Our project DR.sTraTeGY (Drug Resistance mutation Tracking Technology based on Grape Yeast) has developed and test three part collections: Grape Yeast, TU Recorders (the optimal promoter–fluorescent protein combination screening collection), and the homologous arms integration collection for Saccharomyces cerevisiae.
Collection 1: Grape Yeast
ACE2 Knockout and Functional Human GPCR Integration
To create Grape Yeast, we first knocked out the transcription factor gene ACE2, whose function is essential for septum degradation during yeast cell division. The resulting strain fails to release daughter cells after cytokinesis, instead forming multicellular clusters that resemble grape bunches (Ratcliff et al.,2011; Tong et al., 2025)[1][2]. And its morphology and characteristics closely mimic those of pathogenic fungi.[3] Next, we introduced the translational unit of human δ-opioid receptor - HsDOR (BBa_256S6J1M) to the ACE2 deletion locus. Applying SNC80, which is an extracellular ligand that capable of binding to HsDOR, could activate the downstream signaling pathway. As human-derived HsDOR, which is on the membrane, cannot transduce signals inside yeast cells, we introduced Gpa1 (BBa_254K9906), described previously in Brown et al. (2000)[4]. Introduced Gpa1 will serve as the intracellular messenger for HsDOR, thereby achieving a functional human GPCR-yeast coupling. This modification not only endowed the multicellular yeast chassis with drug-ensing capabilities but also demonstrated the feasibility of transplanting human receptors into yeast.
We used the modular assembly yeast toolkit, described by Lee et al. (2015)[5], to construct the parts in our project. An intermediate plasmid (BBa_25EI9P2P) was constructed to integrate different translational units (TU) in ACE2 locus, which is very similar to pYTK096 for URA3 locus integration. The GFP dropout cassette in BBa_25EI9P2P has the BsaI sites oriented in reverse relative to standard parts. When performing the Golden Gate Assembly to produce BBa_25EI9P2P no 55°C digestion nor 80°C heat-inactivation, only ligation — cycling between 37°C and 16°C for 30 cycles. After cycling, the mixture is directly transformed into E. coli, and correct clones are identified by gain of GFP fluorescence with kanamycin resistance, followed by whole-plasmid sequencing confirmation.
This “end-on-ligation” method successfully yielded BBa_25EI9P2P internally we called it 234r-ACE2HRs-G418R. Then, we use it to generate the integration plasmid to put pREV1 driven HsDOR (BBa_25AKJ83S) into ACE2 locus.
Controlled Expression of IME1 and BAX to Modulate Yeast Morphology
To generate morphological diversity of our multicellular yeast, we introduced the IME1 (BBa_250R9OVR) and BAX (BBa_K5441013) genes. IME1 (BBa_25N9YOTD), a key regulator of meiosis (Smith et al.,1990), was integrated into the yeast genome via homologous recombination, which resulted in changes to the size and morphology of daughter cells.[6] BAX (BBa_25KYY8AI), a regulator of apoptosis (Greenhalf et al., 1995), was expressed in the multicellular yeast to accelerate cell death, producing branches of varying sizes and altering the overall morphology of the clusters.[7]
To tightly control the expression of these genes, we implemented the Tet-on/off switch. In the presence of doxycycline or aTc, the pTet2 promoter is active, and transcribes IME1 (BBa_25N9YOTD) and BAX (BBa_25KYY8AI) , thereby leading to either meiosis or apoptosis, which could be the first reported differentiation events in multicellular yeasts. We also developed pCUP1 driven IME1 (BBa_25N9YOTD) or BAX (BBa_25KYY8AI) which could be induced just adding CuSO4.
