iGEM 2022 — Team qdai

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# Notebook

# 2022/08/16

## Kazuma Tsurumaki

1. Agarose gel electrophoresis

   Produced agarose gel & ran gel electrophoresis to verify the pgem-vector. used a nicked pgem-vector.

# 2022/09/08

## Kazuma Tsurumaki

1. Dissolved parts

   Dissolved glu-lac1,glu-lac2, and cell-glu2 in Milli-Q. 10ng/μl of parts are made.
   cell-glu1 is on order.
2. Digest a plasmid with HindIII
   Used HindIII to cut a plasmid.

   > HindIII:0.4㎕
   > pTWV228(0.5㎍/㎕,4039bp) :1㎕
   > NEB Buffer: 1㎕
   > MiliQ:7.6㎕

   pTWV228 cut with HindIII will be 0.019pmol/μg

   The cleavage product is named “pTWV228” and frozen.

# 2022/09/11

## Kazuma Tsurumaki, Kota Toyoda, Gonzalo Sifuentes

1. Gibson assembly
   According to the protocol offered by NEW England Biolabs

   > glc-ace1-　3㎕
   > glc-ace2-　3㎕
   > vector-　　0.3㎕
   > master mix(2x) 10㎕
   > milliQ 3.7㎕

   named the product “gibson 9/11”
2. Made LB broth

   made LB agar medium with ampicillin and liquid medium. Both were autoclaved.

# 2022/09/14

## Kazuma Tsurumaki

1. Transformation
   gibson 9/11 :1μl
   Competent cells:50μl

   Also, transformed 1μl of vector to use as a positive control.
   Following the Single Tube Transformation Protocol provided by iGEM, except for the process of heat shock.

   heat-shock for 30sec at 42°C on a heat block, according to Mr. Tashiro, working in the synbio lab at Kyushu Univ, who offers us cells.
2. Made competent cells
   According to this site
   <https://www.gene.mie-u.ac.jp/Protocol/Original/Transform-Comp-Cell-Freeze.html>

   Protocols

   > Shake at 37℃ to culture.
   > Dispense into tubes .
   > Incubate on ice for 30min.
   > Centrifuge at 2500rpm at 4℃ for 5min .
   > Discard mediums.
   > Add 25 ml of 50 mM CaCl2 and suspend on ice.
   > Incubate on ice for 60min.
   > Centrifuge at 2500rpm at 4℃ for 5min.
   > Discard supernatant.
   > Add 5ml of 50mM CaCl2 / 20% Glycerol and suspend.
   > Dispense 220 µl into one tube and quench at -80℃.

   BL21(DE3) is being cultured in 5ml LB medium.

# 2022/09/15

## Kazuma Tsurumaki, Kota Toyoda

1. Transformation
   checked yesterday’s plates

   Positive control: many colonies,
   Insert : 0 colonies,

   ![](https://static.igem.wiki/teams/4206/wiki/20220915-1-transformation.jpg)

   Therefore, the transformation process was successful, but the INSERT plasmid was not present.
   We” ll try another concentration of DNA in gibson assembly.
2. Inactivation of restriction enzyme
   heat-shocked cleavage products cut with HindIII at 70℃ for 15min.
3. Gibson assembly again

   > glc-ace1: 4㎕,
   > glc-ace2: 4㎕,
   > vector: 0.5㎕,
   > master mix(2x): 10㎕
   > milliQ: 1.5㎕

   named the product “gibson 9/15”
4. Made competent cells

   OD600: 0.9

   This is lower than expected.

   Cultured in centrifuge tubes

   > LB 50ml,
   > Bacterial solution(cultured from 20220914) 1ml

# 2022/09/16

## Kazuma Tsurumaki, Kota Toyoda

1. Determined whether “gibson 9/15” was assembled as designed

* Agarose gel electrophoresis

  Dissolved “gibson 9/15” and the vector with EcoRI.

  Ran gel electrophoresis to verify that “gibson 9/15” was assembled as designed

  Result: the band of control appeared but that of “gibson 9/15” wasn’t observed.

