iGEM 2020 — Team Waseda

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Waseda/Notebook

waseda test

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

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

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Waseda/Engineering

Engineering - iGEM 2020 Team:Waseda

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![](https://static.igem.org/mediawiki/2020/9/9d/T--Waseda--iGEM2020lWasedaLogo.png)

# Engineering success and engineering principles

## Overview

###

In addition to new parts, we have shown many engineering successes, which will be also evaluated in Rubric for judging (Project Section #5 and #8).
Q5 How well were engineering principles used (e.g., design-build-test cycle, use of standards, modularity, etc.)?
Q8 Did the team design a project based on synthetic biology and standard components (BioBricks, software, etc.)?

## New Parts

[BBa\_K3580102](http://parts.igem.org/Part%3ABBa_K3580102) &  [BBa\_K3580200](http://parts.igem.org/Part%3ABBa_K3580200) !

## Design-build-test cycle

###

We have adopted the DBTL(Design-Build-Test-Learn) cycle for a better project. The model was reassembled several times while observing the difference between this ideal graph and the simulation results under the actual parameters.
We especially paid attention to the degradation of protein and the ratio of crosstalk reaction. For the cell-free system, our modeling described above indeed have shown that cell-free toggle switch will not work if the system has low degradation rate.
Therefore we tested the effect of degradation tag, and incorporated the results into the model. Although wet experiments demonstrated the existence of crosstalk, modeling showed that crosstalk would not be a problem in improving the circuit parameters. We eventually succeeded in creating two states simply by changing the concentration of small molecules. Moreover, we launched and worked on some advanced projects based on this circuit.

## Modulality

In monoterpene synthesis, we mixed two E. coli extracts in various ratio, taking advantage of modularity.
Those extracts contained either first 8 or last 2 enzymes of a pathway from Ac-CoA, which is a major intermediate of cell central metabolism.
Through mevalonate pathway, the former extract one (derived from E. coli into which pBbA5c-MevT-MBI has been introduced) can provide IPP and DMAPP, which can also be used as intermediates for other important biosynthesis.
Here, we indeed supplemented only glucose and acetate as carbon sources. We obtained expression system for those eight genes from addgene and have converted this into Biobrick RFC 1000 format by synonymous replacement (BBa\_K3580103).
In order to take advantage of an engineering principle of synthetic biology, we provided two biobrick parts (BBa\_K3580101, BBa\_K3580102) for the source for the latter extract. BBa\_K3580101 has GPP synthase (GPPS) and limonene synthase.

## Standardized measurements

Measurements of fluorescence activity can be assessed within a lab using a common machine. However, it is not possible to directly compare the measurements in different laboratories.
Therefore, we performed a standardized measurement of the fluorescence values in cell-free by calibrating with FITC for each experiment trial, so the measured values can be compared in other labs by following the previous iGEM interlab protocol.
Fluorescein (FITC) is a fluorophore used in iGEM Measurement, and is ideal for the normalization of fluorescence values.

## Comparison between in vivo and in vitro activity

In the degradation tag assay, we took measurements both in vivo and in vitro with the same condition to compare the results.
Such comparison is important when we use cell-free systems as a breadboard for engineering of cellular systems.

2020 — **iGEM Waseda**

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Waseda/Experiments

waseda test

# Experiments

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).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Waseda/Results

waseda test

# Results

You can describe the results of your project and your future plans here.

### 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.

### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)