Replacing Ergosterol with Cholesterol to Mimic Pathogens
Pathogenic fungi differ significantly from Saccharomyces cerevisiae in their membrane lipid composition. The presence of cholesterol components in the cell membranes of pathogenic fungi, which resemble those of human cells, is a key factor in their invasiveness. Therefore, in addition to morphological engineering, we aimed to modify the membrane structure of Grape Yeast by replacing ergosterol with cholesterol to more closely mimic the cell membranes of clinically relevant pathogenic fungi, such as Candida albicans. The products of the ERG5 and ERG6 genes are key enzymes in the S. cerevisiae ergosterol biosynthesis pathway, responsible for catalyzing the formation of the C-22(23) double bond and the C-24 methyl transfer reaction (Bean et al., 2022)[8]. We replaced ERG5 and ERG6 with DrDHCR7 (BBa_25RCU5CB) and DrDHCR24 (BBa_25FOVO4C) respectively, using BBa_25XKAUNH and BBa_25E9K479, BBa_25U7DH3R and BBa_25O1ZVOU for targeted yeast genome integration. This redirection of the metabolic pathway enabled the substitution of ergosterol with cholesterol on the Grape Yeast cell membrane surface.
Fluorescent Timer
Grape Yeast forms complex multicellular clusters as mother cells continuously bud to generate new daughter cell branches. After a period of incubation, this repetitive budding leads to intricate, tangled three-dimensional clusters. This structural complexity makes it extremely difficult to trace the genealogical relationship among individual yeast cells. Consequently, we cannot precisely determine the timing and sequence of drug-resistance mutations within the cluster. To resolve this critical limitation, we introduced a fluorescent timer (BBa_25AT6YR4). By expressing this fluorescent timer within the multicellular yeast, we can accurately determine the time elapsed since a given yeast cell underwent cell division. This enables us to precisely track the temporal accumulation of resistance mutations across different cells and time points.
The core component of the fluorescent timer is modified mCherry (BBa_25TQG9WZ), a monomeric derivative of mCherry whose fluorescence spectrum gradually shifts from blue to red over time. This chromophore maturation rate is temperature-dependent, based on previous finding[9] and our modeling results: at yeast's culture temperature 30∘C, the blue fluorescence reaches its maximum at approximately 0.4 h, while the red fluorescence half-maximum appears at around 12 h.
Leveraging these spectral dynamics, we constructed the fluorescent timer by flanking modified mCherry (BBa_25TQG9WZ) with the ASH1 promoter (BBa_25VHXKNL) and the ASH1 3' UTR (BBa_25AIDL8P). A periodic promoter that drives the rhythmic transcription of color-changing mCherry is needed, otherwise all cells would be mixed with blue and red fluoresence. Because the promoter's choice critically affects the accuracy of time measurement, we developed a mathematical model to evaluate key characters of the promoter. Consistent with a previous study[10], our modeling verified the rationality of using the ASH1 promoter prior to any wet-lab experiments (see our Model page for more details). In addition, ASH1 3' UTR is crucial for spatial regulation. It ensures that the color-changing mCherry mRNA is guided to the daughter cells, and only translated in the daugther cells.[11][12] Together, the continuous and uninterrupted expression of the fluorescent protein in the progeny, guarantees the continuity and accuracy of fluorescence-based timing throughout successive cell divisions.
Table 1. Parts for Grape Yeast Collection
| Part Number | Type | Part Name |
|---|---|---|
| BBa_25MIG4EW | Homologous Region | HR5'_ACE2_Chr12L |
| BBa_259HCU8C | Homologous Region | HR3'_ACE2_Chr12R |
| BBa_25EI9P2P | Plasmid | Plasmid carrying ACE2HRs and G418 resistence |
