Overview

In this year's project, we developed gene circuits and educational tools. The former was to improve the safety and security of synthetic biology, and the latter was to make synthetic biology accessible to a wider audience. To achieve these goals, we went through a "Design-Build-Test-Learn" cycle over and over again, gradually making improvements. Our engineering cycle is detailed below.

Engineering in wet lab: Our journey to create an easily applicable blue light inducible promoter

1. Basic blue light promoter

Design

For our project, we needed a blue light activated promoter system. We decided to start creating the system from a minimum inducible blue light promoter. We tested an existing blue light promoter by putting BBa_K3570021 and BBa_K3927001 together in a plasmid.

Build

A plasmid containing both the C120-trucCYC1 promoter followed by mCherry and its cognate EL222 was transformed into S.cerevisiae. C120-trucCYC1p and EL222 were synthesized by IDT.

Test

An assay of blue light induction was carried out. We compared the expression of mCherry between the two conditions where the strain was exposed to blue light and where it was kept in the dark.

Learn

Expression of mCherry was higher when the strain was placed in the dark. This result contradicted the previous works, which stated EL222 activates C120-trucCYC1 promoter under the existence of blue light. We assumed that this was because the plasmid was eliminated as we used a YPD culture medium instead of a selective medium for the plasmid.

2. Genome integration of EL222 system

Design

In order to prevent the elimination of the plasmid and extend our system with precise control, we decided to integrate the blue light inducible promoter system into the genome. We reshaped the plasmid DNA into a linear DNA strand for genome integration by removing the backbone sequence and adding homologous sequences for the target locus.

Build

We used OE-PCR to add the homologous sequences for YMRWΔ 9 locus and build the linear strand.

Test

We checked the results of OE-PCR several times by electrophoresis.

Learn

OE-PCR for the final linear strand for genome integration was not successful. The information from the wet lab result and the PCR enzyme led us to conclude that the linear strand containing both C120-trucCYC1 promoter with mCherry and its cognate EL222 was too long to construct and amplify by PCR.

3. Improving genome integration of EL222 system part 1

Design

In order to shorten the linear strand for successful OE-PCR and genome integration, we decided to split the strand into two, C120-CYC1 promoter with mCherry and EL222, and integrate each strand into a different genome locus.

Build

We used OE-PCR to build the linear strands. We added homologous sequences for YMRWΔ9 to C120-trucCYC1 promoter with mCherry and YNCRΔ9 to EL222.

Test

We managed to create and amplify the linear strands by OE-PCR for genome integration. Yet, genome integration was not successful.

Learn

We found out that we need to add more insert linear strands to process the genome integration, as genome integration is less effective than plasmid integration. We also learned from our human practice that the optimal length of homologous sequence for genome integration varies. For more details, please refer to our Human Practices page.

4. Improving genome integration of EL222 system part 2

Design

In order to successfully integrate C120-trucCYC1 promoter with mCherry and EL222, we designed different linear strands, one with short homologous sequences of 60 bp and one with long homologous sequences of over 600 bp.

Build

We used OE-PCR to build the linear strands. We integrated C120-trucCYC1 promoter with mCherry into YMRWΔ 9 and then put EL222 into YNCRΔ.

Test

We successfully integrated both C120-trucCYC1 promoter with mCherry, and EL222 into the target genome locus. Blue light induction assay was carried out. We compared the expression of mCherry between conditions when the strain was left in the dark or when it was exposed to blue light for 3 or 6 hours.

Learn

Expression of mCherry was higher when the strain was exposed to blue light. The difference between the control S. cerevisiae which was placed in the dark, was significant when we shone blue light on the S. cerevisiae for 3 hours. For more details, please refer to our Results page.

Engineering in Genochemy

For Genochemy, we repeated the engineering cycle in the form of "versioning up". We developed a version, got feedback from users, and then updated the version. In this way, we updated the version from v0.1, v0.2, v0.3, v0.4, v0.5, v0.6, to v0.7.

For v0.1

Design

Our first motivation was to create a block programming system, like Scratch, for microorganisms. Scratch is a famous block programming system developed by MIT. We thought this is useful to spread the fun of synthetic biology, because people can learn and enjoy creating basic genetic circuits only by accessing the website. First, we designed a system where people can construct genetic circuits by connecting blocks. We prepared a simple activator and repressor.

We developed this app with Vue.js.

Build

Here is the development progress before releasing v0.1.

The state of Genochemy on 3/28/2022, the day we launch Genochemy! The blocks are only placed in the tray.

Within a few days, we implemented a drug-and-drop and block connection feature.

Then we implemented primitive blocks, two promoters, one fluorescence protein, a repressor, and terminator.

