iGEM 2023 — Team tsukuba

{% extends "layout.html" %}
{% 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 %}
{% block page\_content %}

![](https://static.igem.wiki/teams/4871/wiki/the-great-wave-off-kanagawa-1.webp)

NOTEBOOK

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[Download the file instead](https://static.igem.wiki/teams/4871/wiki/igem-notebook.pdf)

{% endblock %}

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

![](https://static.igem.wiki/teams/4871/wiki/the-great-wave-off-kanagawa-1.webp)

ENGINNERING

## 0. Abbreviation

---

CDP: cyclodipeptide
LC-MS: Liquid Chromatography-Mass spectrometry
.
LB: LB medium
M9: M9 minimal culture medium
PBS: Phosphate buffered saline

# 1. Overexpression of cWA synthase in E. coli

## Design 1

---

Our first step is to recombine E. coli to introduce the cWA synthase and show that E. coli can synthesize cWA.

![](https://static.igem.wiki/teams/4871/wiki/results/dna-cwa-synthase.png)

## Build 1

---

![](https://static.igem.wiki/teams/4871/wiki/results/ep-20230531-webp.webp)

First, Streptomyces varsoviensis NRRL B-3589 and Streptomyces monomycini NRRL B-24309 were transferred from RIKEN BRC and cultured in PDA medium. The target genes were then cut out by colony PCR.
The pET28b vector was then cut with restriction enzymes and introduced by Gibson Assembly together with the insert. To confirm that the vector was formed, it was cut again with restriction enzymes and electrophoresed.

## Test 1

---

The vector was then introduced into E. coli DE3 for expression, culture and expression were performed, and the culture medium was collected for LC-MS analysis.

## Learn 1

---

We found that CDP was synthesized at approximately the same rate as in the original paper.

# 2. Separation of main culture and substance production, change of medium for substance production

## Design 2

---

LB medium is the best medium for growth and expression of E. coli, but because it is rich in amino acids in both type and quantity, we wondered if E. coli would take up amino acids other than alanine and tryptophan to produce CDP other than cWA. On the other hand, if we narrowed down the types of amino acids in the main culture stage, growth and production of cWA synthase might be inhibited. Therefore, we decided to separate the main culture and material synthesis stages and use E. coli as a biocatalyst.

## Build 2

---

To separate the main culture and substance production stages and to investigate the effect of changing the composition of the medium for substance production on the amount of synthesis, the following medium compositions, times, and temperatures were used.

## LB main culture 3 hours

---

LB main culture -> Amino acid excess LB for substance synthesis 3 hours
LB main culture -> Amino acid excess LB for substance synthesis 6 hours
LB main culture -> Amino acid excess M9 for substance synthesis 6 hours
LB main culture -> Amino acid excess PBS for substance synthesis 12 hours

## Test 2

---

The reaction solution was subjected to LC-MS. None of the values were higher than those of Test1. In addition, the synthesis was higher in the order of LB, M9, and PBS for the medium used during substance synthesis.

## Learn 2

---

Since none of the media for material synthesis exceeded the Test 1 values, it was inferred that the yield decreased due to the separation of the main culture and material synthesis phases. The highest synthesis was observed in the order of LB, M9, and PBS, suggesting that the survival of E. coli played a major role in the synthesis of substances.

## Design 3

---

Since we found that changing the composition of amino acids in the medium did not change the synthesis ratio of CDP, we wondered if overexpression of native alanine synthase in E. coli and increasing the amount of alanine in the body would increase the amount of synthesis.

![](https://static.igem.wiki/teams/4871/wiki/results/dna-alanine-synthases.png)

## Build 3

---

![](https://static.igem.wiki/teams/4871/wiki/results/ep2-20231002-webp.webp)

The following parts are incorporated into the pCDF vector in different combinations and expressed together with cWA synthase. Currently the vectors are not assembled and we are trying to do so by trial and error.
BBa\_K4871010: avtA
BBa\_K4871011: iscS
BBa\_K4871012: alaD

## Future Prospect

---

・Use more efficient enzyme (BBa\_K4871002)
・Construct a cell-free system (Synthesis of aminoacyl-tRNA synthetase and RNA polymerase)

# 3. cWA Modeling

## Design 4

---

We planned to build and evaluate a molecular model in gauusian to assess the potential of cWA to be used as a paint.Our study also examined what elements are necessary for cWA to become paint.

## Build 4

---

Gauss View was used to actually build a molecular model that could be computed in Gaussian.We also considered whether the bond angles are appropriate when modeling this molecule.

## Test 4

---

Molecular models created by Gauss View were calculated using Gaussian.In addition, by changing the method of calculation when performing this calculation, the time required was much shorter than default planned.This fact has given us time to reflect on our vision for the future.

## learn4

---

Based on the calculation results, it was found that the model is suitable to be used as a paint, so we planned to conduct an experiment to convert it into paint. We also decided to explore the possibility of modifying the amino acids that make up the cyclic dipeptide to make it more efficient as a paint.