| BBa_256S6J1M | coding | HsDOR |
| BBa_25AKJ83S | Translational_Unit | pREV1 driven HsDOR |
| BBa_25XKAUNH | Homologous Region | HR5'_ERG5_Chr13L |
| BBa_25E9K479 | Homologous Region | HR3'_ERG5_Chr13R |
| BBa_25U7DH3R | Homologous Region | HR5'_ERG6_Chr13L |
| BBa_25O1ZVOU | Homologous Region | HR3'_ERG6_Chr13R |
| BBa_25RCU5CB | coding | DrDHCR7 |
| BBa_25FOVO4C | coding | DrDHCR24 |
| BBa_25FU0JM9 | Translational_Unit | pREV1 driven DrDHCR7 |
| BBa_25JA9ZHO | Translational_Unit | pREV1 driven DrDHCR24 |
| BBa_254K9906 | coding | Modified Gpa1 |
| BBa_K3944000 | promoter | pCUP1 |
| BBa_255U7XC8 | Translational_Unit | pCUP1 driven modified Gpa1 |
| BBa_K3944010 | regulatory | rtTA |
| BBa_25JOF7TY | Translational_Unit | pREV1 driven rtTA |
| BBa_252BO17G | promoter | pTet2 |
| BBa_K5441013 | coding | BAX |
| BBa_250R9OVR | coding | IME1 |
| BBa_K5470011 | coding | GSG-E2A |
| BBa_25IB5O7X | coding | mSG |
| BBa_25MTFXKO | Translational_Unit | pTet2 driven BAX-E2A-mSG |
| BBa_25N811P5 | Translational_Unit | pTet2 driven IME1-E2A-mSG |
| BBa_25KYY8AI | Translational_Unit | pCUP1 driven BAX-E2A-mSG |
| BBa_25N9YOTD | Translational_Unit | pCUP1 driven IME1-E2A-mSG |
| BBa_25AIDL8P | 3'UTR | ASH1 3'UTR |
| BBa_25TQG9WZ | coding | modified mCherry |
| BBa_25VHXKNL | promoter | ASH1 AIpro |
| BBa_25AT6YR4 | Translational Unit | ASH1 AIpro driven modified mCherry-ASH1 3'UTR |
We use the following primers to verify the yeast genome intergration
ACE2_5H_F59 5-GCACTTTGGGAAAATTTCAGGACAC common_5H_R56 5-ccatctagatgcgaattcaggg # paired with any _5H_Forward common_3H_F58 5-gtgaatgctggtcgctatactg # paired with any _3H_Reverse ACE2_3H_R58 5-GACCTGCGTGCCCATTGTA ERG5_5H_F58 5-GCAGCTGCTCGTTCGC ERG5_3H_R57 5-GAAGAACATCCTTCTTGGATTGC ERG6_5H_F57 5-CCAAACAGCTAGGGCTGG ERG6_3H_R57 5-CGCAAGAAATCCAATGGCTTTC
Collection 2: TU Recorders using EMS-insensitive Fluorescent Protein
Under the induction of the EMS mutagen, the sequence of the promoter undergoes G/C to A/T base mutations[13], causing a change in the expression level of the fluorescent protein it regulates. This change reflects the degree of promoter sequence mutation. We aim to use this as a gene mutation recorder. However, the fluorescent protein's sequence itself can also change under EMS mutagenesis, leading to alterations in its fluorescence and interfering with the accurate assessment of the promoter's mutation degree. To solve this problem and make the fluorescent protein as stable as possible under EMS, we developed a Software Tool to optimize the fluorescent protein sequence. The tool replaces as many G/C bases with A/T bases as possible to enhance the fluorescent protein's resistance to mutagenesis under EMS induction. Thereby, we have EMS-resist coding sequences of fluorescent proteins (EMSfp) and mutagenic promoters combinations as TU recorders.
To identify the optimal mutation-tracking TU recorder for our project, we selected 4 promoters: pOST1 (BBa_259JX52V), pRNR2 (BBa_K3748013), pSTM1 (BBa_25FQBGJW) and pTDH3 (BBa_K3190001); 7 EMSfps (the number in the name represents the central excitation wavelength of the fluorescent protein): EMSfp383 (BBa_25F6RD26), EMSfp399 (BBa_25M2Z9H7), EMSfp499 (BBa_25IB5O7X), EMSfp506 (BBa_25FAVHQY), EMSfp569 (BBa_25TYRLM9), EMSfp642 (BBa_25GARG3E), EMSfp643 (BBa_2599SI53), and 1 common terminator tENO1 (BBa_K2753051) - yielding a total of 28 TU Recorders. 3 out 4 promoters (pOST1, pRNR2, pSTM1) were inspired by Hodgins-Davis et al. (2019)[13:1], the other pTDH3 (BBa_K3190001) is the strongest promoter test by Lee et al. (2015)[5:1]. By comparing fluorescence changes between the EMS-treated group and the control group, we screened out the combination with the most significant variation as the optimal pair, which was then integrated into the 16 chromosomes of yeast using our homologous arms collection.
