Team:Sao Carlos-Brazil/Design
Project Design
Our Goal
In order to develop an yeast capable of resisting to ultraviolet radiation (UV) incidence, we have planned on creating a way of protecting the cell with some sort of armor composed of melanin. To do this, a display system was developed based on a protein complex consisting of AGA1/AGA2 coupled to a 4D Melanin-Binding Heptapeptide capable of strongly binding to melanin molecules (Ballard, 2011). We have also developed Kill Switches circuits to destroy yeast cells in case of an escape from a contained system. Here, we will demonstrate how we thought about constructing these circuits and which molecular biology strategies were implemented.
Display System
The protein anchoring system on the cell wall of organisms, a technique known as display, has been used by other iGEM teams. Yeast display works basically by expressing a membrane protein Aga2, acting as an anchor to Aga2 protein fused with the peptide of interest. For Astroshield, we needed a molecule that has great affinity to melanin. Therefore, we selected the peptide 4D to be fused with Aga2 by a flexible linker GGGs 4X, creating Part:BBa_K3273006 inspired in Chalmers-Gothenburg team in 2018. As a chassis, we mainly used two strains: the haploid S288C and the diploid industrial yeast PE-2.
In order to produce high levels of AGA1 and improve display system, part BBa_K3273017 was created, with a TDH3 strong promoter Part:BBa_K530008, the same promoter implemented on the display expression system. With AGA1 and AGA2 fused with 4D, we hope that the yeast could be protected as it was wearing a melanin armor.
Circuit
For our project, we have used melanin as a factor to induce UV tolerance on fermenting yeast Saccharomyces cerevisiae through the expression of proteins with a high affinity to it. The circuit is built to express the Aga1 (iGEM Registry Part:BBa_M45091) and Aga2 (BBa_K2027010) proteins, with the last one fused to a viral heptapeptide 4D, (BBa_K2027010) that will bind to melanin.
To ensure high and equivalent levels of Aga1 and Aga2/4D, we designed a transformation cassette that is regulated by the medium force constitutive promoter TDH3 (BBa_K530008).
Kill Switch
To test our yeasts under realistic conditions, a place similar to Mars and especially with high doses of ultraviolet radiation, we planned to conduct high altitude assays using helium balloons capable of reaching the upper atmosphere. As it would be an out of the lab test, all biosafety matters had to be taken into account(more information on our Human Practices page). We then decided to develop a robust set of Kill Switches to prevent any contact of our yeast with the external environment.
Our Kill Switch system is activated by two distinct signals: low glucose concentration and the absence of methionine amino acid. If one or both of these conditions is met, the system activates one or more of three different ways of programmed cell death, mediated by Bax protein, S. aureus nucA, and S. marcescens nucA.
The first system has been designed to sense the glucose concentration by a hybrid promoter developed by Cousens, 1987. In this promoter, the regulatory sequence (UAS) of the ADH2 gene, which is activated in low glucose concentration, was fused to the strong TDH3 promoter, creating the Part:BBa_K3273004. This promoter controls the expression of a Serratia endonuclease that has already proved to be an efficient cellular death method in Saccharomyces cerevisiae (Balan, 1999.). S. marcescens has been chosen as a molecular level Kill Switch due to its capacity of degrading dsDNA ssDNA, dsRNA, ssRNA, and preventing the genetic material from becoming available for environmental organisms
The second system has been developed using the mammalian Bax protein BBa_K3273007 under control of HXT6 promoter Part:BBa_K3273003, which is responsible for activating transcription of the HXT6 transporter on yeast on low glucose concentration. Bax is known for being a pro-apoptotic protein, and there is evidence that it can initiate the apoptosis process in yeast and plant cells (Ligr M et al., 1998; Yoshinaga K et al., 2005). Bax was chosen as a classical Kill Switch method for the project.
For the third system, registry parts were used. We have used Staphylococcus aureus nucA BBa_K1159105, under control of the promoter BBa_K165000 which is activated in low concentrations of methionine. The choice of a promoter sensitive to another stimulus other than glucose was taken into consideration to make the system even more robust. The choice to use another nucA protein was to ensure that the modified genetic material would be degraded.
