlrA + inaK surface display | 1553 bp |

Table 1. Parts Overview

Click on the Part Number to view our Part Registry pages!

#### **Stage 1 Design**

First, we reviewed previous research to set a specific focus for tackling the microcystin pollution issue.

##### **mlr Gene Cluster**

From previous research on the solutions for microcystin pollution (see Description page), we found that biodegradation is an advantageous approach compared to traditional chemical and physical methods. (Ding et al., 2022) We also learned that the mlr gene cluster in natural microcystin degrading bacteria is responsible for the degradation pathway. In order to find a more specific starting point for our project, we carried out an extensive literature review on the mlr gene cluster.

We found that current research on the mlr gene cluster can be roughly categorized into 3 types:

**1) Separation from natural bacterial strains**, where bacteria from different locations are retrieved, identified, and sequenced for their functional gene fragments in the degradation pathway.

**2) Determination of biodegradation pathway**, where reactions and chemical properties of intermediate products of the enzymatic cascade were characterized. The respective roles of the enzymes, catalytic or transporting, were verified through heterologous expression as well. (Shimizu et al., 2012)

**T3) Proposed implementations**, where a functional and mature system of microbial biodegradation is produced for application in a given scenario. Possible troubleshooting and environmental adaptability should also be provided.

In our analysis of 91 articles retrieved from 1994 (when the first natural microcystin degrading bacteria was identified) to May 2022, we found relatively sufficient work in (1) and (2). Dozens of various microcystin degraders have been separated and sequenced, with phylogenetic analysis on their respective mlr gene clusters. The biodegradation pathway has also been primarily clarified, with the reactants, products, and catalysts for each step determined.

However, we found a shortage of implementation-oriented projects. The few current studies took advantage of the efficient nature of the MlrA-catalyzed linearization process, and some added an alternative treatment mainly (chemical or physical) to the less toxic linearized microcystin. (Fionah et al., 2022) The results were promising, and we believe this mlrA-based composite bioremediation could be the research hotspot in the future. These methods can be improved by either optimizing the heterologous expression of mlrA or investigating the secondary treatment.

Considering our team’s interests in iGEM, we decide to explore new ways of expressing mlrA in the recombinant bacteria. The mlrA gene sequence that we used was from Sphingopyxis sp.m6, a strain separated from Lake Taihu by the Zhang Lab that we are experimenting at. It is one of the most efficient types of mlr genes that have been tested yet.

![](https://static.igem.wiki/teams/4125/wiki/engineering/a-pic-1.png)

FIG.1 Structure of MlrA from Sphingopyxis sp. m6

Predicted by Phyre2 with Full Sequence (Kelly et al., 2015)

[View our new Part mlrA here!](http://parts.igem.org/Part%3ABBa_K4125000)

#### **Ice Nucleation Protein (InaK)**

When studying the mlr gene cluster, we found that the expression of MlrABC, the catalytic proteins, depended on the intake of microcystins by MlrD. This complexified the process of degrading toxins present in the environment. Therefore, we were particularly interested in eliminating this step when researching ways of mlrA heterologous expression.

We found abundant literature discussing cell-surface enzyme display systems to degrade chemicals in the environment. Further review led us to the **ice nucleation protein (InaK)** from Pseudomonas syringae, which is a unique surface display motif that is bifunctional: 1) ice nucleation activity; 2) anchoring on the cell surface. Its C-terminal is attached to the cell membrane via the GPI anchor (Glycosylphosphatidylinositol) commonly found in eukaryotic cells only and is stable in the stationary phase of the culture. Furthermore, InaK has been found to be compatible with the protein secretion mechanism of its host cell E. coli. All these qualities make it an ideal choice for an anchoring chassis. (Jung et al., 1998)

![](https://static.igem.wiki/teams/4125/wiki/engineering/a-pic-2.png)

FIG.2 Structure of ice nucleation protein

Predicted by Phyre2 with Full Sequence (Kelly et al., 2015)

[View the Part inaK we used here!](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3986004)

#### **Stage 2 Construction**

Plenty of research has used ice nucleation protein to display specific enzymes that degrade environmental pollutants, including heavy metals (Bae et al. 2002), organophosphates (Shimazu et al., 2001), dye (Gao et al., 2014), and antibiotics erythromycin (Liu et al., 2020). This prompted us to construct an extracellular enzyme display system for MlrA, based on the anchoring InaK.