In the YTK Toolkit developed by Lee et al. (2015)[5:2], pYTK096 is a URA3 integration vector pre-assembled from the following parts: Type 7 (the URA3 3' homology arm), Type 8a (the E. coli replication origin + kanamycin resistance marker + flanking NotI sites), and Type 8b (the URA3 5' homology arm). This design allows the vector to be linearized by NotI digestion prior to transformation. Any TU (Type 2+3+4) put into pYTK096, would be stable, single-copy chromosomal integration at URA3 locus.
All 28 TU Recorders expressing yeast strains were made with the following steps:
- Assembly into pYTK096 integration vector;
- Linearize by NotI digestion, which exposes the URA3 homology arms;
- Transform yeast strain BY4741, followed by integration into the chromosomal URA3 locus via homologous recombination;
- Confirm integration through URA3 phenotypic selection and PCR verification (a fragment of approximately 500 bp can be amplified upon correct integration).
We showcase the results of NotI digest and Phire (a commercial PCR Master Mix) amplification on corressponding iGEM Registry pages.
Table 2. Parts for 28 TU Recorders
| Part Number | Type | Part Name |
|---|---|---|
| BBa_25S7HMJ7 | Translational_Unit | pOST1 driven EMSfp383 (eBFP2) |
| BBa_25DWU82I | Translational_Unit | pOST1 driven EMSfp399 (Bluebonnet2) |
| BBa_25L56M2M | Translational_Unit | pOST1 driven EMSfp499 (mSG) |
| BBa_2584P1I8 | Translational_Unit | pOST1 driven EMSfp506 (mNeonGreen) |
| BBa_251VI6SD | Translational_Unit | pOST1 driven EMSfp569 (mScarlet) |
| BBa_25T5YP8M | Translational_Unit | pOST1 driven EMSfp642 (smURFP) |
| BBa_25BWBNVL | Translational_Unit | pOST1 driven EMSfp643 (miRFP670-2) |
| BBa_25FQWVZE | Translational_Unit | pRNR2 driven EMSfp383 (eBFP2) |
| BBa_25P3CYM6 | Translational_Unit | pRNR2 driven EMSfp399 (Bluebonnet2) |
| BBa_250UY7YR | Translational_Unit | pRNR2 driven EMSfp499 (mSG) |
| BBa_2517PQWR | Translational_Unit | pRNR2 driven EMSfp506 (mNeonGreen) |
| BBa_25GIKETL | Translational_Unit | pRNR2 driven EMSfp569 (mScarlet) |
| BBa_25R85SZ9 | Translational_Unit | pRNR2 driven EMSfp642 (smURFP) |
| BBa_258547VP | Translational_Unit | pRNR2 driven EMSfp643 (miRFP670-2) |
| BBa_25T01PTJ | Translational_Unit | pSTM1 driven EMSfp383 (eBFP2) |
| BBa_25R7QESE | Translational_Unit | pSTM1 driven EMSfp399 (Bluebonnet2) |
| BBa_255T0PHY | Translational_Unit | pSTM1 driven EMSfp499 (mSG) |
| BBa_25P31EK5 | Translational_Unit | pSTM1 driven EMSfp506 (mNeonGreen) |
| BBa_259AX3UO | Translational_Unit | pSTM1 driven EMSfp569 (mScarlet) |
| BBa_259Z131H | Translational_Unit | pSTM1 driven EMSfp642 (smURFP) |
| BBa_25DI5UXV | Translational_Unit | pSTM1 driven EMSfp643 (miRFP670-2) |
| BBa_25PHHOV9 | Translational_Unit | pTDH3 driven EMSfp383 (eBFP2) |
| BBa_258CH7EU | Translational_Unit | pTDH3 driven EMSfp399 (Bluebonnet2) |
| BBa_25L2MY7F | Translational_Unit | pTDH3 driven EMSfp499 (mSG) |
| BBa_25FED5Y1 | Translational_Unit | pTDH3 driven EMSfp506 (mNeonGreen) |
| BBa_25A00YSZ | Translational_Unit | pTDH3 driven EMSfp569 (mScarlet) |
| BBa_257O6RCL | Translational_Unit | pTDH3 driven EMSfp642 (smURFP) |
| BBa_25RJG3B2 | Translational_Unit | pTDH3 driven EMSfp643 (miRFP670-2) |
Collection 3: Sites of Integration to Assess Chromosomal Instability in Saccharomyces cerevisiae
In synthetic biology, efficient and predictable genome integration is fundamental for constructing complex biological systems and accelerating the engineering cycle. To address this need, we have assembled the Homologous Arms Integration Collection for Saccharomyces cerevisiae, covering all 16 chromosomes. This collection not only incorporates previously validated safe integration sites, but also extends to novel replacement-based strategies to another 6 dangerous integration sites, providing future iGEM teams with a powerful, flexible, and standardized solution.
