We present a set of new BioBrick-compatible parts encoding in silico–designed progerin interactors, optimized for use with the SynXpress™ cell-free expression kit.
- BBa_254PC42U: 62aa11_1 optimized for use in SynXpressTM Cell-Free expression kit;
- BBa_252ADNO1: LOGO optimized for use in SynXpressTM Cell-Free expression kit;
- BBa_25KT4K90: n80_02 optimized for use in SynXpressTM Cell-Free expression kit;
- BBa_25E35V1M: Rank_15 optimized for use in SynXpressTM Cell-Free expression kit;
- BBa_253S6Z46: Rank_21 optimized for use in SynXpressTM Cell-Free expression kit
These parts include:
- An N-terminal His tag for straightforward purification and detection;
- A Factor Xa cleavage site inserted between the His tag and the interactor sequence, allowing tag removal if required and minimizing steric hindrance — especially important given the small size of many interactors;
- Codon-optimized DNA sequences ordered from IDT and cloned into the pET-IDT plasmid following the SynXpress™ kit guidelines, ensuring compatibility with in vitro expression workflows
Why These Parts Matter to the iGEM Community
Bridging in silico predictions and experimental validation
These parts provide a direct path for teams aiming to test potential progerin-binding partners. They enable immediate validation through Microscale Thermophoresis (Monolith) or pull-down assays, without the need for de novo design and optimization.
Modular foundation for chimeric fusion proteins and Progeria research
Once validated, these interactors can serve as versatile building blocks for constructing functional fusion proteins such as degraders, fluorescent reporters, or therapeutic effectors.
Because they specifically target progerin’s C-terminal region, they open a new experimental avenue for exploring laminopathies and aging-related mechanisms within a standardized synthetic biology framework.
Future iGEM teams can reuse and adapt these interactors across expression systems — from yeast to mammalian cells — simply by adjusting expression context or codon optimization. This cross-platform compatibility turns our parts into a modular toolkit for both applied and fundamental research on progeria.
In summary, these interactor parts are not merely test components — they represent foundational building blocks for synthetic biology interventions centered on Progerin. Their modularity, compatibility, and disease relevance make them a valuable and reusable contribution to the iGEM community.
We provide two BioBrick-compatible parts designed for in vitro expression and interaction testing within the SynXpress™ Cell-Free Expression Kit framework:
- BBa_25BEEQ7F: SpyTag optimized for cell-free expression;
- BBa_259R437F: SpyCatcher optimized for cell-free expression
Both parts were codon-optimized for the SynXpress™ system, ordered from IDT, and cloned into the pET-IDT plasmid as described in the kit’s instructions for use.
These parts include:
- An N-terminal His tag for purification and detection;
- A Factor Xa cleavage site to allow tag removal if required
Why These Parts Matter to the iGEM Community
Versatile modules for modular protein assembly
SpyTag and SpyCatcher form a robust covalent bond upon interaction, allowing researchers to assemble multi-protein complexes, create modular fusion proteins, or link separate subunits in a controlled way. By providing these optimized parts, we enable teams to integrate the SpyTag/SpyCatcher system directly into cell-free workflows or recombinant expression platforms without additional cloning steps.
Ready-to-use components for interaction assays
These constructs can be expressed directly in cell-free reactions and tested in binding assays such as Microscale Thermophoresis (Monolith) or pull-downs to measure dissociation constants (Kd) and validate interaction efficiency. Since the fusion context can influence binding affinity, having standardized and validated standalone SpyTag and SpyCatcher parts allows rapid assessment of how different fusion partners affect complex formation.
Expanding modular protein design capabilities
With these optimized and standardized parts, teams can easily incorporate a reliable, covalent interaction pair into their synthetic biology designs — whether for biosensors, protein scaffolds, multimeric enzyme assemblies, or therapeutic constructs. Ready for in vitro use, they provide a flexible and immediate resource for building and characterizing protein–protein interactions within the iGEM community.
Progerin is the protein responsible for Hutchinson–Gilford Progeria Syndrome (HGPS), a rare premature aging disorder. Like all lamins, in humans it is composed of four distinct domains:
- an N-terminal head domain;
- a central α-helical rod domain consisting of four coiled-coil segments separated by flexible linkers that enable dimerization;
- a nuclear localization signal (NLS);
- a nuclear localization signal (NLS);
- a nuclear localization signal (NLS);
- an Ig-like domain followed by an intrinsically disordered C-terminal tail
The C-terminal tail contains the CaaX motif recognized by protein farnesyltransferase (FTase), which is crucial for the protein’s proper anchoring to the nuclear membrane.
