iGEM 2019 — Team ASIJ_Tokyo

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

# Team:ASIJ Tokyo/Notebook

![logo](https://static.igem.org/mediawiki/2019/b/b3/T--ASIJ_Tokyo--logo1.png)

* [Home](https://2019.igem.org/Team%3AASIJ_Tokyo)
* Team

  [Members](https://2019.igem.org/Team%3AASIJ_Tokyo/Team)
  [Collaborations](https://2019.igem.org/Team%3AASIJ_Tokyo/Collaborations)
* Project

  [Description](https://2019.igem.org/Team%3AASIJ_Tokyo/Description)
  [Design](https://2019.igem.org/Team%3AASIJ_Tokyo/Design)
  [Experiments](https://2019.igem.org/Team%3AASIJ_Tokyo/Experiments)
  [Notebook](https://2019.igem.org/Team%3AASIJ_Tokyo/Notebook)
  [Results](https://2019.igem.org/Team%3AASIJ_Tokyo/Results)
  [Demonstrate](https://2019.igem.org/Team%3AASIJ_Tokyo/Demonstrate)
  [Improve](https://2019.igem.org/Team%3AASIJ_Tokyo/Improve)
  [Attributions](https://2019.igem.org/Team%3AASIJ_Tokyo/Attributions)
* [Model](https://2019.igem.org/Team%3AASIJ_Tokyo/Model)
* Parts

  [Parts Overview](https://2019.igem.org/Team%3AASIJ_Tokyo/Parts)
  [Basic Parts](https://2019.igem.org/Team%3AASIJ_Tokyo/Basic_Part)
  [Composite Parts](https://2019.igem.org/Team%3AASIJ_Tokyo/Composite_Part)
  [Part Collection](https://2019.igem.org/Team%3AASIJ_Tokyo/Part_Collection)
* [Safety](https://2019.igem.org/Team%3AASIJ_Tokyo/Safety)
* Human Practices

  [Human Practices](https://2019.igem.org/Team%3AASIJ_Tokyo/Human_Practices)
  [Education & Engagement](https://2019.igem.org/Team%3AASIJ_Tokyo/Public_Engagement)
* [Judging Form](https://igem.org/2019_Judging_Form?team=ASIJ_Tokyo)

# Notebook

#### Documenting our methods and results

## Our main lab notebook

## General Protocols Notebook

![](https://static.igem.org/mediawiki/2019/7/7b/T--ASIJ_Tokyo--PyMOL.png)

![](https://static.igem.org/mediawiki/2019/7/70/T--ASIJ_Tokyo--BiomodelingCo..jpg)

![](https://static.igem.org/mediawiki/2019/5/55/T--ASIJ_Tokyo--SwissModel.jpg)

![](https://static.igem.org/mediawiki/2019/2/2a/T--ASIJ_Tokyo--SwissModel1.jpg)

![](https://static.igem.org/mediawiki/2019/8/8f/T--ASIJ_Tokyo--ThermoFisher.jpg)

![](https://static.igem.org/mediawiki/2019/9/9b/T--ASIJ_Tokyo--TUAT.jpg)

![](https://static.igem.org/mediawiki/2019/7/78/T--ASIJ_Tokyo--Chiba.jpg)

![](https://static.igem.org/mediawiki/2019/9/98/T--ASIJ_Tokyo--AIST.png)

![](https://static.igem.org/mediawiki/2019/1/19/T--ASIJ_Tokyo--ClusPro.png)

![](https://static.igem.org/mediawiki/2019/a/ac/T--ASIJ_Tokyo--New_England_Biolabs_Logo.jpg)

![](https://static.igem.org/mediawiki/2019/b/bb/T--ASIJ_Tokyo--.jpg)

![](https://static.igem.org/mediawiki/2019/3/35/T--ASIJ_Tokyo--IDSM.jpg)

#### Follow Us:

[![](https://static.igem.org/mediawiki/2019/a/a5/T--ASIJ_Tokyo--Twitter.png)](https://twitter.com/asijigem)

[![](https://static.igem.org/mediawiki/2019/1/15/T--ASIJ_Tokyo--Instagram.png)](https://www.instagram.com/igem.asij)