There two groups homologous arms in this collection:
10 Safe Integration Sites
Shaw et al. (2023) designed 10 sets of safe integration sites located on different chromosomes of the S288C strain Saccharomyces cerevisiae. The selection and design of these integration sites were rigorously based on four standards[14]:
- Minimize Host Cell Impact: To ensure that the integration of the foreign DNA into the host genome has the least possible disruptive effect on the yeast cell's essential physiology.
- Maximize Integration Efficiency and Stability: To increase the likelihood of achieving highly efficient and stable integration of the genetic constructs.
- Highly Conserved in Common Strains: To guarantee that the loci are well-conserved across common laboratory strains of S. cerevisiae, thereby ensuring the broad utility and portability of the toolkit.
- Directly Amenable to CRISPR-Cas9 Manipulation: To make these sites readily accessible and targetable for precise editing using CRISPR-Cas9 technology. Please note in our project, no gRNA nor Cas9 were introduced into yeast.
To uphold these principles, Shaw et al. (2023) picked 10 sites because they reside in true intergenic regions (non-coding DNA), being located more than 1 kb from any known gene's start codon and more than 0.5 kb from its stop codon.
Furthermore, we recognized that these integration sites exhibit varying distances from the centromere across the chromosomes. We hypothsize that during yeast growth, sites located farther from the centromere undergo more frequent recombination events, leading to greater genetic variation. The question of whether these variations can be recorded was part of the inspiration behind our design of the TU Recorders.
Table 3. Integration Sites with varying distances to the centromere
| Part number | Part name | Integration Loci | Chr. Arm | Distance to Centromere |
|---|---|---|---|---|
| BBa_250U3B6G | HR5'_Chr1L | ChrI: 169,422-169,940 | Right | 30% |
| BBa_25GQIZIK | HR3'_Chr1R | ChrI: 169,942-170,478 | Right | 26414 bp |
| BBa_253K1B8N | HR5'_Chr4L | ChrIV: 359,868-360,355 | Left | 20% |
| BBa_25P0EZQP | HR3'_Chr4R | ChrIV: 360,356-360,897 | Left | 90061 bp |
| BBa_2552AC6E | HR5'_Chr6L | ChrVI: 10,278-10,913 | Left | 93% |
| BBa_25IULUBT | HR3'_Chr6R | ChrVI: 10,914-11,424 | Left | 140333 bp |
| BBa_25ZYL3GW | HR5'_Chr7L | ChrVII: 12,472-12,982 | Left | 97% |
| BBa_25H1VEJW | HR3'_Chr7R | ChrVII: 12,983-13,498 | Left | 483414 bp |
| BBa_25FYK3TX | HR5'_Chr8L | ChrVIII: 191,015-191,539 | Right | 21% |
| BBa_25PJP4LG | HR3'_Chr8R | ChrVIII: 191,540-192,044 | Right | 96388 bp |
| BBa_25FUWBU7 | HR5'_Chr9L | ChrIX: 340,431-340,933 | Left | 5% |
| BBa_25PDQZ8Z | HR3'_Chr9R | ChrIX: 340,935-341,523 | Left | 17943 bp |
| BBa_25DIWZUL | HR5'_Chr11L | ChrXI: 24,931-25,451 | Left | 94% |
| BBa_257IAIOG | HR3'_Chr11R | ChrXI: 25,452-25,963 | Left | 428273 bp |
| BBa_25F4SCLY | HR5'_Chr13L | ChrXIII: 408,123-408,657 | Right | 23% |
| BBa_25GCY7VQ | HR3'_Chr13R | ChrXIII: 408,658-409,161 | Right | 154324 bp |
| BBa_25B7ZBSD | HR5'_Chr15L | ChrXV: 686,950-687,450 | Right | 50% |
| BBa_25Q3F2B3 | HR3'_Chr15R | ChrXV: 687,451-687,964 | Right | 369943 bp |
| BBa_25RRB2RQ | HR5'_Chr16L | ChrXVI: 569,995-570,541 | Right | 9% |
| BBa_25U7CTYJ | HR3'_Chr16R | ChrXVI: 570,542-571,023 | Right | 33350 bp |
6 Dangerous Integration Sites
To provide comprehensive coverage across the yeast genome, we analyzed genome-wide stability data to identify locus whose perturbation could potentially disrupt chromosomal integrity. The rationale for selecting these six dangerous integration sites was guided by quantitative findings from Puddu et al. (2019)[15], which systematically characterized genomic instability across Saccharomyces cerevisiae deletion strains.