  Ecorl could not work properly. If that's the case, gibson 9/15 which we dissolve with EcoRI is in a plasmid state

2. Nanodrop

   > gibson 9/15
   > DNA Concentration: 1360ng/㎕
   > 260/280:2.20

   Overall, we determined that gibson 9/15 is in a plasmid state.
3. Made competent cells

   made successfully. frozen at -80℃.

# 2022/09/20

## Kazuma Tsurumaki

1. Gibson assembly

   > aatA2:3μl,
   > poxB:3μl,
   > vector:1.5μl,
   > miliQ:2.5μl,
   > mastermix:10μl,

   named the product “gib9/20”
2. Transformation

   Transformed gib9/20 and con9/15 into competent cells we made.
   Made 2 versions. “Low-concentration version” is transformed with DNAs as they are. “High-concentration version” is transformed with DNAs centrifuged and the supernatant removed.
   We” ll check at 9:00 AM, tomorrow.

# 20220921

## Kazuma Tsurumaki, Kota Toyoda

1. Transformation
   observed colonies (9:40)

   ![](https://static.igem.wiki/teams/4206/wiki/20220921.png)

   > Number of colonies:
   > insert(High concentration):6
   > insert(Low concentration):1
   > control(High concentration):many
   > control(Low concentration):85 (¼ of area)

   Picked colonies inoculated it into LB-Cam and cultured overnight.

# 2022/09/22

## Kazuma Tsurumaki

1. Measured OD600 of cam cultured from yesterday

   OD600: 0.416 at 10:10
2. Dispended IPTG
   5 g of IPTG dissolved in 21 ml of water.
   IPTG(1mol/l) was dispensed in 1.4ml portions and stored frozen.
3. IPTG induction and culturing
   Added 1.5μL IPTG and ampicillin to LB medium and cultured with 30㎕ of the culture fluid of gib9/20.
   at the same time, cultivated without IPTG as control

> Procedure
>
> Add 1mol/L IPTG and ampicillin(100x) to 1.5ml of LB medium
> Add 30μl of the culture fluid to the LB medium.
> Cultured for 4-5h at 37℃ in culture tubes in a shaking water bath.

Result: OD600

![](https://static.igem.wiki/teams/4206/wiki/20220922.png)

Since the values at the 1-hour-point was too different, we” ll carried out the same experiment.

4. Prepared standard curve for acetic acid determination
   According to the protocol, made 20,16,12,8,4,0mM acetate, and measured absorbance.
   Protocol: <https://www.bioassaysys.com/datasheet/EOAC.pdf>

   To measure absorbance, used an absorbance spectrophotometer
   (USHIO Picoscope <https://www.ushio.co.jp/jp/technology/lightedge/201708/500214.html>)

   Measured absorbance of 455-630nm.
   However, because the data is too irregular, we” ll try again tomorrow. This might be because we failed to centrifuge samples to remove particles.

# 2022/09/23

## Kazuma Tsurumaki, Kota Toyoda

1. Prepared standard curve for acetic acid determination

   According to the protocol, made 0,4, 8, 12, 16, and 20 mM acetate standards in water, and made a standard curve. To remove particles, centrifuge the samples and use the supernatant as a test object.
2. Miniprep

   Following Promega's protocol, miniprep 2 ml of the concentrated medium cultured yesterday.
   Named the product “9/23 Plasmid”.

# 2022/09/24

## Kazuma Tsurumaki

1. IPTG induction and cultured

   Added 1.5μl of IPTG(1mol/l) and ampicillin(100x) to 1.5ml of LB medium and cultured with 30㎕ of cultured fluid from 13:30 just like 20220922.
   Measured od600 and concentration of acetate.

   Result:
   ![](https://static.igem.wiki/teams/4206/wiki/20220924.png)
2. Analyzed 9/23 Plasmid

   Nanodrop: 33ng/㎕ 260/280:1.77
3. Digested 9/23 Plasmid with EcoRI

   > EcoR1 1㎕
   > CutSmartbuffer 5㎕
   > 9/23 Plasmid 20㎕
   > Milli-Q 24㎕

# 2022/09/26

## Kazuma Tsurumaki, Kota Toyoda, Gonzalo Sifuentes, Akito Shima

1. IPTG induction and cultured

   Prepared 3ml of LB medium containing 0mM,0.5mM,1mM,1.5mM of IPTG and cultured E.Coli picked at 20220921 in it, followed to 20220922 - IPTG induction and culturing - procedure for 5 hours.
   Measured concentration of acetate and OD600.

