Overview

Our Optopass system aimed to use the order of different colors of light stimuli as a cryptogram and control the target gene and kill switch of S. cerevisiae. This system has three key components: light inducible promoter system, site-specific recombination system, and kill switch. We performed experiments for each element and successfully implemented the core parts of Optopass: light and order. Since we tested each part separately and together, we could benefit from our modularity to identify the problematic part of our system and make room for adding more parts. This process established the foundation of Optopass, providing proof of concept.

Light: optogenetic promoter system

In our system, we intended to use blue, red, green, and UV-B light to regulate gene expression. As a first step to lay this framework for Optopass, we focused on a blue light inducible promoter, as we found more previous works on it. We carried out multiple Engineering cycles and successfully constructed and tested a blue light inducible system in S.cerevisiae BY4741. The results demonstrated that we could control gene expression with blue light, suggesting the possibility of using light as a stimulus for Optopass. For more detail, please check our Results page.

Order: site-specific recombination system

To control S. cerevisiae by order, we introduced site-specific recombination system in Optopass. As an example of recombinase, we verified how the Cre-loxP system works. The result indicated that the Cre-loxP system worked in BY4741, cutting out the sequence between two loxP. With this successful result of Cre-loxP, we could see a high potential for a recombination system for storing the order of experienced stimulus on DNA sequence. This is an essential step toward the realization of our project. For more detail, please check our Results page.

Light and Order

Controlling S.cerevisiae by order of different colors of light is one of the most distinctive and significant characteristics of Optopass. We created this system in BY4741, placing Cre gene under the blue light inducible promoter. We then tested if Cre proceeds recombination of loxP sites. This result showed that Cre was expressed and caused recombination under the blue light illumination. This result provided the foundation of our concept: an optogenetic passcode system.

This result also showed that the leakage of the blue light inducible promoter is crucial since a non-activated promoter which was left in the dark can also result in sufficient levels of Cre expression for recombination. We thus combined Modeling of blue light inducible promoter and recombination to understand how much leakage is allowed in this system and how we can minimize it. From these modeling, we discovered that by reducing the ease of recombinase attachment to the recognition sequence, we could raise the recombination threshold and make the system more tolerant to leakage.

Our accomplishment this year

• We created a blue light inducible promoter system and tested the characteristics.
• We implemented Cre-loxP system for S. cerevisiae.
• Recombination occurred by Cre which was expressed under the control of blue light inducible promoter.
• We created a system for S. cerevisiae to store the memory of blue-light stimuli on DNA. This is our foundational step for the implementation of Optopass, as storing the light stimuli enables us to control S. cerevisiae by the order of light. This alone even opens up the possibility for precise yet safer control of S. cerevisiae, as we can now use the order of the lights as stimuli instead of combination of lights or chemicals.
• We found reducing the leakage of promoters and raising the threshold for the minimum amount of Cre to proceed recombination is a key to making the Optopass system accurate.
• We learned that reducing the ease of recombinase attachment to the recognition sequence could reduce the effect of leakage.

Future prospect

• Reduce the effect of leakage by using the less effective recombination site, and improving blue light inducible promoter.
• Engineer different types of light inducible promoters and recombination systems taking advantage of the modularity of our system.
• Implement kill switch system in BY4741, which is the last one of three key elements we were not able to successfully engineer this year.