Although it remains uncertain whether these structural domains are fully conserved when progerin is expressed in Saccharomyces cerevisiae, our goal was to recreate the pathological phenotype in this simple and cost-effective eukaryotic model. To achieve this, we derived the full-length progerin mRNA sequence from the ENA database (AY357727.1) and codon-optimized it for expression in S. cerevisiae. To facilitate future work and ensure compatibility with the iGEM Registry, we standardized the entire sequence according to RFC10 and RFC1000 standards. This provides the community with the first fully standardized, yeast-compatible version of progerin.
Building on our proposed best part BBa_25NDL8N0, we also designed two functional fragments derived from progerin’s C-terminal domain for use in protein–protein interaction studies:
- Amino acids 430–614: BBa_25HM35ST;
- Amino acids 545–614: BBa_25IF4C4Z
These fragments were created to investigate specific interactions within the lamin network and between progerin and potential interactors using the yeast two-hybrid (Y2H) assay.
Why This Part Matters to the iGEM Community
Establishing yeast as a model for progeria research
This construct enables the use of S. cerevisiae as a simplified, genetically tractable model to study progerin biology. Yeast offers a eukaryotic environment where cellular processes such as protein folding, post-translational modification, and nuclear envelope organization can be observed with minimal complexity and low cost. By transferring progerin expression to yeast, we provide a platform where the molecular effects of this pathological lamin variant can be dissected using genetic and biochemical tools that are not easily applicable in mammalian systems.
Enabling new studies on aging and nuclear lamina dysfunction
The standardized yeast-compatible progerin sequence allows teams to explore the impact of progerin accumulation on cellular aging, oxidative stress, and genome stability. Yeast has long been a cornerstone model for aging research, and introducing human lamin pathologies into this context bridges the gap between molecular mechanisms of aging and human disease. These parts offer a scalable system for screening modifiers of progerin toxicity, studying farnesylation-related defects, or testing potential therapeutic interventions in a rapid and ethically accessible way.
In summary, our standardized and yeast-optimized progerin sequence — together with its C-terminal fragments — transforms S. cerevisiae into a new experimental model for studying laminopathies and aging mechanisms. It bridges synthetic biology and biomedical research, enabling low-cost, modular exploration of one of the most iconic aging-related proteins in a genetically tractable eukaryotic system.
We derived the progerin mRNA sequence from the ENA database (AY357727.1) and we codon optimised it in order to express it in S. cerevisiae.
Phenotype characterization progerin+pYES2
We expressed BBa_25NDL8N0 in pYES2 in a galactose dependent manner (GAL1 promoter) in order to study progerin phenotype on yeasts.
Progerin is responsible for a cytotoxic effect on S. cerevisiae CEN.PK as we confirmed by spot test and growth curve.
Spot test result:
Growth curves:
Our results pose the base for further studies about the modelling of progeria in S. cerevisiae and could be used as a tool to study phenotype rescue in the context of a chemical library to find new possible therapeutic molecules.
Progerin for yeast two hybrid (Y2H)
When we first implemented progerin in order to express it in the Y2H plasmids, we cloned its whole sequence (a version non complied with RFC10), in both PGADT7 and pGBKT7 and transformed it into S. cerevisiae Y190 cells.
Unfortunately, we were unable to obtain consistent cell growth and we confirmed no progerin expression (through Western blot). This was probably due to the fact that pGADT7 and pGBKT7 express protein of interest under the ADH1 constitutive promoter, making the progerin cytotoxic effect that we witnessed when pYES2+progerin was transformed in S. cerevisiae CEN.PK cells stronger.
For this reason we decided to express only the C-term of progerin to limit its toxicity to the cell. This is the sections of protein we selected based on previous studies[1]:
- From aa 430 to 614 (BBa_25HM35ST); this sequence includes the Ig-like part of the protein;
- From aa 545 to 614 (BBa_25IF4C4Z); this sequence does not include the Ig-like part of the protein
After expressing the truncated version of progerin in S. cerevisiae Y190, we were able to obtain transformed cells that expressed progerin.
We employed these cells in co-transformation with pGAD+BUBR1 and pGAD+interactors in order to perform the Y2H test on selective medium SD Glucose -Leu -Trp -His +3AT 30mM.
BUBR1 is a core component of the spindle assembly checkpoint that was proved to bind selectively to progerin C-terminus by pull down assay and co-immunoprecipitation in mammalian cell culture[1]. For this reason, we chose to express it in our Y2H system to find out more about the possibility of finding other progerin interactors with the system. We did not focus on its natural role as it was out of our interest in our work.
