Overview

iGEM is part of the Science and Technology curriculum at University of Tartu. For that, Tartu_TUIT human practices is a whole year round quest. Talking to the current president of Estonia Kersti Kaljulaid about synthetic biology right after the previous Jamboree, we discussed the potential of synthetic biology to become a strategic development field for our country. Thus, human practices was one of the priorities for us. We set our aim to communicate science not only to academia but to our society. Our efforts ranged from scientific collaborations to public engagement events that target different age groups. From designing our project according to the feedback from industry giants such as Microsoft Research to integrating solutions from innovative startups as Synthex Labs. From closely working with a specialist within our university to reaching out to leading scientists all around the world. We strive to make change with everyone’s contribution and make synbio more appealing to the world. We demonstrate our efforts in different sections below. You can click to learn more.

Integrated Human Practices

Introduction

The extraction of the majority of valuable bioactive compounds from yeast cells requires cell wall disruption. Taking into account the fact that yeasts possess a complex cell wall composed of a layered meshwork of β-glucans, chitin, and mannoproteins, the cell wall disruption is one of the most challenging and expensive parts of extraction of the desired substance from yeast [4]. To overcome this issue, many cell wall disruption methods including physical, chemical and enzymatic have been developed. However, the cell wall disruption remains to be either expensive (enzymatic digestion), time-consuming or environmentally unfriendly (physical and chemical approaches). Based on these concerns, the goal of our project is to develop an autolytic yeast strain. The use of such strain as a platform for a yeast cell factory will ease the extraction of the valuable compounds from cells. Besides that, the employment of this strain into scientific research will simplify laboratory protocols involving yeast lysis (for example, cell wall disruption for plasmid rescue from yeast cells). During our project implementation, we talked to bioengineers, molecular biologists and other related specialists from academic laboratories and companies to collect useful information about this topic and improve our project. Initially, we wanted to check, what problems, connected to the current cell lysis methods people deal with in the lab. To collect this information, we sent a cell lysis survey to other iGem teams. Unfortunately, we could not manage to collect reliable data since only a few iGem teams are using yeast cell lysis for their projects. However, one team, Amazonas, has responded that one of the researchers from Amazonas University is interested to know more about our project! So, we had a skype call with Dr. Edson Do Carmo and he described common cell lysis problems he is facing quite often in his usual workflow. He uses one of the most popular cell lysis methods - zymolyase enzymatic degradation, and he mentioned the following issues:

○ Price: one set of zymolyase enzymes costs ~300 euros, and that is enough for 200 cell lysis procedures (1.33 euros per procedure);

○ Time-consuming, long experiment preparation.

Dr. Edson mentioned that he would be willing to try our strains once they are ready. He inspired our team with his feedback and it encouraged us to work even harder!

Cultivation method

There are different types of cultivation of yeast cells. To decide on a cultivation method that suites our goal the most and to model our project we asked for advice from Dr. Rainis Venta from the University of Tartu Institute of Technology. Dr. Venta has an extensive research background on the yeast cell cycle. At the same time, he was helping on the validation stage of the initial idea of our project and he was well familiar with the concept. His advice was to focus on a batch production model and choose a suitable compound to integrate it into the model at the end. Batch fermentation has several advantages. It allows a defined growth period and is cheaper. Moreover, for research purposes, S. cerevisiae cultures are usually grown in batch cultures. Dr. Venta gave us come suggestions concerning future modeling. Based on his recommendations, we decided to model the glucose uptake of our yeast strain in correlation with its growth rate and compound production rate in a single batch.

Product choice

To demonstrate how to choose the cell lysis point of our yeast strain, we were looking for a suitable compound. Its production rate should be visualized together with the growth rate to determine the time point at which we would like to have the majority of cells lysed. Since we want our yeast strain to be used not only by academia but also in industry, the selection of compounds should also be answering industry needs. The compound should be readily synthesized during the yeast growth phase and it should be a value-added product. To get suggestions on this stage we reached out to Dr. Nemailla Bonturi from European Research Area Chair in Synthetic Biology at the University of Tartu. She is an expert in microbial cell factories. She has worked on the production of microbial oil in oleaginous yeasts and now is dealing with protein turnover in yeast to increase the efficiency of valuable strains. Her experience with industry proved to be vital for us. With her suggestion, we chose β-carotene. Although it is not native for budding yeast, β-carotene synthesis is well-studied, and its production was previously successfully employed and modeled in Saccharomyces cerevisiae. Also, carotenoids are easy to separate from solutions and quantify using HPLC [1] [2] [3].