{% 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 %}

## Basic Operations

---

## Cultivation

1. Write information such as gene, vector, strain name on a conical tube.
2. Add 7 mLof liquid medium to the conical tube.
3. Poke one colony on the place with a tip and drop the tip with colony into the liquid medhium.
4. Shaking culturing at 37°C for 3 hours or 25°C for 12 hours.

## Wastewater treatment

Collect the medium containing recombinant E. coli in a Falcon or triangular flask covered
with silicone plug, and place the medium in a bag as specified and then autoclave it to kill the
bacteria.
Dispose ethyl acetate and organic solvents discharged by the LCMS safety in accordance with
the waste disposal regulations set forth by the university.

## Autoclave

1. Wrap aluminum foil around micropipette tips placed in a container, and place the medium
   in a container less than 1/3 of the container, and cover it with a silicone lid to ensure air
   permeability.
2. Open the lid of the autoclave sterilizer (Tommy Seiko LBS-325) and pour tap water so that
   the liquid level is above the bottom.
3. Place the items to be autoclaved in a mesh basket, and place the basket in the sterilizer.
4. Close the lid tightly, start autoclaving at 121°C for 15 minutes, and wait for 90 minutes.
5. Cool down the temperature of the can sufficiently, after that open the lid. And place the chips and other items to be dried more in the oven at 60°C for several hours.

## Cloning

---

## PCR, electrophoresis, and gel extraction

1. Mix as follows. Modify the values according to the concentrations of the DNA or the template.

   | compounds | μL | Final Concentration |
   | --- | --- | --- |
   | DW | X |  |
   | KOD one Master Mix | 12.5 |  |
   | Primers | 0.75 each | 0.3 μM each |
   | Template | Y | Plasmid DNA: ~1ng/μL cDNA: 15ng/μL |
2. Amplify by adjusting the temperature in 2steps ((92°C/5sec→63°C/20sec)・30 ).
3. Mix 1% agarose with TAE buffer, heat to dissolve, and solidify to form a gel, pour the sample into wells and electrophoresed at 100 V for 25 min.
4. Cut out a thin slice of the desired band and extract DNA using the Japan Genetics FastGene™ Gel / PCR Extraction Kit.
5. Measure DNA concentration using Nanodrop 2000.

## Double digestion by restriction enzyme

1. Mix as follows and incubate at 37°C for 2 hours.

   | compounds | μL |
   | --- | --- |
   | CutSmart Buffer x10 | 5 |
   | vector | 6 |
   | Enzymes | 1 each |
   | DW | X |
2. Electrophorese with the uncut vector to confirm that it was correctly cleaved (only one band in the middle of the two bands of the uncut vector), and then cut the gel out and extract the gel.

## Double digestion by restriction enzyme

1. Mix as follows and incubate at 37°C for 2 hours.

   | compounds | μL |
   | --- | --- |
   | CutSmart Buffer x10 | 5 |
   | vector | 6 |
   | Enzymes | 1 each |
   | DW | X |
2. Electrophorese with the uncut vector to confirm that it was correctly cleaved (only one band in the middle of the two bands of the uncut vector), and then cut the gel out and extract the gel.

## Gibson Assembly

1. Mix as follows and incubate at 50°C for 30 minutes.

   | compounds | μL |
   | --- | --- |
   | Master Mix | 3 |
   | Vector | 1 |
   | inserts | 2 each |

## Transformation

1. Place competent cells on ice for 15 minutes to thaw in a microtube. During this time, set the heat block to 56°C.
2. Add all of the Gibson Assembly reaction solution to the competent cells.
3. Conduct heat shock placing the cells. 0°C for 5 min, 56°C for 35 sec, and 0°C for 5 min.
4. Add 100 μL of antibiotic-free liquid LB medium, and recover at 37°C for 1 hour.
5. Spread the cells on antibiotic-added LB agar medium and incubate at 37°C overnight.

## Plasmid extraction

1. Poke One colony on plate with a sterilized pipette tip.
2. Put the tip into antibiotic-added liquid LB medium and incubate with shaking until the medium became turbid.
3. Extract plasmids according to FastGene Plasmid Mini.
4. Measure DNA concentration using Nanodrop 2000.

## Culturing, Compounds Analysis

---

## Pre-culture and main culture

1. Add one drop of the inoculum to a 15 mL test tube containing antibiotic-added liquid LB medium and incubate at 37°C with shaking.
2. When the culture medium became turbid, transfer it to a baffled Erlenmeyer flask containing 100 mL of antibiotic-added liquid LB medium and incubate at 37°C, 120 rpm for 3 hours with shaking.

## Substance Synthesis

1. Centrifuge the culture medium and concentrate the bacteria to make a pellet. Collect the supernatant in an appropriate container and autoclaved to sterilize.
2. Add saline solution to the pellet, vortex to suspend it, and measure OD.
3. Add saline so that the OD was 10.
4. To a 50 mL Falcon, add 4.5 mL of medium for material synthesis and 0.5 mL of E. coli suspension. After that, shake for the specified time.