Result:
![](https://static.igem.wiki/teams/4206/wiki/20220926.png)

Since the amount of reagent was wrong in the third hour and there was a significant discrepancy, the test will be conducted again tomorrow.

# 20220928

## Kazuma Tsurumaki, Kota Toyoda

1. Made standard carve of to assay concentration of acetate

   Prepared 0,4, 8, 12, 16, and 20 mM acetate standards in LB
   Measured concentration according to the protocol by the acetate assay kit
2. Gibson assembly

   > master mix(2x):10㎕
   > cell-glu1:3㎕
   > cell-glu2:3㎕
   > vector:2㎕
   > H2O:2㎕
   > Named the product 9/28 cell gib
3. IPTG induction and cultured again

   OD600 and the concentration of acetate were too low.

# 2022/09/29

## Kazuma Tsurumaki

1. Transformation

   > 9/28 cell gib: １㎕
   > Competent cell: 50 ㎕
   > Transformed with DNAs centrifuged and the supernatant removed.
   > We'll centrifuge DNAs from now on before transformation and will not make a “Low-concentrate version”
2. IPTG induction and cultured again same as 20220922

   Reduced IPTG: 0,1mM.
   We” ll check the pH of the medium tommorow.

# 2022/09/30

## Kazuma Tsurumaki

1. Transformation
   Checked mediums made yesterday
   No colony is observed. The same experiment will be conducted.
2. Measured pH of the medium
   Cultured overnight
   pH: 8

   ![](https://static.igem.wiki/teams/4206/wiki/20220930.jpg)
3. IPTG induction and cultured again the same as 20220922
   Only the concentration of IPTG was differ from the procedure of 20220922

   IPTG: 0,1 mM

   pH test will be conducted tomorrow.

# 2022/10/01

## Kazuma Tsurumaki, Kota Toyoda

1. pH of the medium with acetogenic bacteria

   pH: 8-9 (both 0mM and 1mM IPTG)

   ![](https://static.igem.wiki/teams/4206/wiki/20221001.jpg)
2. Gibson assembly
   According to the protocols offered by NEB.

   > glc-ace1:2:8 ㎕,
   > vector:3 ㎕,
   > milliQ:1 ㎕,
   > mastermix:20 ㎕,

   Named the product “10/1 cell”
3. Transformation
   Transformed “9/28 cell gib” and control on each plate since 16:05.
   We” ll check whether the colony appears or not tomorrow.

# 2022/10/02

## Kazuma Tsurumaki, Kota Toyoda, Gonzalo Sifuentes

1. Checked plates
   No colony was observed
2. Restriction Enzyme Treatment of Vectors Once More
   named the product “V”
3. Gibson assembly

   > glc-ace1:3.5㎕,
   > glc-ace2:3.5㎕,
   > vector 2.5㎕,
   > milliQ:0.5㎕,
   > master mix(2x):10㎕,

   Named the product “gib10/02”
4. Transformation
   Transformed gib10/02 and control on each plate
   Both insert and control are cultured in each Petri plate.
   The Petri plates will be checked tomorrow.

# 2022/10/03

## Kazuma Tsurumaki

1. Checked the Petri plates.
   No colony is observed.
2. Transformation
   Transformed gib10/02 and control.

> Competent cells: 50 ㎕,
> Gib10/02 or Vector: 5㎕

```
Both insert and control are cultured in each Petri plate.
The Petri plates will be checked tomorrow.
```

# 2022/10/05

## Kazuma Tsurumaki

1. Checked the Petri plates
   Colonies were observed.

   ![](https://static.igem.wiki/teams/4206/wiki/20221005.jpg)
2. Culturing Acid-producing bacteria

   > LB mediums: 3ml,
   > IPTG: 3μl,
   > Culture fluid for acetic acid-producing strains: 30μl

   Cultured overnight

# 2022/10/06

## Kazuma Tsurumaki

1. Made Glycerol stock of acid-producing bacteria
   mixed 500㎕ of culture fluid cultured from yesterday for acetic acid-producing strains and 20% glycerol.
   frozen at -80℃.
   named the products “aatA-poxB”.

