iGEM 2022 — Team kuas-korea

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{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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## What should this page contain?

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* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

## Inspirations

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* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

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## **Engineering**

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### **Design**

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At the design stage, we first did the preliminary research for our project. Through this, necessary enzymes and substances were identified, and expression vectors and genes were selected.
The four enzymes we selected are as follows. Nitrite Reductase, Hydrazine Synthase, and Hydrazine Oxideneductase will be used for Nitrite (NO2-) and Ammonium (NH4+) removal.
And superoxide dismutase enzymes will be used to perform H2O2 production.
And we used this to construct parts. First, BBa\_K4371000 (Anammox cluster: HZS + HZO + NIR) was created by clustering three enzymes used in Nitrite (NO2-) and Ammonium (NH4+) removal.
It was inserted into pEXP5-NT, a specific cell free expression (CFE) vector.
Similarly, BBa\_K38007 (SOD1 protein), a parts of Superoxide dismutase, an enzyme that conducts H2O2 production, was inserted into the expression vector pEXP5-NT.

### **Build**

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In order to obtain the sequence found in the previous design stage, we ordered the sequence through Twist Bioscience. However, there was a problem that the sequence of the alpha subunit of the hydrazine synthase was longer than 1.8 kb. So we decided to get the gene by sequencing in the plasmid method.
And we construct cell free expression system's experiment methods.

### **Test**

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The T7 promoter shows the most active protein expression at 25°C.

![](https://static.igem.wiki/teams/4371/wiki/ic07-cfspt7.png)

Figure. pT7 maximum Temperature (http://parts.igem.org/Help:Cell-free\_chassis/Modelling#References)

When checked through modeling, it can be seen that it is generated to the maximum at 25°C.

### **Learn**

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In order to obtain more production, it is necessary to replace it with plasmid origin with a higher CN value or with a stronger RBS. Through this, more enzymes can be obtained and it can have advantages in producing filters.

### **Reference**

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* http://parts.igem.org/Help:Cell-free\_chassis/Modelling#References

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## 1. Experimental Design

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### 1.1. CFE(Cell Free Expression) system selection

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* Our team has decided to use CFE system of E.Coli for following reasons:

1) Anammox is originated from Bacteria, which implied that using cell extracts from E.coli would be unlikely to produce erroneous result.

2) E.coli cell free Expression System is the quickest and most efficient way of CFE.

* The kit we have selected for the experiment is  from Thermo Fisher (catalog nos. K9901-00, K9900-96, K9900-97).

### 1.2. DNA sequences selection

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* The selection of the target DNA sequence(parts)and design has been demonstrated on the ["DEISGN"](https://2022.igem.wiki/kuas-korea/design) page.

### 1.3. DNA template design

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* We have decided to use the expression vector pEXP5-NT/TOPO® which is provided by the Expressway™ Cell-Free E. coli Expression System kit of Thermo Fisher.

* DNA template will be built by ligating insert sequence(see also 1.2) with the vector plasmid.

## 2. DNA Template Generation

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### 2.1. DNA fragment order

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* We have placed an order for 6 target sequences(Superoxide Dismutase; Hydrazine Synthase\_alpha; Hydrazine Synthase\_beta; Hydrazine Synthase\_gamma; Hydroxylamine oxidoreductase; Nitrite reductase, see also ["parts"](https://2022.igem.wiki/kuas-korea/parts)) with Twist Bioscience.

### 2.2. DNA fragment preparation: PCR amplification

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* Through this step, we can add homologous overlaps to DNA fragments(or inserts) and vectors, wihch is required for processing Gibson Assembly.

#### 2.2.1. Materials

Component | Volume || Insert or Vector DNA (100 pg/μL – 1 ng/μL in TE) | 0.5 μL |
| 10 μM Forward Primer |  |
| 10 μM Forward Primer | 2.5 μL |
| 10 μM Reverse Primer | 2.5 μL |
| 10 mM dNTPs | 1 μL |
| 10 mM dNTPs | 1 μL |
| 5X Phusion HF Buffer | 10 μL |
| Phusion® DNA Polymerase (2 U/ μL) | 0.5 μL |
| Nuclease-free Water | 33 μL |
| Total | 50 μL |

#### 2.2.2. Methods

a. Operation of Thermocycle

Step | Temperature | Duration | Number of cycles || Initial denaturation | 98°C | 30 seconds | 1 cycle |
|  | 98°C | 10 seconds |  |
| Amplification | Primer Tm(60°C to 70°C) | 20 seconds | 25–30 cycles |
|  | 72°C | 30 seconds per kb |  |
| Final extension | 72°C | 5 minutes | 1 cycle |
| Hold | 4°C | - | 1 cycle |

b. Purification

* Analyze the assembly reaction by performing gel electrophoresis with 5–10 μL of the reaction on an 0.8–2% agarose gel.

### 2.3. Gibson Assembly

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* We have followed the protocol provided with .

#### 2.3.1. Materials

* GA 1-Step Master Mix (2X)
* GA Positive Control (2X)

#### 2.3.2. Methods

1. Thaw GA 1-Step Master Mix (2X) on ice.
2. In PCR tubes, prepare DNA fragments in nuclease-free using the following formula.
[pmol DNA = [ng DNA/(660 x # of bases)] x 1000]
3. Vigorously vortex the master mix for 15 seconds immediately before use after it is thawed.
4. In a tube on ice, combine 5 μL of DNA fragments and 5 μL of GA 1-Step Master Mix (2X). Mix the reaction by pipetting.
5. For the positive control, combine 5 μL of the Positive Control (2X) and 5 μL of GA 1-Step Master Mix (2X) in a tube on ice. Mix the reaction by pipetting.
6. Vortex and spin down all reactions.
7. Incubate the reactions at 50°C for 1 hour.
8. After the incubation is complete, store the reactions at −20°C or dilute reactions for the downstream application, CFE.
9. Analyze the assembly reaction by performing gel electrophoresis with 5–10 μL of the reaction on an 0.8–2% agarose gel.