[![](https://static.igem.org/mediawiki/2019/4/4f/T--ASIJ_Tokyo--YouTube.png)](https://www.youtube.com/channel/UCSI1wk_zyxVKn-rs31qqhnw)

---

#### Contact Us:

![](https://static.igem.org/mediawiki/2019/4/4e/T--ASIJ_Tokyo--email.png)

###### asijigem@gmail.com

![](https://static.igem.org/mediawiki/2019/a/a2/T--ASIJ_Tokyo--MenuBarLogo.png)


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

# Team:ASIJ Tokyo/Experiments

![logo](https://static.igem.org/mediawiki/2019/b/b3/T--ASIJ_Tokyo--logo1.png)

* [Home](https://2019.igem.org/Team%3AASIJ_Tokyo)
* Team

  [Members](https://2019.igem.org/Team%3AASIJ_Tokyo/Team)
  [Collaborations](https://2019.igem.org/Team%3AASIJ_Tokyo/Collaborations)
* Project

  [Description](https://2019.igem.org/Team%3AASIJ_Tokyo/Description)
  [Design](https://2019.igem.org/Team%3AASIJ_Tokyo/Design)
  [Experiments](https://2019.igem.org/Team%3AASIJ_Tokyo/Experiments)
  [Notebook](https://2019.igem.org/Team%3AASIJ_Tokyo/Notebook)
  [Results](https://2019.igem.org/Team%3AASIJ_Tokyo/Results)
  [Demonstrate](https://2019.igem.org/Team%3AASIJ_Tokyo/Demonstrate)
  [Improve](https://2019.igem.org/Team%3AASIJ_Tokyo/Improve)
  [Attributions](https://2019.igem.org/Team%3AASIJ_Tokyo/Attributions)
* [Model](https://2019.igem.org/Team%3AASIJ_Tokyo/Model)
* Parts

  [Parts Overview](https://2019.igem.org/Team%3AASIJ_Tokyo/Parts)
  [Basic Parts](https://2019.igem.org/Team%3AASIJ_Tokyo/Basic_Part)
  [Composite Parts](https://2019.igem.org/Team%3AASIJ_Tokyo/Composite_Part)
  [Part Collection](https://2019.igem.org/Team%3AASIJ_Tokyo/Part_Collection)
* [Safety](https://2019.igem.org/Team%3AASIJ_Tokyo/Safety)
* Human Practices

  [Human Practices](https://2019.igem.org/Team%3AASIJ_Tokyo/Human_Practices)
  [Education & Engagement](https://2019.igem.org/Team%3AASIJ_Tokyo/Public_Engagement)
* [Judging Form](https://igem.org/2019_Judging_Form?team=ASIJ_Tokyo)

# Experiments

#### Time for lab work!

## Veryfying the effectiveness of unmodified 0.19 α-amylase inhibitor

#### Gibson Assembly & Protein Purificaion

###### We assembled the three types of constructs for protein production. Once the yeast cell grew, the cells without secretion tag construct were lysed to extract and purify the protein. For the ones with secretion tag, media just above the cell pellets were taken for purification. The purified proteins were then used in all of the test mentioned below.

#### Gel Electrophoresis

###### We conducted the electrophoresis as a way to collect more evidence to establish that the 0.19 albumin alpha-amylase inhibitor was produced successfully in each construct. By comparing the known size of the 0.19 protein from literature to the proteins isolated and collected from our samples, we were able to evaluate whether or not 0.19 had been correctly produced. The product produced by our yeast was expected to travel the same distance and show the same molecular weight. This method was chosen because the materials were readily available to us and many members of the team have performed it before.

#### Bradford Assay

###### The purpose of this is to test whether our modifications were produced by measuring the concentration of 0.19. This concentration value would be used in math modeling as well. However, this assay did not give consistent results (some protein concentration was negative), but we do not know the reason behind this.

## Improving the 0.19 amino acid sequence

#### Starch Solution Inhibition Assay with Iodine

###### In this experiment, we measured the concentration of starch with alpha-amylase and 0.19 overtime to see if the modified 0.19s successfully inhibited the breakdown of starch by the amylase. The iodine solution was used as a indicator for the presence of starch, which can then be anaylzed with absorbance reader. This Assay gave us unexpected results, as some colors in the 96 well plate did not fit in the gradient. Therefore, we did not analyze the absorbance values from this Assay.

