Abstract
Sophisticated genetic engineering tasks such as protein domain grafting and multi-gene fusions are hampered by the lack of suitable vector backbones. In particular, many restriction sites are in the backbone outside the polylinker region (multiple cloning site; MCS) and thus unavailable for use, and the overall length of a plasmid correlates with poorer ligation efficiency. To address this need, we describe the design and validation of a collection of six minimal integrating or centromeric shuttle vectors for Saccharomyces cerevisiae, a widely used model organism in synthetic biology. We constructed the plasmids using de novo gene synthesis and consisting only of a yeast selection marker (HIS3, LEU2, TRP1, URA3, KanMX, or natMX6), a bacterial selection marker (ampicillin resistance), an origin of replication, and the MCS flanked by M13 forward and reverse sequences. We used truncated variants of these elements where available and eliminated all other sequences typically found in plasmids. The MCS consists of ten unique restriction sites. To our knowledge, at sizes ranging from ~2.6 to 3.5 kb, these are the smallest shuttle vectors described for yeast. Further, we removed common restriction sites in the open reading frames and terminators, freeing up ~30 cut sites in each plasmid. We named our pLS series in accordance with the well-known pRS vectors, which are on average 63% larger: pLS400, pLS410 (KanMX); pLS403, pLS413 (HIS3); pLS404, pLS414 (TRP1); pLS405, pLS415 (LEU2); pLS406, pLS416 (URA3); and pLS408, pLS418 (natMX6). This resource substantially simplifies advanced synthetic biology engineering in S. cerevisiae.
Keywords: Saccharomyces cerevisiae, minimal shuttle vector, multi-gene fusion, domain insertion, domain grafting, restriction sites, de novo synthesis
Graphical Abstract
Graphical Abstract.
Introduction
Plasmids are critical tools for molecular and synthetic biology [1–3]. Yeast shuttle vectors generally range from 4 to 10 kb in size [4–15] and harbour superfluous elements and restriction sites outside the multiple cloning site (MCS) that complicate experiments. Larger plasmids tend to be more difficult to manipulate, e.g. for cloning multiple genes into the same backbone [16] or for site-directed mutagenesis. Certain applications, e.g. DNA break repair assays, require avoiding the same DNA sequences around engineered genomic modifications [17]. Critically, restriction sites that are present in the backbone cannot be used to modify insert DNA. For example, for grafting a protein domain into another, e.g. for light or chemical control [18, 19], dozens of insertion sites typically need to be tested in the target protein [20], requiring ideally no restriction sites to be used up in the vector backbone so they can be introduced by silent mutations in the target gene. Similarly, the design of scarless gene editing strategies such as pop-in-pop-out becomes more challenging the fewer restriction sites are available [21].
To address these limitations, there has been a growing interest in developing size-reduced plasmids. This minimalistic philosophy aims to create compact, precisely engineered vectors that only contain essential elements. By reducing plasmid size and eliminating unnecessary sequences, minimal plasmids have been shown to enhance cloning efficiency, gene transfer, and genetic manipulation [22–24]. Size-reduced cloning vectors have been previously constructed through the progressive miniaturization of existing commercial plasmids, but an aggressive miniaturization strategy has not been applied to shuttle vectors for Saccharomyces cerevisiae [25–28].
In the present study, we leveraged de novo gene synthesis to create minimal shuttle vectors for S. cerevisiae. The pLS plasmids only contain a yeast selection marker (HIS3, LEU2, TRP1, URA3, KanMX, or natMX6), the bacterial selection marker AmpR conferring ampicillin resistance, an origin of replication (ORI), and a MCS flanked by M13 forward and reverse sequencing regions (Fig. 1A–F). We used truncated variants of these elements where available and avoided unnecessary DNA sequences. Furthermore, we recoded the yeast selection markers and AmpR, and we mutated terminators to enhance the availability of restriction sites for insert manipulation. We introduced, on average, 68 mutations per plasmid, removing most cut sites in the vector backbone while preserving the original amino acid sequences. Recoding the selectable markers recovered ~30 restriction sites recognized by widely used Type IIP and IIS restriction endonucleases in each plasmid. Furthermore, we developed and functionally validated both integrating and centromeric versions of each pLS minimal shuttle vector.
Figure 1.
Maps of the integrating pLS minimal shuttle vectors.