# 2022/10/08

## Kazuma Tsurumaki, Kota Toyoda

1. Made LB medium
   Since the LB medium made last month was muddy, we determined that this was contaminated. Therefore, we made new mediums.
   Both ager and liquid medium are 200ml.
2. Miniprep cellulose-degrading bacteria
   Following Promega's protocol, minipreped samples of cellulose-degrading strain picked at 20221005.
   named the product “glc-plasmid10/8”.
3. Restriction Enzyme Treatment of glc-plasmid10/8
   For Agarose gel electrophoresis, cut glc-plasmid10/8 with EcoRI, according to the protocol offered by New England Bio Labs
4. Gibson Assembly

> glu-ace2: 5㎕,
> glue: 0.5㎕,
> vector: 1.5㎕,
> milliQ:3㎕,
> master mix(2x):10㎕

5. Transformation of aatA-poxB
   Culture fluid: 100㎕
   Both insert and control are cultured in each Petri plate
6. Dissolved cellulose

> Procedure
>
> Dispense LB medium, cellulose powder, culture fluid, and ampicillin (100x) into each culture tube.
> Culture in shaking water bass at 37℃ for 5 hours or more.

```
Made 3 samples. Each tube contains

> LB medium: 6ml
> Cellulose: 0.06g
> Culture fluid: 30μl
> Ampicillin: 6μl
```

Cultured overnight from 17:00, 20221008.

# 2022/10/09

## Kazuma Tsurumaki, Kota Toyoda

1. Agarose gel electrophoresis
   Ran gel electrophoresis to determine whether glc-plasmid10/8 was as designed.
   Used glc-plasmid cut at 20221008 with EcoRI.
2. Dissolved cellulose

   > Each tube contains:
   > Cellulose: 0.03g,
   > Ampicillin(100x): 0.3μl,
   > LB medium:3ml,
   > Culture fluid : 3μl

   Three controls and three inserts each were prepared.
   Cultured overnight in a shaking water bath.

# 2022/10/10

## Kazuma Tsurumaki, Kota Toyoda

1. Dissolved cellulose

   > Glucose assay protocol:
   > According to the protocol offered by the manufacturer.
   > <https://www.dojindo.co.jp/products/G264/img/g264_manual.pdf>
   >
   > To remove particles, centrifuge 80μL of samples at 10000rpm, 27℃ for 1min and use 50μL of supernatant fluid.
2. Determine the concentration of glucose by assay kit

   11:45 Sampling
   12:15 Mix working reagent and samples
   12:45 Measure absorbance

   Abosorbrances are much lower than only LB

   13:02 Mix working reagent and samples which are not centrifuged

   Desided not to centrifuge samples for that time.

   Result: (mM)
   ![](https://static.igem.wiki/teams/4206/wiki/20221010.png)

   The transformed E.coli will have been cultured overnight.

# 2022/10/11

## Kota Toyoda

1. Determine the concentration of glucose by assay kit at 9:15
   Result(mM);

   ![](https://static.igem.wiki/teams/4206/wiki/20221011.png)

   Much lower than yesterday

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# Engineering

DBTL(Design-Build-Test-Learn) cycle
We adopted the DBTL cycle and tried to make it reproducible by others by showing the basic and composite parts used and the lab notebook.

# **1-1.Design**

We designed a pathway to artificially produce methane in three steps: to breakdown of cellulose in cattle manure to glucose, to generate of acetic acid from glucose, and to produce of methane from it.
We focused on creating acetic acid that is needed as a reaction substance for methanogens of these three processes.

# **1-2. Build**

Next, we designed the parts that hydrolyze cellulose to glucose.
We introduced the genes of four enzymes, BBa\_K3394000:Endo5a cellulase
, BBa\_K1499501:Endo-1,4-beta-glucanase, BBa\_K2449012:exo-1, 4-glucanase
, BBa\_K1131002:B-Grucpsodase, which is needed for hydrolysis into pH1 T7 Flexi® Vectors and transformed E. coli.
Then we added cellulose samples to the culture medium, and verified that glucose could be detected by TLC. We added cow manure containing cellulose to the culture medium as well.