#### CNP Assay

###### The CNP (2-chloro-4-nitrophenyl maltotrioside) test was used to measure the enzymatic activity of alpha-amylase. CNP demonstrates a color change from a transparent blue to colorless as it is cleaved by alpha-amylase, thus acting as an indicator that allowed us to observe amylase inhibition via absorption measurements. The CNP assay allowed us to measure inhibition with greater precision than the starch-iodine assay.

## iGEM parts improvement for the effective production of alpha-amylaseGibson assembly, protein purificationFor assembling parts, we used the same method as above, except the cells were E.coli competent cells, not yeast. This was because we improved already-existing E.coli part from the iGEM registry.CNP AssayThe results of the CNP from testing 0.19 proved to have a consistent measurements, therefore we adopted the same method for measuring amylase activity.

![](https://static.igem.org/mediawiki/2019/7/7b/T--ASIJ_Tokyo--PyMOL.png)

![](https://static.igem.org/mediawiki/2019/7/70/T--ASIJ_Tokyo--BiomodelingCo..jpg)

![](https://static.igem.org/mediawiki/2019/5/55/T--ASIJ_Tokyo--SwissModel.jpg)

![](https://static.igem.org/mediawiki/2019/2/2a/T--ASIJ_Tokyo--SwissModel1.jpg)

![](https://static.igem.org/mediawiki/2019/8/8f/T--ASIJ_Tokyo--ThermoFisher.jpg)

![](https://static.igem.org/mediawiki/2019/9/9b/T--ASIJ_Tokyo--TUAT.jpg)

![](https://static.igem.org/mediawiki/2019/7/78/T--ASIJ_Tokyo--Chiba.jpg)

![](https://static.igem.org/mediawiki/2019/9/98/T--ASIJ_Tokyo--AIST.png)

![](https://static.igem.org/mediawiki/2019/1/19/T--ASIJ_Tokyo--ClusPro.png)

![](https://static.igem.org/mediawiki/2019/a/ac/T--ASIJ_Tokyo--New_England_Biolabs_Logo.jpg)

![](https://static.igem.org/mediawiki/2019/b/bb/T--ASIJ_Tokyo--.jpg)

![](https://static.igem.org/mediawiki/2019/3/35/T--ASIJ_Tokyo--IDSM.jpg)

#### Follow Us:

[![](https://static.igem.org/mediawiki/2019/a/a5/T--ASIJ_Tokyo--Twitter.png)](https://twitter.com/asijigem)

[![](https://static.igem.org/mediawiki/2019/1/15/T--ASIJ_Tokyo--Instagram.png)](https://www.instagram.com/igem.asij)

[![](https://static.igem.org/mediawiki/2019/4/4f/T--ASIJ_Tokyo--YouTube.png)](https://www.youtube.com/channel/UCSI1wk_zyxVKn-rs31qqhnw)

---

#### Contact Us:

![](https://static.igem.org/mediawiki/2019/4/4e/T--ASIJ_Tokyo--email.png)

###### asijigem@gmail.com

![](https://static.igem.org/mediawiki/2019/a/a2/T--ASIJ_Tokyo--MenuBarLogo.png)

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

# Team:ASIJ Tokyo/Results

![logo](https://static.igem.org/mediawiki/2019/b/b3/T--ASIJ_Tokyo--logo1.png)

* [Home](https://2019.igem.org/Team%3AASIJ_Tokyo)
* Team

  [Members](https://2019.igem.org/Team%3AASIJ_Tokyo/Team)
  [Collaborations](https://2019.igem.org/Team%3AASIJ_Tokyo/Collaborations)
* Project

  [Description](https://2019.igem.org/Team%3AASIJ_Tokyo/Description)
  [Design](https://2019.igem.org/Team%3AASIJ_Tokyo/Design)
  [Experiments](https://2019.igem.org/Team%3AASIJ_Tokyo/Experiments)
  [Notebook](https://2019.igem.org/Team%3AASIJ_Tokyo/Notebook)
  [Results](https://2019.igem.org/Team%3AASIJ_Tokyo/Results)
  [Demonstrate](https://2019.igem.org/Team%3AASIJ_Tokyo/Demonstrate)
  [Improve](https://2019.igem.org/Team%3AASIJ_Tokyo/Improve)
  [Attributions](https://2019.igem.org/Team%3AASIJ_Tokyo/Attributions)
* [Model](https://2019.igem.org/Team%3AASIJ_Tokyo/Model)
* Parts