![](https://static.igem.wiki/teams/4125/wiki/engineering/a-pic-3.png)

FIG.3 Schematic of Recombinant Plasmid

Created with SnapGene 6.1.1

The recombinant plasmid pET-23b-inaK+mlrA is then transformed into E.coli BL21 (DE3). We hypothesized an extracellular enzyme display system that allows our MlrA enzymes to be immobilized on the surface of the bacteria cell. This way, they can directly degrade the toxins present in the environment. (Liu et al., 2020)

![](https://static.igem.wiki/teams/4125/wiki/engineering/a-pic-4.png)

FIG.4 Schematic of Extracellular Enzyme Display System

Created with Biorender

[View our new composite Part here!](http://parts.igem.org/Part%3ABBa_K4125001)

#### **Stage 3 Verification**

We designed a set of experiments to verify our theoretical design. They can be mainly categorized into 3 steps:

1.Identification of plasmid construction by restrictive endonuclease digestion and agarose gel electrophoresis

2.Identification of protein location by cell fractionation with ultra-centrifuge, followed by SDS-PAGE western blotting (Shi and Su, 2001; Li et al., 2004)

3.Deriving the standard curve for cyclic microcystin-LR concentration, and degradation assay in high performance liquid chromatography

![](https://static.igem.wiki/teams/4125/wiki/engineering/a-pic-5.png)

FIG.5 Schematic of Cell Fractionation

Created with Biorender

From these experiments, we successfully verified that our design has been realized, and that our cell can efficiently degrade microcystin-LR.

[View more details in Proof of Concept page!](https://2022.igem.wiki/nanjing-nfls/proof-of-concept)

#### **Stage 4 Further Analysis**

After succeeding with the initial system, we proceed to analyze its qualities and implementations.

We first utilized unstructured kinetics to model the enzyme-substrate interaction and coupled substrate depletion. (Manheim et al., 2019) From statistical analysis, we found that both models demonstrated excellent compatibility with out experimental data, and derived the fitted parameters. We also found that our bacteria can degrade microcystin-LR at rather low concentrations that are more environmentally-relevant. We believe these models will assist in the bacteria’s bioremediation applications.

[View more details in Model page!](https://2022.igem.wiki/nanjing-nfls/model)

Through learning stakeholders’ attitudes in using GMO for bioremediation (Sayler and Ripp, 2000), and field research in potential launching sites, we found it necessary to study the artificial bacteria’s influence on the environment. We decided to co-culture the bacteria with microcystis aeruginosa (a strain of microcystin-producing algae) and analyze their interactions in the following 3 aspects:

1.The surviving population of algae. This would be measured by directly counting the algae cells in a small sample from the culture.

2.The metabolic activity of algae. This would be measured by performing quantitative PCR on gene sequences 16S, psaB, psbD, rbcL, fbp.

3.The microcystin production of algae. The concentration of microcystin produced by algae at experimental scope is rather low, and is unsuitable for HPLC detection. The production would also be measured by performing quantitative PCR on genes mcyA, mcyB, mcyD, mcyG that are responsible for microcystin synthesis. (Nishizawa et al., 1999)

After monitoring these indicators for 7 days, we discovered that our bacteria has no influence on any of these aspects. Their compatibility with algae populations is verified. Moreover, many novel algae technologies are constructed based on their metabolism, and our bacteria can degrade toxins without interfering. They have the potential of becoming a component of a larger system of algae technology.