## Solvent extraction and LC/MS of CDP

1. To 3 mL of the reaction solution, add 3 mL of ethyl acetate, shake well, and centrifuge at 10,000 rpm for 1 minute.
2. Reserve 2.5 mL of the oil layer, add 2.5 mL of ethyl acetate to the remainder, and repeat the same mixing, separation, and preparative procedure twice.
3. Evaporate 7.5 mL of ethyl acetate solution of the sample in a centrifugal evaporator (Sakuma Seisakusho EC100) at reduced pressure.
4. Dissolved the remaining solid in 100 μL of methanol, transfer it to a micro tube, and centrifuge at 15,000 rpm for 10 min. collect 40 μL of the supernatant carefully and place in an ampoule to make a sample.
5. Conduct LC/MS analysis (SHIMADZU LCMS-8045) using the following settings
   Organic solvent: acetonitrile
   Inorganic solvent: formic acid
   time: 26 minutes

## Culture Recipe

---

## LB

| compounds | g |
| --- | --- |
| Bacto Tryptone | 2 |
| Bacto Yeast Extract | 1 |
| NaCl | 2 |
| DW | up to 200 mL |

## M9

| compounds | g |
| --- | --- |
| Na2HPO4 | 0.72 |
| KH2PO4 | 0.6 |
| NH4Cl | 0.2 |
| NaCl | 0.1 |
| DW | up to 100 mL |

## PBS

| compounds | g |
| --- | --- |
| Na2HPO4 | 0.04 |
| KH2PO4 | 0.01 |
| NaCl | 0.9 |
| DW | up to 100 mL |

{% 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 %}

![](https://static.igem.wiki/teams/4871/wiki/the-great-wave-off-kanagawa-1.webp)

RESULTS

## 0. Abbreviation

---

CDP: cyclodipeptide
LC-MS: Liquid Chromatography-Mass spectrometry
.
LB: LB medium
M9: M9 minimal culture medium
PBS: Phosphate buffered saline

# 1. Overexpression of cWA synthase in E. coli

## Design 1

---

Our first step is to recombine E. coli to introduce the cWA synthase and show that E. coli can synthesize cWA.

![](https://static.igem.wiki/teams/4871/wiki/results/dna-cwa-synthase.png)

## Build 1

---

![](https://static.igem.wiki/teams/4871/wiki/results/ep-20230531-webp.webp)

First, Streptomyces varsoviensis NRRL B-3589 and Streptomyces monomycini NRRL B-24309 were transferred from RIKEN BRC and cultured in PDA medium. The target genes were then cut out by colony PCR.
The pET28b vector was then cut with restriction enzymes and introduced by Gibson Assembly together with the insert. To confirm that the vector was formed, it was cut again with restriction enzymes and electrophoresed.

## Test 1

---

The vector was then introduced into E. coli DE3 for expression, culture and expression were performed, and the culture medium was collected for LC-MS analysis.

## Learn 1

---

We found that CDP was synthesized at approximately the same rate as in the original paper.

# 2. Separation of main culture and substance production, change of medium for substance production

## Design 2

---

LB medium is the best medium for growth and expression of E. coli, but because it is rich in amino acids in both type and quantity, we wondered if E. coli would take up amino acids other than alanine and tryptophan to produce CDP other than cWA. On the other hand, if we narrowed down the types of amino acids in the main culture stage, growth and production of cWA synthase might be inhibited. Therefore, we decided to separate the main culture and material synthesis stages and use E. coli as a biocatalyst.

## Build 2

---

To separate the main culture and substance production stages and to investigate the effect of changing the composition of the medium for substance production on the amount of synthesis, the following medium compositions, times, and temperatures were used.

## LB main culture 3 hours

---

LB main culture -> Amino acid excess LB for substance synthesis 3 hours
LB main culture -> Amino acid excess LB for substance synthesis 6 hours
LB main culture -> Amino acid excess M9 for substance synthesis 6 hours
LB main culture -> Amino acid excess PBS for substance synthesis 12 hours

## Test 2

---

The reaction solution was subjected to LC-MS. None of the values were higher than those of Test1. In addition, the synthesis was higher in the order of LB, M9, and PBS for the medium used during substance synthesis.

## Learn 2

---

Since none of the media for material synthesis exceeded the Test 1 values, it was inferred that the yield decreased due to the separation of the main culture and material synthesis phases. The highest synthesis was observed in the order of LB, M9, and PBS, suggesting that the survival of E. coli played a major role in the synthesis of substances.

## Design 3

---

Since we found that changing the composition of amino acids in the medium did not change the synthesis ratio of CDP, we wondered if overexpression of native alanine synthase in E. coli and increasing the amount of alanine in the body would increase the amount of synthesis.

![](https://static.igem.wiki/teams/4871/wiki/results/dna-alanine-synthases.png)

## Build 3

---

![](https://static.igem.wiki/teams/4871/wiki/results/ep2-20231002-webp.webp)

The following parts are incorporated into the pCDF vector in different combinations and expressed together with cWA synthase. Currently the vectors are not assembled and we are trying to do so by trial and error.
BBa\_K4871010: avtA
BBa\_K4871011: iscS
BBa\_K4871012: alaD

## Future Prospect

---

・Use more efficient enzyme (BBa\_K4871002)
・Construct a cell-free system (Synthesis of aminoacyl-tRNA synthetase and RNA polymerase)

{% endblock %}