We derived the BUBR1 N-term sequence we used from the same previous studies[1] and codon optimised it for expression in S. cerevisiae.
The sequence was expressed in the plasmid pGADT7 (“prey” plasmid, expressing the activation domain “AD”). Check here how the yeast two hybrid test works.
The Y2H test was carried out by co-transforming S. cerevisiae Y190 with pGAD+BUBR1 N-term and pGBK+progerin C-term (430).
The test was carried out by spot test in SD Glucose -Leu -Trp -His +3AT 30 mM medium; the results of a 72 h long growth at 28°C are shown in the picture:
| Position | Plate 1 | Plate 2 |
|---|---|---|
| 1 | CTR (+) | CTR (+) |
| 2 | CTR (-) | CTR (-) |
| 3 | pGBK_∅ + pGAD_BUBR1 #1 | pGBK_∅ + pGAD_BUBR1 #1 |
| 4 | pGBK_∅ + pGAD_BUBR1 #2 | pGBK_progerin 430 + pGAD_∅ #1 |
| 5 | pGBK_progerin 430 + pGAD_∅ #1 | pGBK_progerin 430 + pGAD_BUBR1 #1 |
| 6 | pGBK_progerin 430 + pGAD_∅ #2 | pGBK_progerin 430 + pGAD_BUBR1 #3 |
| 7 | pGBK_progerin 430 + pGAD_BUBR1 #1 | pGBK_progerin 430 + pGAD_BUBR1 #4 |
| 8 | pGBK_progerin 430 + pGAD_BUBR1 #2 | pGBK_progerin 430 + pGAD_BUBR1 #5 |
Despite the results of previous studies, we were unable to confirm BUBR1-progerin interaction in our system; this means that this kind of interaction seems to not occur in the yeast two hybrid systems.
We derived the mature lamin A sequence from UniProt (P02545) by removing the amino acids that are in vivo ceased through post-translational modification (processing at aa 646-647).
We then codon optimised the amino acid sequence in order to obtain the DNA sequence suitable for expression in S. cerevisiae.
This sequence was employed in the Y2H system and expressed in pGBKT7 (the bait plasmid, expressing the DNA-binding domain BD). The results were in line with our expectations and confirmed that the interactors show no off-target interaction with lamin A.
| No. | PLATE I | PLATE L | PLATE M |
|---|---|---|---|
| 1 | pGBK_∅ + pGAD_interactor 1 | pGBK_∅ + pGAD_interactor 4 | pGBK_∅ + pGAD_interactor 5 |
| 2 | pGBK_laminA + pGAD_∅ | pGBK_laminA + pGAD_∅ | pGBK_laminA + pGAD_∅ |
| 3 | pGBK_laminA + pGAD_interactor 1 #1&2 | pGBK_laminA + pGAD_interactor 4 #1&2 | pGBK_laminA + pGAD_interactor 5 #1&2 |
| 4 | pGBK_laminA + pGAD_interactor 1 #3&4&5 | pGBK_laminA + pGAD_interactor 4 #3&4&5 | pGBK_laminA + pGAD_interactor 5 #3&4&5 |
We believe that other than testing this sequence in the Y2H system, it would be interesting to understand if it responsible for a phenotype similar to progerin’s one, since yeast nuclei, like many other lower eukaryotes, do not express lamins proteins[2] and are characterised by a different nuclei structure.
Our bioinformatically predicted progerin interactors were obtained by a thoughtful analysis (you can read about our work in the modelling page) and then expressed in our Y190 chassis S. cerevisiae and human cell lines. In Y190 we created different codon optimised sequences.
We expressed the interactors in the pGAD plasmid (“prey” plasmid, expressing the activation domain “AD”) in order to test them through Y2H.
Only our top 5 interactors in terms of binding were tested through Y2H due to time limitation:
- BBa_25R6U8N1: LOGO optimised for S. cerevisiae;
- BBa_25RLZ3X4: n80_02 optimised for S. cerevisiae;
- BBa_25G9QB6S: Rank_15 optimised for S. cerevisiae; BBa_25UZI9YM: Rank_21 optimised for S. cerevisiae;
- BBa_256U2NU6: 62aa11_1 optimised for S. cerevisiae
Unfortunately, we were able to verify that the interaction with progerin C-term ( 430 and/or 545 fragments) does not occur.