In this landmark work, the authors analyzed over one thousand yeast isolates and knockout mutants to explore the causes of chromosomal aneuploidy and genome instability. Their results showed that deletion of certain genes markedly increases genome instability, often leading to chromosomal gain or loss, genome duplication, and altered karyotypes.[15:1] These rearrangements were strongly associated with antifungal drug resistance. Their finding indicates that perturbations leading to genome duplication can also create opportunities for evolutionary innovation, as the extra genomic content allows cells to rewire gene expression and acquire new functions. Consistently, a recent study by Tong et al. (2025)[2:1], observed whole-genome duplication upon daily selection for larger size, over 1000 days.
Building on these insight, we hypothesized that integrating our TU Recorders into locus associated with genome instability could allow us to record mutational events while simultaneously observing the potential emergence of adaptive phenotypes. In other words, while the 10 safe integration sites reported by Shaw et al. (2023) ensure genome stability[14:1], the 6 dangerous integration sites we picked, provide a complementary perspective — a framework to study how instability itself may drive mutation, adaptation, and genomic evolution.
Using the dataset from Puddu et al. (2019)[15:2], we examined indicators such as chromosomal copy number variation, genome duplication frequency, and inter-chromosomal effects. Based on these parameters, we selected 6 representative locus — CAF, SWI4, DPB3, FEN2, SOD1, and BDF1 — each located on a distinct chromosome, where gene knockout was correlated with either:
- Instability of other chromosomes;
- Aneuploidy of the affected chromosome itself.
These loci represent genomic regions prone to rearrangement and serve as a useful contrast to the safe integration sites described by Shaw et al.. (2023)[14:2]. Although we integrate TU Recorders rather than mutagenic drivers, including these instability-prone loci completes whole-genome chromosomal coverage and allows exploration of how genomic context impacts mutation capture and recording fidelity.
To support this rationale, we provide an reorganized summary table derived from Puddu et al.. (2019)[15:3] supplementary information showing chromosomal copy number changes and genome stability indicators for CAF, SWI4, DPB3, FEN2, SOD1, and BDF1 (Supplementary Table).