  [Parts Overview](https://2019.igem.org/Team%3AASIJ_Tokyo/Parts)
  [Basic Parts](https://2019.igem.org/Team%3AASIJ_Tokyo/Basic_Part)
  [Composite Parts](https://2019.igem.org/Team%3AASIJ_Tokyo/Composite_Part)
  [Part Collection](https://2019.igem.org/Team%3AASIJ_Tokyo/Part_Collection)
* [Safety](https://2019.igem.org/Team%3AASIJ_Tokyo/Safety)
* Human Practices

  [Human Practices](https://2019.igem.org/Team%3AASIJ_Tokyo/Human_Practices)
  [Education & Engagement](https://2019.igem.org/Team%3AASIJ_Tokyo/Public_Engagement)
* [Judging Form](https://igem.org/2019_Judging_Form?team=ASIJ_Tokyo)

# Results

#### Outcomes of our experiments

## Confirming the Function of Secretion Tag

Figure 1.1

![](https://static.igem.org/mediawiki/2019/thumb/0/0b/T--ASIJ_Tokyo--Bluelight_2.jpg/1600px-T--ASIJ_Tokyo--Bluelight_2.jpg)

Figure 1.2

![](https://static.igem.org/mediawiki/2019/5/5e/T--ASIJ_Tokyo--ori_0.19_bluelight_1.jpg)

###### Figure 1.2 shows the qualitative fluorescence results of our constructs with unmodified 0.19 tagged with GFP and a secretion tag. Figure 1.1 shows our modified 0.19 constructs, also tagged with GFP and a secretion tag. A seen in the photographs above, our modified 0.19 constructs are fluorescent, showing that our modificaitons have not prevented the secretion tag from functioning, thus enabling the modified 0.19 to be secreted from yeast cells.

## Confirming the Molecular Weight of 0.19

Figure 2

![](https://static.igem.org/mediawiki/2019/thumb/a/a1/T--ASIJ_Tokyo--Electrophoresis.jpg/1435px-T--ASIJ_Tokyo--Electrophoresis.jpg)

###### This is the electrophoresis with 0.19 protein solution(Figure 2).The protein marker in lane 1 and 5 (counting from left) show bands of molecular weight at 10, 13, 22, 32, 67, and 94 kDa. In lane 2 and 3 we have GFP-tagged 0.19 with combined molecular weight of around 40kDa and the electrophoresis results show that the two bands are between 32 and 67 kDa as expected. To confirm the molecular weight of 0.19 by itself, we put the purified protein solution collected from the cell pellet containing [Construct 1](https://2019.igem.org/Team%3AASIJ_Tokyo/Design#section2) in lane 6 and 7. Although the bands are faint, the results show the correct molecular weight of 0.19, which is 13kDa. We assumed that the faint bands are due to the low protein concentration.

## 2-chloro-4-nitrophenyl maltotrioside (CNP) Assay

Figure 1

![](https://static.igem.org/mediawiki/2019/b/be/T--ASIJ_Tokyo--ResultsGraph.png)

Figure #

![](https://static.igem.org/mediawiki/2019/2/2f/T--ASIJ_Tokyo--ExampleCalculation.jpg)

###### As you can see from the data above, the α-amylase activity for all of the 0.19 variations α-amylase activity over a 30-minute period was measured. Regarding the modifications made to improve 0.19’s bonding strength to α-amylase (Gly to Glu and Ile to Asn modifications), their α-amylase activity was consistently lower than the original 0.19 throughout the 30-minute period which means the modification that increased the affinity with α-amylase was successful.

###### The modification with the intention of preventing dimerization, Arg to Glu with and without secretion tag had an α-amylase activity was similar to the unmodified 0.19 from t~3.5 to t=5 throughout the 30-minute period; however, it had a lower α-amylase activity overall, so we considered the modification to be positive. Regarding the modification with the same intention, but for Cys to Arg, there was a higher α-amylase activity than the original 0.19 from t=4 to t=8, but we also consider this to be insignificant according to the overall data, therefore, this modification had a positive result as well.