| No. | PLATE A | PLATE B | PLATE C |
|---|---|---|---|
| 1 | pGBK_progerin 430 + pGAD_∅ | pGBK_progerin 430 + pGAD_∅ | pGBK_progerin 430 + pGAD_∅ |
| 2 | pGBK_progerin 430 + pool pGAD_interactor 1 #1 | pGBK_progerin 430 + pool pGAD_interactor 2 #1 | pGBK_∅ + pool pGAD_interactor 3 |
| 3 | pGBK_progerin 430 + pool pGAD_interactor 1 #2&3 | pGBK_progerin 430 + pool pGAD_interactor 2 #2&3 | pGBK_progerin 430 + pool pGAD_interactor 3 #1 |
| 4 | pGBK_progerin 430 + pool pGAD_interactor 1 #4&5 | pGBK_progerin 430 + pool pGAD_interactor 2 #4&5 | pGBK_progerin 430 + pool pGAD_interactor 3 #2&3 |
| 5 | pGBK_∅ + pool pGAD_interactor 1 | pGBK_∅ + pool pGAD_interactor 2 | pGBK_progerin 430 + pool pGAD_interactor 3 #4&5 |
| No. | PLATE D | PLATE E | PLATE F |
|---|---|---|---|
| 1 | pGBK_progerin 545 + pGAD_∅ | pGBK_progerin 545 + pGAD_∅ | pGBK_progerin 545 + pGAD_∅ |
| 2 | pGBK_progerin 545 + pool pGAD_interactor 1 #1 | pGBK_∅ + pool pGAD_interactor 2 | pGBK_∅ + pool pGAD_interactor 3 |
| 3 | pGBK_progerin 545 + pool pGAD_interactor 1 #2&3 | pGBK_progerin 545 + pool pGAD_interactor 2 #1 | pGBK_progerin 545 + pool pGAD_interactor 3 #1 |
| 4 | pGBK_progerin 545 + pool pGAD_interactor 1 #4&5 | pGBK_progerin 545 + pool pGAD_interactor 2 #2&3 | pGBK_progerin 545 + pool pGAD_interactor 3 #2&3 |
| 5 | pGBK_∅ + pool pGAD_interactor 1 | pGBK_progerin 545 + pool pGAD_interactor 2 #4&5 | pGBK_progerin 545 + pool pGAD_interactor 3 #4&5 |
Also for the last two interactors the interaction with progerin C-term (progerin 545 fragment) does not occur in the Y2H system.
| No. | PLATE G | PLATE H |
|---|---|---|
| 1 | pGBK_∅ + pGAD_interactor 4 | pGBK_∅ + pGAD_interactor 5 |
| 2 | pGBK_progerin 545 + pGAD_∅ | pGBK_progerin 545 + pGAD_∅ |
| 3 | pGBK_progerin 545 + pGAD_interactor 4 #1&2 | pGBK_progerin 545 + pGAD_interactor 4 #1&2 |
| 4 | pGBK_progerin 545 + pGAD_interactor 4 #3&4&5 | pGBK_progerin 545 + pGAD_interactor 4 #3&4&5 |
BBa_25B01XHB HA-progerin
This construct expresses HA-tagged progerin, allowing straightforward detection through anti-HA immunofluorescence or Western blot. Given the challenges of working directly with HGPS patient-derived cells, this plasmid provides a reliable and accessible way to study progerin expression and localization in human cells. It reproduces the same cytotoxic effects observed with untagged progerin, making it a faithful model while enabling rapid, antibody-based analysis of expression and nuclear accumulation. This construct thus serves as a foundational tool for studying the mechanisms of progeria and the cellular consequences of progerin expression.
BBa_25IIEVZB SpyTag-link-progerin
This construct expresses SpyTag-tagged progerin, enabling its covalent interaction with any SpyCatcher-fused protein. Like HA-Progerin, it faithfully replicates the cytotoxic effects of untagged progerin, while the SpyTag addition opens the door to numerous applications. By fusing SpyCatcher to a protein of choice—such as fluorescent markers, degradation domains, or molecular sensors—teams can directly test the modulation of progerin levels, localization, or stability. This construct transforms progerin into a versatile molecular hub, supporting an unlimited range of experiments in targeted degradation or protein–protein interaction studies.
BBa_25O5I756 SpyTag-laminA
As mentioned above for SpyTag-progerin, this construct provides the same modular SpyTag interface but applied to wild-type lamin A. It preserves the structural functionality of lamin A while enabling covalent binding to SpyCatcher-fused proteins. Beyond serving as a control for studies with SpyTag-progerin, this construct also allows the study of lamin A behavior under controlled modification. It thus provides a flexible platform to explore nuclear lamina assembly, remodeling, and interaction with engineered protein partners.