In the team meeting the screen layout is drastically changed, which remains to today. The tray came to the bottom of the screen. Then, by adding one more promoter, a fluorescence protein and an activator, we finally deployed v0.1, three months after the launch. (In this version, only Japanese is available language)

Test

We used v0.1 in the class at a senior high school. For the details, see Education & Communication page. We gained feedback.

Learn

Many students said they enjoyed programming microorganisms. However, some students said they do not feel as if they are writing a program for "real" life. Hearing their voices, we struggled to improve Genochemy.

For v0.2

Design

In v0.1, some users said it was difficult to feel as if they were programming real life. For this point, we too simplified the concept of biology for the implementation into Genochemy. Thus, we increased the number of blocks and made it possible to create more complex circuits that are close to those designed in real synthetic biology. Actually, it became possible to create this year's our iGEM project, by implementing recombinases and more kinds of activators.

Also, we found the need to explain gene circuits in an easy to understand way, which can be resolved by the visualization of Genochemy. We made it possible to load our project's constructs to Genochemy.

Build

Two months after v0.1, we released v0.2. (In this version, only Japanese is available language)

In the load tab in the right bottom of the screen, there is a button for loading the UTokyo 2022 project. When clicked, our project constructs are loaded. It helps a lot when we explain our design to others.

Test

We used Genochemy v0.2 for the summer synbio lecture with Waseda_Tokyo team. For details please see our Education & Communication page. Then we gained feedback.

Learn

Many participants answered that it was interesting. They enjoyed using recombinases and light induced activators. However, most said the three questions prepared for the lecture were too difficult.

For v0.3 and later

Until v0.2, we struggled to implement more kinds of blocks, enabling people to create complex circuits. However, from v0.3, we focused on making Genochemy easier to use for wider people, hearing their feedback.

Digests of engineering cycle

Since it is redundant to show our engineering cycle 5 times (v0.3 to v0.7) here, we summarized the important points.

• From what we learned in v0.2, we set up 9 questions, whose level is gradually increased, and the final question is mimicking our project. The questions are shown in a short sentence and illustration like below, in the bottom right of the screen. For some questions we prepared the answers. In the future we would like to implement the evaluation system, which calculates whether the circuit created by the user is correct or not.

The above are examples of questions.

• Hearing the feedback for v0.2, we implemented the graph feature, in which the time variation of fluorescence strength of mCherry and GFP can be seen. It was said sometimes it is difficult to judge the fluorescence strength from the cell appearance. We developed this with a student from the Waseda_Tokyo team (see Collaborations1 page).
  • We found that this feature may be useful as a software for modeling. In the future, we would like to make it possible to adjust more detailed parameters, and develop Genochemy as a software which enables strict simulations.
• Hearing the users' voice who used v0.3, we set up the tutorial which consists of 12 slides, shown in the bottom right tab. It describes how to create genetic circuits from scratch.
• A person who is unfamiliar with biology and used v0.4 said the block names were difficult. At the time we used protein names for block names to have users feel the reality from Genochemy, but it was hard to grasp for people unfamiliar with biology. Thus we changed block names. They are now named after their features, from v0.5. For example, the previous EL222 block is now named Blue-light-sensor.
• Since Genochemy uses various colors for each kind of block, we consulted color-blind people to hear whether the colors are difficult to tell from, and gained the opinion that most parts are distinguishable and two blocks whose colors look similar have quite different shapes so it is OK. But they said it is more reassuring if we fill blocks of the same shape in different patterns, like dots or lines. This point is updated in v0.7. For more information, please visit Inclusivity page.

"Build and Test" Histories

We continued developing and updating versions, from v0.3, v0.4, v0.5, v0.6, and v0.7, hearing users' feedback.

v0.3: Added questions and graph features. Questions are shown in the bottom right of the screen. This version was tested in the event "Transintellisession conference". Please see here for details. (In this version, only Japanese is available language)

From v0.4, we published it on Twitter, held an online competition and tested, gaining feedback from ordinary people.

v0.4: Added tutorials and hints. Tutorials are shown in the bottom right of the screen. (In this version, only Japanese is available language)

v0.5: Simplified block names. Minor fixes like fixing tutorial explanations. (In this version, only Japanese is available language)

v0.6: Changed the block system and supported English.

And showed more detailed help in the tooltips.

v0.7: Supported showing vDNA (virtual DNA), which is a DNA sequence converted from blocks. Also added explanation of the details of vDNA sequences and how the real DNA sequences can be ordered from these "text" sequences. This feature enables users to imagine the real process in synthetic biology by learning how to convert Genochemy blocks to virtual DNA sequences and how the sequences are ordered and used.

Also added different patterns to blocks with the same shape.