[View more details in Implementation page!](https://2022.igem.wiki/nanjing-nfls/implementation)

##### References:

[1].Ding, Q., Song, X., Yuan, M., Sun, R., Zhang, J., Yin, L. and Pu, Y., 2022. Removal of microcystins from water and primary treatment technologies – A comprehensive understanding based on bibliometric and content analysis, 1991–2020. Journal of Environmental Management, 305, p.114349.

[2].Shimizu, K., Maseda, H., Okano, K., Kurashima, T., Kawauchi, Y., Xue, Q., Utsumi, M., Zhang, Z. and Sugiura, N., 2012. Enzymatic pathway for biodegrading microcystin LR in Sphingopyxis sp. C-1. Journal of Bioscience and Bioengineering, 114(6), pp.630-634.

[3].Fionah, A., Hackett, C., Aljewari, H., Brady, L., Alqhtani, F., Escobar, I. and Thompson, A., 2022. Microcystin-LR Removal from Water via Enzymatic Linearization and Ultrafiltration. Toxins, 14(4), p.231.

[4].Kelley, L.A., Mezulis, S., Yates, C.M., Wass, M.N. and Sternberg, M.J., 2015. The Phyre2 web portal for protein modeling, prediction and analysis. Nature protocols, 10(6), pp.845-858.

[5].Jung, H.C., Lebeault, J.M. and Pan, J.G., 1998. The surface display of Zymomonas mobilis levansucrase using the ice-nucleation protein of Pseudomonas syringae. Nature Biotechnology, 16(6), pp.576-580.

[6].Bae, W., Mulchandani, A. and Chen, W., 2002. Cell surface display of synthetic phytochelatins using ice nucleation protein for enhanced heavy metal bioaccumulation. Journal of inorganic biochemistry, 88(2), pp.223-227.

[7].Shimazu, M., Mulchandani, A. and Chen, W., 2001. Cell surface display of organophosphorus hydrolase using ice nucleation protein. Biotechnology Progress, 17(1), pp.76-80.

[8].Gao, F., Ding, H., Feng, Z., Liu, D., and Zhao, Y., 2014. Functional display of triphenylmethane reductase for dye removal on the surface of Escherichia coli using N-terminal domain of ice nucleation protein. Bioresource Technology, 169, pp.181-187.

[9].Liu, M., Feng, P., Kakade, A., Yang, L., Chen, G., Yan, X., Ni, H., Liu, P., Kulshreshtha, S., Abomohra, A.E.F. and Li, X., 2020. Reducing residual antibiotic levels in animal feces using intestinal Escherichia coli with surface-displayed erythromycin esterase. Journal of hazardous materials, 388, p.122032.

[10].Shi, H. and Su, W.W., 2001. Display of green fluorescent protein on Escherichia coli cell surface. Enzyme and microbial technology, 28(1), pp.25-34.

[11].Li, L., Gyun Kang, D. and Joon Cha, H., 2004. Functional display of foreign protein on surface of Escherichia coli using N‐terminal domain of ice nucleation protein. Biotechnology and bioengineering, 85(2), pp.214-221.

[12].Manheim, D.C., Detwiler, R.L. and Jiang, S.C., 2019. Application of unstructured kinetic models to predict microcystin biodegradation: Towards a practical approach for drinking water treatment. Water research, 149, pp.617-631.

[13].Sayler, G.S. and Ripp, S., 2000. Field applications of genetically engineered microorganisms for bioremediation processes. Current opinion in biotechnology, 11(3), pp.286-289.

[14].Nishizawa, T., Asayama, M., Fujii, K., Harada, K.I. and Shirai, M., 1999. Genetic analysis of the peptide synthetase genes for a cyclic heptapeptide microcystin in Microcystis spp. The journal of biochemistry, 126(3), pp.520-529.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

## Inspirations

---

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

## 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.

## Inspirations

---

* [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)

{% endblock %}