Our goal with our Kill Switches is to inhibit all three promoters while the yeast cell remain in the fermentation medium, making the S. cerevisiae cell still functional since there will be no Kill Switch activity. If the cell escapes from these conditions, the promoters will be activated, leading to the expression of endonucleases and sequent degradation of the cell DNA. Furthermore, mammal Bax protein should be expressed activating the apoptosis cascade in the cell. The Kill Switches all belong to a NAND logic gate, as illustrated in the above image. Moreover, the following image represents the overall synthetic construct of our project, that shall be integrated into the yeast's genome.
With our circuit, we hope it'll be applicable to the food and fuel production on space and Earth, as well as on a UV-based sterilization process in the fermentation industry, enhancing the efficiency of this sector and avoiding waste and generation of harmful byproducts.
Molecular Biology Strategy
The chosen approach to execute the yeast transformation consisted of the integration of four expression cassettes on Saccharomyces cerevisiae’s chromosome V. In order to execute this procedure, the chosen technique was integration by homologous recombination. The selected screening method was based on the use of 5-FOA and in URA3 expression cassette, which can either be used as a positive or negative selection method. URA3 gene produces orotidine 5-phosphate decarboxylase protein, this enzyme converts orotidine 5-phosphate into uridylic acid. In the presence of 5-FOA the enzyme converts it into 5-fluorouracil, a toxic compound that is lethal for the yeast, selecting the cells without URA3 gene. The other screening method based on URA3 gene is related to auxotrophy, in the gene absence, the yeast becomes auxotrophic for uracil and can only survive on supplemented medium. Cultivating in in normal medium, only the yeast that presents the gene survives. Each transformation step removes or inserts the URA3 gene, alternating the screening method used.
The first transformation round was the deletion URA3 gene by insertion of Part:BBa_K3273000 and select ΔURA3 in 5-FOA medium. The objective of this first transformation was to prevent the yeast from staying auxotrophic on the last transformation round, as the yeast becomes very sensitive and fragile when auxotrophic. The second transformation was the insertion of Display system BBa_K3273016 in the same locus and recovery URA3 phenotype. Next step was the insertion of AGA1 gene Part:BBa_K3273017 in URA3 locus, removing again this gene and select ΔURA3 in 5-FOA medium. The last transformation was the insertion of the Kill switch cassette and recovery of URA3 gene, finishing the construction of Astroshield.
Cassette Construction
All sequences implemented on our project were obtained by de novo synthesis, this has been carried out by Integrated DNA Technologies (IDT) and Twist Bioscience. During this step, most of the sequences implemented had been codon-optimized for yeast. Due to the synthesis method limitations, like difficulty in synthesizing repetitive sequences and with high GC content, some parts had to be subdivided into smaller parts, that some were subsequently joined by fusion PCR. All sequences synthesized by Twist Bioscience® had adapters, which were removed with specific primers and PCR was carried out.
References
BALAN, Andrea; SCHENBERG, Ana Clara G. A conditional suicide system for Saccharomyces cerevisiae relying on the intracellular production of the Serratia marcescens nuclease. Yeast, v. 22, n. 3, p. 203-212, 2005.
BALLARD B., JIANG Z., SOLL C.E., et al. In vitro and in vivo evaluation of melanin-binding decapeptide 4B4 radiolabeled with 177Lu, 166Ho, and 153Sm radiolanthanides for the purpose of targeted radionuclide therapy of melanoma. Cancer Biother Radiopharm. 2011.
BALAN, A.; SCHENBERG, A.C.G.. Sistema de contenção para Saccharomyces cerevisiae visando biossegurança. Anais, 1999.
LIGR, M. et al. Mammalian Bax triggers apoptotic changes in yeast. Febs Letters, v. 438, n. 1-2, p.61-65, 30 out. 1998. Wiley.
MERCK. Yeast Transformation Protocols.
PULSCHEN, André Arashiro et al. Survival of extremophilic yeasts in the stratospheric environment during balloon flights and in laboratory simulations. Appl. Environ. Microbiol., v. 84, n. 23, p. e01942-18, 2018.
Stanford-Brown 2016 iGEM Team. Biomembrane UV protection.
YOSHINAGA, K. et al. Mammalian Bax initiates plant cell death through organelle destruction. Plant Cell Reports, v. 24, n. 7, p.408-417, 7 abr. 2005. Springer Science and Business Media LLC.