Table 4. Sites of Integration to Assess Chromosomal Instability in Saccharomyces cerevisiae
| Part number | Part name | Integration Loci |
|---|---|---|
| BBa_25ATGCHY | HR5'_DPB3_Chr2L | ChrII: 735469-735988 |
| BBa_25Y6BTXZ | HR3'_DPB3_chr2R | ChrII: 736574-737141 |
| BBa_25Q67BOD | HR5'_FEN2_Chr3L | ChrIII: 165465-165991 |
| BBa_25TEB42Q | HR3'_FEN2_Chr3R | ChrIII: 163418-163951 |
| BBa_25TMD7MC | HR5'_SWI4_Chr5L | ChrV: 390514-391024 |
| BBa_255A36IQ | HR3'_SWI4_Chr5R | ChrV: 386704-387244 |
| BBa_25CLCLXX | HR5'_SOD1_Chr10L | ChrX: 601161-601690 |
| BBa_2533RATE | HR3'_SOD1_Chr10R | ChrX: 600171-600723 |
| BBa_25MZORVY | HR5'_BDF1_Chr12L | ChrXII: 867546-868044 |
| BBa_25R5XTI0 | HR3'_BDF1_Chr12R | ChrXII: 864985-865484 |
| BBa_25U88LU3 | HR5'_CAF_Chr14L | ChrXIV: 82768-83297 |
| BBa_25O0GI56 | HR3'_CAF_Chr14R | ChrXIV: 84390-84957 |
| BBa_25U7CTYJ | HR3'_Chr16R | ChrXVI: 570,542-571,023 |
In addition to Shaw's primers for verifying genome integration, we use the following primers as well
SOD1_5H_F56 5-CTGCACAAGATAAACTGAGATGACT common_5H_R56 5-ccatctagatgcgaattcaggg # paired with any _5H_Forward common_3H_F58 5-gtgaatgctggtcgctatactg # paired with any _3H_Reverse SOD1_3H_R56 5-CAGTTCTACAGAATTTTTGGACGAG CAF_5H_F56 5-CCAAGTGCATTTATTGAATGTTTTTGG CAF_3H_R57 5-CTTTTGTCCTATCCGTATTTGTTTAGTTATTTTG SWI4_5H_F56 5-CGCGTTTGAAGTGACGC SWI4_3H_R58 5-CAAAGTTTGCTCAAGTTGACTTAGAAATTG DPB3_5H_F56 5-CAGGTATCGCCCTCTCTATGAA DPB3_3H_R57 5-CTGTTGAATTAGATTAGTGACGTTTGTTTTG FEN2_5H_F58 5-CTTCAGAATAATTGTAATATTTACGCTTGCTTATTT FEN2_3H_R57 5-CTTTTCATGGCCTGGCAATT BDF1_5H_F59 5-GAATCTCGCAAGTCGCCT BDF1_3H R58 5-GAACTGCTACTGAATCCCTCAGAC
Summary
Together, these three collections establish a comprehensive toolkit for engineering, recording, and evaluating genomic behavior in multicellular Saccharomyces cerevisiae.
Starting from the Grape Yeast chassis, which mimics pathogenic fungal morphology and membrane composition, we created a medically relevant and modular foundation for synthetic design. Building on this, the TU Recorders enable direct visualization and quantification of mutation events through EMS sensitive promoters driven EMSfp (EMS-resistant coding sequencs of fluorescent proteins), forming the core of our mutation-tracking tool. Finally, by integrating these recorders into both safe and dangerous integration sites, we extended our investigation to the chromosomal level — examining how genome stability, duplication, and structural variation influence multicellular yeasts' adaptive evolution.
This multi-layered design not only expands the experimental versatility of yeast synthetic biology but also bridges the gap between stable genetic engineering and dynamic evolutionary processes, providing future iGEM teams with a powerful framework for studying mutation, adaptation, and genomic resilience.