## 3. Protein Synthesis Reaction

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### 3.1. Protocol

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* given for 100 µl of reaction volume
* refer to [[invitrogen: Expressway Cell-Free E.coli Expression System](https://static.igem.wiki/teams/4371/wiki/expressway-system-man.pdf)] mannual provided by Thermo Fisher

#### 3.1.1. Materials

* Expression construct or other suitable DNA template (purified; resuspended in TE or water at a concentration greater than 500 ng/µl)
* For each reaction, sterile, RNase-free 1.5 ml Eppendorf tubes or other suitable reaction vessels
* RNase-free pipette tips and microcentrifuge tubes
* Standard shaking incubator (set to 30°C or 37°C and 300 rpm)
* Expressway™ E. coli slyD- Extract (thaw on ice)
* Expressway™ 2.5X IVPS E. coli Reaction Buffer (-A.A.) (thaw on ice)
* Expressway™ 2X IVPS Feed Buffer (thaw on ice)
* T7 Enzyme Mix (keep on ice; store at -20°C after initial use)
* 50 mM Amino Acids (-Met)
* 75 mM Methionine
* DNase/RNase-free distilled water (supplied with the kit)

#### 3.1.2. Methods

1. For each sample, add the following reagents to the appropriate reaction vessel:

Reagent | Amount || E. coli slyD- Extract | 20 µl |
| 2.5X IVPS E. coli Reaction Buffer (-A.A.) | 20 µl |
| 50 mM Amino Acids (-Met) | 1.25 µl |
| 75 mM Methionine\* | 1 µl |
| T7 Enzyme Mix | 1 µl |
| DNA Template | 1 µg |
| DNase/RNase-free Distilled Water | To a final volume of 50 µl |

2. Close the tube and incubate sample in a standard shaking incubator (300 rpm) at 30°C for 30 minutes. If the protein you are synthesizing is known to be soluble, you may incubate the sample at 37°C.

3. During the 30 minute incubation, prepare the Feed Buffer. For each sample, add the following reagents to a sterile, RNase-free microcentrifuge tube. For multiple samples, you may scale up the volume of reagents used accordingly and prepare one master mix.

Reagent | Amount || 2X IVPS Feed Buffer | 25 µl |
| 50 mM Amino Acids (-Met) | 1.25 µl |
| 75 mM Methionine\* | 1 µl |
| DNase/RNase-free Distilled Water | To final volume of 50 µl |

4. After 30 minutes of incubation (from Step 2 above), add 50 µl of the Feed Buffer to the sample (total volume = 100 µl).

5. Cap the tube and return the sample to the shaking incubator (300 rpm). Incubate for 3-6 hours at 30-37°C as appropriate.

6. Place the reaction on ice and proceed to Analyzing Samples, or store the sample at -20°C for future processing or analysis.

## 4. Measurement and Analysis

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### 4.1. Analysis

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#### 4.1.1. Materials

* Acetone (room temperature)
* 1X SDS-PAGE sample buffer

1) Combine the following reagents:

Reagent | Amount || 0.5 M Tris-HCl, pH 6.8 | 2.5 ml |
| Glycerol (100%) | 2 ml |
| β-mercaptoethanol | 0.4 ml |
| Bromophenol blue | 0.02 g |
| SDS | 0.4 g |

2) Bring the volume to 20 ml with sterile water.

3) Aliquot and freeze at -20°C until needed.

* Appropriate polyacrylamide gel to resolve your protein of interest
* Appropriate protein stain

#### 4.1.2. Methods

a. Acetone Precipitation

1. Add 5 µl of the protein reaction product from Step 6, previous page, to 20 µl of acetone. Mix well.
2. Centrifuge for 5 minutes at room temperature in a microcentrifuge at 12,000 rpm.
3. Carefully remove the supernatant, taking care not to disturb the protein pellet.
4. Resuspend pellet in 20 µl of 1X SDS-PAGE sample buffer.
5. Heat at 70-80°C for 10-15 minutes and centrifuge briefly. Proceed to Polyacrylamide Gel Electrophoresis on the next step.

b. Polyacylamide Gel Electrophoresis

1. Load 5-10 µl of the sample from Step 6, previous page on an SDS-PAGE gel and electrophorese at 120V. You may save your sample by storing at -20°C, if desired.
2. Stain gel with Coomassie blue stain or other stain.

### 4.2. Protein Yield Determination

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#### 4.2.1. Methods

a. Total Count Determination

1. Mix and spot 5 µl of each radiolabeled reaction from Step 6 above on a glass microfiber filter (Type GF/C; Whatman).
2. Set aside and let dry. Do not wash or TCA precipitate these filters.

b. TCA Precipitation

1. Mix and spot 5 µl of each radiolabeled reaction from Step 6, page 21 on a separate set of individual glass fiber (GF/C) filters and allow to air dry for approximately 5-10 seconds.
2. Place filter in a beaker and wash once with cold 10% TCA for 10 minutes at room temperature while shaking gently (use approximately 10-20 ml per filter).
3. Wash with 5% TCA for 5 minutes at room temperature while shaking gently. Repeat wash.
4. Rinse filters with methanol to facilitate drying.
5. Allow filters to dry, place in scintillation vials, and add scintillation fluid. Count samples in a scintillation counter.
6. Proceed to Calculating Protein Yield on the next step.

c. Protein Yield Calculation

* Use the equations below to calculate the yield of protein obtained.

![](https://static.igem.wiki/teams/4371/wiki/analyze.png)

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