###### Looking into the results between the modifications with high affinity to α-amylase, as expected from our results from the dry lab, the Gly to Glu modifications were more effective than the Ile to Asn modifications regarding our modification for strengthening the affinity between 0.19 and α-amylase (solid lines); this can be inferred by comparing the α-amylase activity at t=2 (Gly to Glu without secretion tag was 0.005137 mol/L/min, Gly to Glu with secretion tag was 0.004840 mol/L/min, Ile to Asn without secretion tag was 0.007767 mol/L/min, and Ile to Asn with secretion tag was 0.01354 mol/L/min).

###### Another point to be mentioned is the increasing slopes in the Arg to Glu modifications from t=2 to t=6 which was different from all the other 0.19s which had a decreasing slope. We were not able to interpret this specific characteristic, so further research is necessary.

###### The last point that should be noted is the average speed of α-amylase activity in Gly to Glu modification with the secretion tag. Despite the initial α-amylase activity being relatively low compared to the original 0.19 with secretion tag, the average speed of α-amylase activity for Gly to Glu with secretion tag was nearly identical to the average speed of α-amylase activity in the original 0.19 with secretion tag from t=0 to t=2 (Gly to Glu with secretion tag was −0.01499 mol/Lmin, 0.19 with secretion tag was −0.02610 mol/Lmin). This is significant data because the average speed of α-amylase activity is another important factor to consider, and it was interesting to see that only the Gly to Glu with secretion tag had a relatively large average speed of α-amylase activity considering it to start at a relatively low α-amylase activity to the original 0.19s. We do not know the reason behind this, so future investigation is necessary. In conclusion, all of our modifications were overall more successful at inhibiting α-amylase compared to the original 0.19s.

![](https://static.igem.org/mediawiki/2019/7/7b/T--ASIJ_Tokyo--PyMOL.png)

![](https://static.igem.org/mediawiki/2019/7/70/T--ASIJ_Tokyo--BiomodelingCo..jpg)

![](https://static.igem.org/mediawiki/2019/5/55/T--ASIJ_Tokyo--SwissModel.jpg)

![](https://static.igem.org/mediawiki/2019/2/2a/T--ASIJ_Tokyo--SwissModel1.jpg)

![](https://static.igem.org/mediawiki/2019/8/8f/T--ASIJ_Tokyo--ThermoFisher.jpg)

![](https://static.igem.org/mediawiki/2019/9/9b/T--ASIJ_Tokyo--TUAT.jpg)

![](https://static.igem.org/mediawiki/2019/7/78/T--ASIJ_Tokyo--Chiba.jpg)

![](https://static.igem.org/mediawiki/2019/9/98/T--ASIJ_Tokyo--AIST.png)

![](https://static.igem.org/mediawiki/2019/1/19/T--ASIJ_Tokyo--ClusPro.png)

![](https://static.igem.org/mediawiki/2019/a/ac/T--ASIJ_Tokyo--New_England_Biolabs_Logo.jpg)

![](https://static.igem.org/mediawiki/2019/b/bb/T--ASIJ_Tokyo--.jpg)

![](https://static.igem.org/mediawiki/2019/3/35/T--ASIJ_Tokyo--IDSM.jpg)

#### Follow Us:

[![](https://static.igem.org/mediawiki/2019/a/a5/T--ASIJ_Tokyo--Twitter.png)](https://twitter.com/asijigem)

[![](https://static.igem.org/mediawiki/2019/1/15/T--ASIJ_Tokyo--Instagram.png)](https://www.instagram.com/igem.asij)

[![](https://static.igem.org/mediawiki/2019/4/4f/T--ASIJ_Tokyo--YouTube.png)](https://www.youtube.com/channel/UCSI1wk_zyxVKn-rs31qqhnw)

---

#### Contact Us:

![](https://static.igem.org/mediawiki/2019/4/4e/T--ASIJ_Tokyo--email.png)

###### asijigem@gmail.com

![](https://static.igem.org/mediawiki/2019/a/a2/T--ASIJ_Tokyo--MenuBarLogo.png)