Reference
Ratcliff, W. C., Denison, R. F., Borrello, M., & Travisano, M. (2012). Experimental evolution of multicellularity. Proceedings of the National Academy of Sciences of the United States of America, 109(5), 1595–1600. DOI: 10.1073/pnas.1115323109 ↩︎
Tong, K., Datta, S., Cheng, V., Haas, D. J., Gourisetti, S., Yopp, H. L., Day, T. C., Lac, D. T., Khalil, A. S., Conlin, P. L., Bozdag, G. O., & Ratcliff, W. C. (2025). Genome duplication in a long-term multicellularity evolution experiment. Nature, 639(8055), 691–699. DOI: 10.1038/s41586-025-08689-6 ↩︎ ↩︎
Saputo, S., Chabrier-Rosello, Y., Luca, F. C., Kumar, A., & Krysan, D. J. (2012). The RAM network in pathogenic fungi. Eukaryotic cell, 11(6), 708–717. DOI: 10.1128/EC.00044-12 ↩︎
Brown, A. J., Dyos, S. L., Whiteway, M. S., White, J. H., Watson, M. A., Marzioch, M., Clare, J. J., Cousens, D. J., Paddon, C., Plumpton, C., Romanos, M. A., & Dowell, S. J. (2000). Functional coupling of mammalian receptors to the yeast mating pathway using novel yeast/mammalian G protein alpha-subunit chimeras. Yeast (Chichester, England), 16(1), 11–22. PMID: 10620771 ↩︎
Lee, M. E., DeLoache, W. C., Cervantes, B., & Dueber, J. E. (2015). A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS synthetic biology, 4(9), 975–986. DOI: 10.1021/sb500366v ↩︎ ↩︎ ↩︎
Smith, H. E., Su, S. S., Neigeborn, L., Driscoll, S. E., & Mitchell, A. P. (1990). Role of IME1 expression in regulation of meiosis in Saccharomyces cerevisiae. Molecular and cellular biology, 10(12), 6103–6113. DOI: 10.1128/mcb.10.12.6103-6113.1990 ↩︎
Greenhalf, W., Stephan, C., & Chaudhuri, B. (1996). Role of mitochondria and C-terminal membrane anchor of Bcl-2 in Bax induced growth arrest and mortality in Saccharomyces cerevisiae. FEBS letters, 380(1-2), 169–175. DOI: 10.1016/0014-5793(96)00044-0 ↩︎
Bean, B. D. M., Mulvihill, C. J., Garge, R. K., Boutz, D. R., Rousseau, O., Floyd, B. M., Cheney, W., Gardner, E. C., Ellington, A. D., Marcotte, E. M., Gollihar, J. D., Whiteway, M., & Martin, V. J. J. (2022). Functional expression of opioid receptors and other human GPCRs in yeast engineered to produce human sterols. Nature communications, 13(1), 2882. DOI: 10.1038/s41467-022-30570-7 ↩︎
Subach, F. V., Subach, O. M., Gundorov, I. S., Morozova, K. S., Piatkevich, K. D., Cuervo, A. M., & Verkhusha, V. V. (2009). Monomeric fluorescent timers that change color from blue to red report on cellular trafficking. Nature chemical biology, 5(2), 118–126. DOI: 10.1038/nchembio.138 ↩︎
Kari, H., Bandi, S. M. S., Kumar, A., & Yella, V. R. (2023). DeePromClass: Delineator for Eukaryotic Core Promoters Employing Deep Neural Networks. IEEE/ACM transactions on computational biology and bioinformatics, 20(1), 802–807. DOI: 10.1109/TCBB.2022.3163418 ↩︎
Bertrand, E., Chartrand, P., Schaefer, M., Shenoy, S. M., Singer, R. H., & Long, R. M. (1998). Localization of ASH1 mRNA particles in living yeast. Molecular cell, 2(4), 437–445. DOI: 10.1016/s1097-2765(00)80143-4 ↩︎
Brodsky, A. S., & Silver, P. A. (2000). Pre-mRNA processing factors are required for nuclear export. RNA (New York, N.Y.), 6(12), 1737–1749. DOI: 10.1017/s1355838200001059 ↩︎
Hodgins-Davis, A., Duveau, F., Walker, E. A., & Wittkopp, P. J. (2019). Empirical measures of mutational effects define neutral models of regulatory evolution in Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences of the United States of America, 116(42), 21085–21093. DOI: 10.1073/pnas.1902823116 ↩︎ ↩︎
Shaw, W. M., Khalil, A. S., & Ellis, T. (2023). A Multiplex MoClo Toolkit for Extensive and Flexible Engineering of Saccharomyces cerevisiae. ACS synthetic biology, 12(11), 3393–3405. DOI: 10.1021/acssynbio.3c00423 ↩︎ ↩︎ ↩︎
Puddu, F., Herzog, M., Selivanova, A., Wang, S., Zhu, J., Klein-Lavi, S., Gordon, M., Meirman, R., Millan-Zambrano, G., Ayestaran, I., Salguero, I., Sharan, R., Li, R., Kupiec, M., & Jackson, S. P. (2019). Genome architecture and stability in the Saccharomyces cerevisiae knockout collection. Nature, 573(7774), 416–420. DOI: 10.1038/s41586-019-1549-9 ↩︎ ↩︎ ↩︎ ↩︎