Modeling

One of the best options to predict the possible results of the experiments is to build the model to simulate the ongoing processes. The accurate model not only may predict the results but also point to the specific issues our team could face. To mathematically demonstrate our model proved to be a challenge and we encountered various difficulties. We successfully tackled these obstacles by incorporating valuable advice not just within our university but from research labs around the globe and corporations such as Microsoft Research and Synthex Labs. With questioned, concerning modeling, we reached out to a past IGEMer Dr. Sander Wuyts who performed modeling for a gold medal-winning IGEM project. Currently, he is a postdoctoral researcher at the European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany. His research focused on applied microbiology. His expertise in industrial microbiology and modeling was very valuable for us. Through multiple online video communications, Dr.Wuyts acted as a mentor for our modeling team. He advised us to include in our model estimation for an ideal lysis time of yeast cells in the culture. This would help to reduce the cost associated with the production process. As a result, our model predicts the time period of maximum production and calculates the most suitable time point for cell lysis. Another vital suggestion was to incorporate wet-lab data into our model to optimize it. We decided to use the growth curve for our strain in our model. This allowed a more precise estimation of time when we want cells to pop in our culture. We wanted to further optimize our model by tuning our parameters to closely represent our yeast strain. We acquired the help of Dr. Oleg Košik, a mathematician working in system biology at the University of Tartu, to mathematically validate our model. With his assistance, we calculated growth parameters for our strain based on available experimental data for the W303 strain. He also suggested using the results of microscopy to estimate the time point when to initiate cell lysis. To accomplish that, we observed how long it takes for the cells to be destroyed after inducing the glucanase expression under the microscope and fed the data into our model. This allowed us to determine the time point for induction of glucanases.

Inducible knockouts

After formulating the project idea, we wanted to discuss it with different specialists to illuminate potential issues and drawbacks before we face them. That is why the conversation with Sadig Niftullayev, PhD student from the University of Cologne, who studies yeast aging, really proved useful. He suggested that it might be not sufficient just to overexpress glucanases inside the yeast because the cell wall assembly enzymes will compensate for the destructive effect. He offered us to use auxin-inducible knock-outs system to eliminate the genes participating in the cell wall synthesis. His advice led us to search for the possible candidates to knock out. We have chosen the GAS1 gene as its deletion does not affect the growth of the cell but makes it more sensitive to zymolyase treatment. We are currently working on the process of yeast strain formation with GAS1 gene deletion.

Proof of concept

We were looking for a simple yet accurate method to validate our designed system and demonstrate its efficiency. Dr. Maria Soloveychik, a co-founder of SyntheX inc., suggested a simple method to check whether cell lysis was efficient: it is possible to measure GFP fluorescence in the supernatant after centrifugation of yeast culture after induction of glucanase expression. Following her advice, we introduced the pTDH3-EGFP cassette into four strains carrying different glucanase variants and performed an experiment to measure GFP fluorescence in the cell culture supernatant. Dr. Paul Grant, a synthetic biologist in the Biological Computation Group at Microsoft Research suggested us to use Bradford assay to measure glucanases in the medium. We implemented his advice in our work and performed protein measurements of cell culture supernatant after glucanase induction.

References:

1. S. Yamano, H. Ikenaga, N. Misawa, T. Ishii, and M. Nakagawa, “Metabolic Engineering for Production of β-Carotene and Lycopene in Saccharomyces cerevisiae,” Biosci. Biotechnol. Biochem., 1994.
2. N. Lange and A. Steinbüchel, “β-Carotene production by Saccharomyces cerevisiae with regard to plasmid stability and culture media,” Appl. Microbiol. Biotechnol., 2011.
3. M. C. Ordoñez, J. P. Raftery, T. Jaladi, X. Chen, K. Kao, and M. N. Karim, “Modelling of batch kinetics of aerobic carotenoid production using Saccharomyces cerevisiae,” Biochem. Eng. J., 2016.
4. G. Lesage, H. Bussey, Cell Wall Assembly in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev., 2016.

[1] 1. S. Yamano, H. Ikenaga, N. Misawa, T. Ishii, and M. Nakagawa, “Metabolic Engineering for Production of β-Carotene and Lycopene in Saccharomyces cerevisiae,” Biosci. Biotechnol. Biochem., 1994.

[2] 2. N. Lange and A. Steinbüchel, “β-Carotene production by Saccharomyces cerevisiae with regard to plasmid stability and culture media,” Appl. Microbiol. Biotechnol., 2011.

[3] 3. M. C. Ordoñez, J. P. Raftery, T. Jaladi, X. Chen, K. Kao, and M. N. Karim, “Modelling of batch kinetics of aerobic carotenoid production using Saccharomyces cerevisiae,” Biochem. Eng. J., 2016.

[4] 4. G. Lesage, H. Bussey, Cell Wall Assembly in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev., 2016.

Team:Tartu TUIT/Human Practices