# **1-3. Test**

Although we have not actually experimented with it, an expert who represents a thermostable enzyme laboratory company which is involved in synthetic biology in Japan told us to create a more efficient cellulase preparation instead of using the plasmid. In addition, he advised us on the direction of this project.

# **1-4. Learn**

We learned that the demand for cellulase mass production was not so great, whereas an efficient synthesis of acetic acid has not been established yet. Therefore, we decided to focus on how the acetic acid worked as one of the three processes.

# **2-1Design**

We decided to create an E. coli which produces acetic acid.
In the process, we introduced a gene, PoxB, which would make more acetic acid in vivo and to attach another gene, AatA, as a pump to the E. coli to discharge acetic acid.

# **2-2Build**

Acetic acid was presumed to be toxic to E. coli.
According to Stéphane Pinhal et al. (2019), high concentrations of acetate inhibit growth of E. coli because it causes the perturbation of acetate metabolism. Therefore, we decided to create the modeling.

# **2-3Test**

Next, we predicted that the E. coli died depending on the amount of gene expression from the results of modeling.

# **2-4Learn**

Since it was suggested that IPTG-induced preparation of Pox B expression might be necessary, we eventually thought it would be better to create a vector with an ever-expressing promoter and created a plasmid that enabled PoxB to be adjusted with design IPTG.

# **3-1. design**

We created an E. Coli producing acetate.
PoxB is regulated by IPTG and aatA always expresses.
To find the optimal promoter strength, adjust the IPTG concentration and measure the amount of acetic acid produced and the growth rate of E. coli.

# **3-2. build**

Incorporate poxB and aatA into the expression plasmid, pTWV228, and express in E.coli.
Pox B is placed downstream of the lac promoter.
AatA is placed downstream of the constitutive promoter.
![スクリーンショット 2022-10-11 164604.png](https://static.igem.wiki/teams/4206/wiki/screenshot-2022-10-11-164604.png)

# **3-3. test**

We cultured E. Coli at different iptg concentrations and determined OD 600 and acetic acid concentrations.
OD 600 increased rapidly at higher IPTG concentrations(graph.1), and acetate was detected by acetate assay kit in the samples with IPTG 1,5mM(graph.2).
![OD600 time.png](https://static.igem.wiki/teams/4206/wiki/od600-time.png)
![graph.1.png](https://static.igem.wiki/teams/4206/wiki/graph-1.png)

# **3-4. learn**

Millard et al. (2022) have argued that acetate acts as a co-substrate of glycolytic nutrients and boosts E. coli growth at low glycolytic fluxes.
The change in OD600 with the addition of IPTG may be due to increased intracellular acetate concentration and growth promotion by the expression of poxB.
we have confirmed the production of acetic acid, we need to use these data to explore the optimal promoter strength of poxB.

# **References**

Millard, P., Uttenweiler-Joseph, S., & Enjalbert, B. (2022). From toxic waste to beneficial nutrient: acetate boosts Escherichia coli growth at low glycolytic flux. BioRxiv. https://doi.org/10.1101/2022.09.20.506926

{% endblock %}

{% extends "layout.html" %}
{% block title %}Home{% endblock %}
{% block lead %}**Welcome to iGEM 2022!** Your team has been approved and you are ready to start the iGEM season!{% endblock %}
{% block page\_content %}

# Experiments

# **Cellulose degradation**

The complete cellulose hydrolysis reaction require Endo5a cellulase, B-Glucosidase, exo-β-1,4-glucanase and Endo-1,4-beta-glucanase.

Procedures

1. Gibson assembly

   Forming the plasmid contains cell-glu1and cell-glu2.
2. Transformation

   Following the Single Tube Transformation Protocol provided by iGEM, except for the process of heat shock.
   We conducted heat-shock for 30sec at 42°C on a heat block, according to Mr. Tashiro, working in the synbio lab at Kyushu Univ, who offers us cells.
3. Culturing

   Picking the colonies and cultured them in LB mediums with ampicillin. The shaking culture was conducted in a shaking water bath at 168 min^-1 overnight.