BBa_25Y190DU mEGP-progerin
This construct expresses mEGFP-progerin, enabling immediate visualization of its expression and localization via fluorescence microscopy. It serves as a key reporter tool for monitoring the nuclear accumulation of progerin without sample manipulation. Moreover, when combined with additional tags such as SpyTag or degradation domains, this construct can be repurposed to study mislocalization, aggregation, or targeted degradation of progerin under various conditions, making it a versatile probe for laminopathy research.
BBa_252QSG7W mEGFP-NLS
This construct encodes mEGFP fused with a nuclear localization signal (NLS), providing a fluorescent control for nuclear import. It allows verification that mEGFP tagging does not alter the nuclear localization of lamin A or progerin. This construct is essential for comparative studies aimed at understanding whether observed mislocalization is due to pathological mutation or to tagging artifacts.
BBa_256804GM RING-NLS-link-mEGFP
This construct encodes the RING domain fused to a nuclear localization signal and mEGFP through a flexible linker. Designed as both an expression and localization control, it enables the direct observation of RING domain distribution within the nucleus and facilitates studies on its potential role in nuclear protein turnover. When fused to other domains or interactors, this construct can also be used to trace RING localization in real time or explore its effect on nuclear targets.
BBa_255KDS7V RING-NLS-link-SpyCatcher
This construct represents a highly modular platform for studying RING-mediated degradation. By merging the catalytic activity of the RING domain with the covalent SpyTag/SpyCatcher system, it enables targeted protein degradation in eukaryotic cells. Any SpyTag-fused protein can serve as a substrate: the SpyCatcher domain binds covalently to the tagged target, bringing it into proximity with the RING domain, which promotes ubiquitination and subsequent proteasomal degradation. This system has vast potential for studying loss-of-function phenotypes and can be applied in diverse research contexts ranging from synthetic biology to therapeutic design.
BBa_25ZFZPVO 62aa11_1 optimized for the use in N196 pBiT1.1-C [TK/LgBiT] Vector
BBa_25SY5RH3 62aa11_1 optimized for the use in N198 pBiT1.1-N [TK/LgBiT] Vector
BBa_25J9L03P LOGO optimized for the use in N196 pBiT1.1-C [TK/LgBiT] Vector
BBa_25AT078M LOGO optimized for the use in N198 pBiT1.1-N [TK/LgBiT] Vector
BBa_25ZP4XF1 n80_02 optimized for the use in N196 pBiT1.1-C [TK/LgBiT] Vector
BBa_25J7MVES n80_02 optimized for the use in N198 pBiT1.1-N [TK/LgBiT] Vector
BBa_25AVIIKW Rank_15 optimized for the use in N196 pBiT1.1-C [TK/LgBiT] Vector
BBa_25SYRBHM Rank_15 optimized for the use in N198 pBiT1.1-N [TK/LgBiT] Vector
All these constructs encode potential in silico designed progerin interactors, specifically optimized for compatibility with both the N196 pBiT1.1-C [TK/LgBiT] and N198 pBiT1.1-N [TK/LgBiT] NanoBiT expression vectors. This dual compatibility allows flexible cloning in either the N- or C-terminal configuration of the Large BiT fragment, enabling comparative testing of binding efficiency and orientation effects.
These interactors are designed to evaluate their capacity to associate with progerin expressed from the N199 pBiT2.1-N [TK/SmBiT] vector within the NanoBiT Protein–Protein Interaction System. When co-expressed, successful interaction between progerin and its interactor restores NanoLuc luciferase activity, generating a quantifiable luminescent signal directly proportional to the strength of their binding.
To ensure synthesis reliability and proper cloning performance, additional bases were included at the 5′ and 3′ ends of each coding sequence in accordance with TWIST Bioscience synthesis specifications. For NanoBiT assay preparation, each construct can be digested with XhoI and EcoRI, and the resulting fragment ligated into the corresponding NanoBiT vector linearized with the same restriction enzymes.
Altogether, these optimized constructs provide the iGEM community with ready-to-use tools for quantitative analysis of progerin–interactor binding in mammalian cells, streamlining the transition from in silico prediction to live-cell interaction validation.
- [1] Zhang, N., Hu, Q., Sui, T., Fu, L., Zhang, X., Wang, Y., Zhu, X., Huang, B., Lu, J., Li, Z., & Zhang, Y. (2023). Unique progerin C-terminal peptide ameliorates Hutchinson-Gilford progeria syndrome phenotype by rescuing BUBR1. Nature aging, 3(2), 185–201. https://doi.org/10.1038/s43587-023-00361-w
- [2] Dittmer, T. A., & Misteli, T. (2011). The lamin protein family. Genome biology, 12(5), 222. https://doi.org/10.1186/gb-2011-12-5-222