Team:BHSF/Poster

Poster: BHSF

Team:BHSF

Authors:

Poster design: Reina He

Wiki code: Kevin Yan

Content: Reina He, Annika Shi, Michael Li, Steven Mei, Kevin Yan

Proofread: Zhe Feng, Kevin Yan

Instructor: Zhe Feng, Tianze Zhu

Abstract

    When baking at home, people always have their bread over-fermented due to the of experience and disturbance from other matters. However, fermentation is the most important process in baking. Therefore, we modified the genes in auxotroph baking yeast and designed a circuit based on toggle switch to stop fermentation at appropriate times. Before the timing begins, the switch is kept at “off” state, where a protein that complements the auxotroph gets to express, after an inducer is added, marking the initiation of timing, the switch is turned on by accumulation of the previously repressed gene in the switch, the expression of the other gene is then shut down, along with the gene of the complimentary protein, leading to a drop in their concentrations and terminating fermentation over a certain period of time.

Team

Introduction

    This year, out team strive to designed an engineered yeast which can stop the fermentation process by itself at a precise moment, by using a delicately designed circuit which contains a toggle switch as a key element. After discovering a need for a solution to over-fermentation from human practices, we set our goal and carried out a preliminary design. Then we developed our design by several suggestions from human practices and communication with other teams and professionals. Then we build a model for calculating the fermentation time of our yeast, according to different strengths of promoters we use in the toggle switch. In the meantime, we engaged ourselves in the promotion of synthetic biology to a range of audiences. In the future, we will keep our focus on creating a method of letting our yeast perform the secondary fermentation process automatically, making it more versatile in making different kinds of bread.

Inspiration

    When people bake at home, fermentation is always a difficult step in the process of bread making. The most unmanageable factor which influences the degree of fermentation is the time. Annika, the team leader and a baking lover herself, explored the problem that every baker may face—over fermentation. In the survey we had with the public, we also found that insufficient fermentation is also a common problem for most home bakers. Through human practices, we learned that there is also a need for yeast that goes into dormancy after fermentation in the pre-made dough industry, for keeping the yeast inactivate by freezing the dough damages their flavor. Our design would help home-bakers control fermentation time by programming the yeast to stop fermenting by themselves.

Design

    We use a toggle switch to turn the yeast from an active state into an inactive state. The toggle switch is made of a pair of mutually repressing promoters, P lac and P lambda. These promoters are constructed by adding prokaryotic operators downstream of TATA boxes in eukaryotic promoters, which has proven to be an effective way of constructing inducible eukaryotic promoters.
    Outside the switch, a galactose promoter controls the expression of an integrase. In the beginning, the complement gene for auxotrophic yeast begins to express, and allows it to perform its function. When galactose is added into the system, integrase is expressed and inverts the sequence of the promoter upstream of the lacI, so that lacI begins to accumulate, even after galactose is removed. Then, after the toggle switch is triggered, the expression of the complement gene stops, leading to a drop in its concentration, and putting the yeast into dormancy for lack of the nutrient.
    The use of toggle switch allows us to develop yeasts of different fermentation time by changing promoters in the switch. This will make our yeast fitful for different types of bread.

Model

    In order to find out the promoters with the most suitable intensity to construct the toggle switch that best suits the time needed for fermentation, we created a model with the intensity of promoter as independent variable, and the concentration time course of two protein as dependent variable.
    In our model, u is the concentration of repressor 1, v is the concentration of repressor 2, a1 is the effective rate of synthesis of repressor 1, a2 is the effective rate of synthesis of repressor 2, b is the cooperativity of repression of promoter 2 and g is the cooperativity of repression of promoter 1.
    In our project, we will randomly first selected one or several pairs of promoters and test the transition time of each pair. Then, by plugging the experiment data, we will able to fix the formula and get a more precise relation between transition time and promoter strengths, therefore find appropriate combinations to meet different needs of fermentation time.

Engineering

    Our project is based on the engineering design of genetic circuit. To validate such a complex system as we designed, we separated the genetic circuit into different parts to verify if the parts are successfully operating, step by step.

Toggle Switch

    Toggle switch consists of two promoters that are placed in different directions with their downstream genes. Each side of the downstream genes can be translated into protein can inhibit the promoter on the opposite side.

    In order to test whether the toggle switch can operate normally in eukaryotic cells, we first separated the toggle switch into two parts, each of which consists only one promoter and a reporter.
    If the single promoter can function properly, we will then integrate two promoters of such kind to form the two-sided toggle switch and verify the validity of the toggle switch.

Integrase

    In the verification of the integrase to reverse DNA sequence, we put a constitutive promoter in front of an inverted sequence of fluorescent protein. On either side of the inversed sequence we added the recognition sites of integrase, expecting the integrase to inverse the sequence. After the alignment of the fluorescent protein to the constitutive promoter, fluorescent protein would be produced, glowing green light.

Galactose Promoter

    In order to solve the problem of having to add galactose to the system periodically, we combined galactose promoter with integrase to make a toggle switch function properly in our timer yeast. The promoter sequence before the inhibitor gene is inversed, making the polymerase unable to start transcription. Galactose would trigger the promoter to express the integrase, then integrase would turn the promoter sequence over and start transcription. This would be a stable system that does not require extended addition of galactose.