![](https://static.igem.wiki/teams/4206/wiki/experiment.jpg)

4. Dissolving cellulose

   Culturing like "3. Cultureing" with cellulose. Added 0.03-0.06mg of cellulose powder (Wako 28μm) to the working mixture, containing 3μl of culture medium, ampicillin and 3ml of LB medium.
5. Assay consentration of glucose

   * According to the protocol offered by assay kit's manifacture, make the standard curve. Since LB medium contains glucose and absorbrance, glucose samples used in calibration was made by dliting 10mmol/L Glucose Standard with LB mediums instead of water.
   * Sampleing culture medium containing cellulose and mix with the glucose assay kit.
   * Mesuring absorbrance of 400-540nm by spectrometer (USIO Picoscpe).
   * The protocol is here; <https://www.dojindo.co.jp/products/G264/>

# **Acetate production**

AatA is an ABC transporter found in *Acetobacter acetii* which moves acetate out of the cell. It is known that this gives resistance to high levels of acetic acid in the cellular environment and drives the acetate production.
PoxB,Pyruvate dehydrogenase, is a peripheral cell membrane enzyme that catalyzes the oxidative decarboxylation of pyruvate to form acetate and CO2.
In this project, poxB is regulated by lac promoter and aatA is constitutively expressed.
Cultivate transformed E. coli in medium supplemented with IPTG and measure OD600 and acetic acid concentration every hour.

Procedures

1. Gibson assembly

   Forming the plasmid contains aatA and poxB.
2. Transformation

   Please refer to "cellulose degradation - Transformation"
3. Culturing

   Pick colonies and culture them in LB-cam containing ampicillin overnight.
4. IPTG induction

   Add 30μl of culture fluid to 3 ml of LB-medium containing ampicillin and 0-1.5μmol of IPTG. Cultured for 4-5h at 37℃ in culture tubes in a shaking water bath at 168 min^-1
5. Measure the cocentration of acetic acid every hour

   * According to the protocol offered by assay kit's manifacture, make the standard curve. Since LB medium might contains acetate and absorbrance, acetate samples used in calibration was made by dliting acetate Standard with LB mediums instead of water.
     The protocols is here; <https://www.bioassaysys.com/datasheet/EOAC.pdf>
   * Sampleing culture medium and mix with the working reagent of assay kit.
   * Mesure absorbrance of 455-630nm by spectrometer (USIO Picoscpe) every hour to determine the concentration of acetic acid based on the standard curve.
   * OD600 was measured too by spectometer.

# **Measuring the pH of the culture**

To prove the production and transportation of acetate ion, pH tests were performed on cultures containing E. coli transformed with AatA and PoxB.

Procedures

1. Culture the acetobacteria

   Propagate the E.Coli transformed in "Acetate production".
   Add the 30μl of culture fluid and ampicillin to 3ml of LB medium.
   Concurrently, IPTG induction is coducted; each tube contain 0-1.5μM of IPTG.
   Under the circumstance, the E.Coli have been cultured overnight in culture tubes in shaking water bath at 168min^-1, 37℃.
2. Measure pH of the culture fluid

   After culturing overnight, pH tests are conducted based on pH test paper.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Home{% endblock %}
{% block lead %}**Welcome to iGEM 2022!** Your team has been approved and you are ready to start the iGEM season!{% endblock %}
{% block page\_content %}

# Results

# **acetate production**

![acetate concentration with 1.5mM IPTG (1).png](https://static.igem.wiki/teams/4206/wiki/acetate-concentration-with-1-5mm-iptg-1.png)
As shown in the graph, acetic acid was synthesized when 1.5 mM IPTG was added.

![OD600 time.png](https://static.igem.wiki/teams/4206/wiki/od600-time-1.png)
OD600 has a higher growth rate at higher IPTG concentrations.

# **cellulose degradation**

![20221011.png](https://static.igem.wiki/teams/4206/wiki/20221011-1.png)
In insert, there is a trend toward increased glucose levels up to 6 hours, while there is no significant trend in CONTROL
After one night of incubation, glucose levels in both cases have decreased significantly.
This is assumed that the rate of cellulose degradation exceeded the rate of glucose consumption, as a result of E. coli growth and more glucose being used as a nutrient source, while cellulose was precipitated and was not degraded as much.

# **Measuring the pH of the culture**

![figure.1.jpg](https://static.igem.wiki/teams/4206/wiki/figure-1-1.jpg)
ph increased regardless of whether iptg was added.

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