Integrated System

    In order to verify the final system, we again added the gene of fluorescent protein to the downstream of each promoter in the toggle switch. Expectation would be: the yeast gives out green light when galactose is added to the system and gives out no green light when galactose is absent. Finally, the whole system is successfully built and verified step by step, ensuring each element is operating well.

Human Practices

Goal Setting

    After the idea of inventing a timer yeast first came to us, we interviewed the bakers of ‘Palette’ and ‘Farina’, two famous bakeries in Beijing, to investigate possible implementations for our project from different perspectives.
    From the interviews, we learned that professional bakers use time as the measure of fermentation process, and that fermentation time varies a lot among breads. Both of the bakers thought our idea to be interesting and unique. They also suggested that apart from home baking, our project could also be of value to frozen dough industries, where doughs are fermented and then frozen, ready for baking.

    From these investigations, we set our goal to engineer a yeast strain that uses time as a measure for fermentation process, and goes into dormancy after a certain period of time.

Project Refinement

    We contacted professor Jianfen Liang from China Agricultural University and asked for her advice on our preliminary design of the project.

    Professor Liang pointed out that the toxin in our design may cause problem in food safety and that our present design can cause repulsion among the public. In addition, professor Liang suggested that we find alternatives to trigger the switch, for it requires a consistent accumulation of lacI protein. If we used galactose promoter to fulfill the function, it would be difficult to control the amount of galactose to be appropriate for baking.
    Inspired by her advice, we abandoned our original design of killing the yeast by toxic proteins, rather, we chose to use auxotroph yeast so that we will only need to stop the expression of its complementary gene in order to terminate fermentation when the time comes.

Future

Secondary fermentation

    We know that a lot of bread requires secondary fermentation, but the system we're working with is now designed to kill the yeast after it completes a fermentation process. Therefore, if we can set the yeast into a "dormant" stage after the first fermentation process, and then start a second fermentation automatically after a period of time, we will be able to enlarge our consumer group. So far, we've made a good beginning toward this direction, because instead of killing the yeast with toxins to complete the fermentation stage, our way to terminating fermentation is to stop the expression of the complementary gene of the auxotroph

Human Practices

    Our initial design focused on the application in home baking. And now it can be extended to industrial transportation; as we mentioned before, the frozen yeast dough will affect the taste of bread, if our system can make yeast deactivation, there would be no need for a refrigerated transport, that will definitely and greatly reduce the baker and the factory’s cost. Must be a big step forward. Of course, mass applications in industrial production also require our designs to be more reliable and controllable, and also require lower manufacturing costs.

Collaboration

    Our team collaborated with Keystone Academy to hold a Syn BioFair where We introduced our projects and promoted synthetic biology on the fair. We also exchanged opinions on our projects with other teams and collected suggestions from the visitors.
    The fair also helped us figure out the big problem of the application of our projects especially for the teams with their project related to food: the untrust of genetic engineering in the public.
    We attended 4th Southern China Regional Online Meeting and the Beijing International iGEMer Collaboration Seminar. We got lots of suggestions and support from other teams and built up close relationships with some teams.
    We also collaborated with Keystone, QHFZ-China and BJ101HS on various aspects.

References and Acknowledgements

-BAKING

1. The Bread Builders: Hearth Loaves and Masonry Ovens:
https://books.google.co.kr/books?id=VA6y1EMnkpYC&pg=PA34&lpg=PA34&redir_esc=y#v=onepage&q&f=false
2. Microbial re-inoculation reveals differences in the leavening power of sourdough yeast strains
https://zenodo.org/record/1065934#.X3722pMzbeo
3. Should I put less yeast in my bread & what happens when I add too much?
https://www.busbysbakery.com/should-i-put-less-yeast-in-my-bread/
4. Bakerpedia--fermentation
https://bakerpedia.com/processes/fermentation/#:~:text=Fermentation%20is%20an%20anaerobic%20biological,causes%20the%20dough%20to%20rise.
5. <面包科学> ——吉野精一
6. Advertisement of Angleyeast:
https://www.angelyeast.com//contents/3328/65569.html

-DESIGN

1. Negative Feedback Regulation of Fatty Acid Production Based on a Malonyl-CoA Sensor–Actuator
https://pubs.acs.org/doi/pdf/10.1021/sb400158w

-MODELING

1. Model of Duke univeristy’s team in 2013:
https://2013.igem.org/Team:Duke/Modeling/Thermodynamic_Model_Application
2. Edelstein-Keshet, L. Mathematical Models in Biology (McGraw-Hill, New York, 1988).
2. Kaplan, D. & Glass, L. Understanding Nonlinear Dynamics (Springer, New York, 1995).
3. Yagil, E. & Yagil, G. On the relation between effector concentration and the rate of induced enzyme synthesis. Biophys. J. 11, 11卤27 (1971).
4. Rubinow, S. I. Introduction to Mathematical Biology (Wiley, New York, 1975).