We named the pLS plasmids in accordance with the well-known pRS400 series [13]: pLS400 and pLS410 (KanMX), pLS403 and pLS413 (HIS3), pLS404 and pLS414 (TRP1), pLS405 and pLS415 (LEU2), pLS406 and pLS416 (URA3), and pLS408 and pLS418 (natMX6) [29–31]. To our knowledge, the pLS plasmids are the smallest shuttle vectors for yeast, ranging from ~2.6 to 3.5 kb in size. We have deposited all plasmids with Addgene. We anticipate that this resource will substantially simplify previously challenging genetic engineering tasks in S. cerevisiae.
Materials and methods
Plasmid construction
The DNA sequence of pLS405 was synthesized by GenScript. After validating the functionality of pLS405, we constructed the other minimal shuttle vectors pLS400, pLS403, pLS404, pLS406, and pLS408 through Gibson Assembly (New England Biolabs, USA). The common plasmid backbone was amplified from pLS405, while the recoded yeast selection marker sequences (KanMX, HIS3, TRP1, URA3, and natMX6) were synthesized by Twist Bioscience and subsequently cloned into the amplified backbone. For the generation of the centromeric pLS plasmids, the centromere/autonomously replicating sequence (CEN/ARS) (504 bp) was amplified from pRS416. Simultaneously, the backbone of the pLS vectors was amplified using divergent primers located between the ORI and M13 reverse. The primers used to amplify the CEN/ARS region included 20 bp tails with homology to the region of the pLS backbone that was being amplified. These products were then assembled using Gibson Assembly, resulting in the development of the centromeric versions of the pLS minimal shuttle vectors. DNA digestions were performed using restriction endonucleases (New England Biolabs, USA). All PCRs were performed using high-fidelity Phusion Polymerase (New England Biolabs, USA). The DNA sequences of the constructs were verified by Sanger sequencing (Microsynth AG, Switzerland). The DNA sequences of the minimal shuttle vectors pLS400, pLS403, pLS404, pLS405, pLS406, and pLS408 are provided in Supplementary Notes 1–6. The DNA sequences of the integrative minimal shuttle vectors pLS400, pLS403, pLS404, pLS405, pLS406, and pLS408 containing sequences from the non-essential gene YCR051W for single-copy genomic integration (Supplementary Fig. S1, Supplementary Table S1) are provided in Supplementary Notes 7–12. The DNA sequences of the centromeric pLS minimal shuttle vectors pLS410, pLS413, pLS414, pLS415, pLS416, and pLS418 are provided in Supplementary Notes 13–18.
Bacterial strains and media
Plasmids were propagated in XL10 Escherichia coli cells cultured in LB medium, which was supplemented with ampicillin to select for plasmid-containing bacteria.
Yeast strains and media
The experimental validation of the minimal shuttle vectors was conducted using the wild-type haploid W303 yeast strain background (MATa ade2-1 leu2-3 ura3-1 trp1-1 his3-11,15 can1-100). Yeast transformations were performed using the LiAc/DNA carrier/PEG (polyethylene glycol) protocol [32]. Transformed strains were selected on drop-out plates lacking histidine, tryptophan, leucine, or uracil, or on plates supplemented with antibiotics G418 or ClonNat for the selection of KanMX or natMX6 markers, respectively. Synthetic complete (SC) media without methionine (–Met), supplemented with 2% (w/v) glucose (D) and 0.1% (w/v) monosodium glutamate (instead of ammonium sulphate), was used for culturing [33]. For preparing solid SC plates, 2% agar and 0.17% yeast nitrogen base were added to the medium.
Experimental validation
To validate the functionality of the recoded yeast selection markers and the usability of the minimal shuttle vectors for genetic manipulation of S. cerevisiae, we inserted sequences from the non-essential gene YCR051W into the MCS of pLS400, pLS403, pLS404, pLS405, pLS406, and pLS408, which allowed single-copy integration (Supplementary Fig. S1, Supplementary Table S1). Cloning was performed using the restriction endonucleases BssHII and SalI (New England Biolabs, USA), while ligation was performed with T4 ligase (New England Biolabs, USA). Prior to yeast transformation, plasmid linearization was achieved using the restriction endonuclease AfeI, which is present within the YCR051W sequence. For yeast transformation, 1 μg of DNA was used, either in linearized form for integrative plasmids or circular form for centromeric plasmids. The concentration of G418 used for pLS400 and pLS410 selection is 150 μg/ml in Fig. 2 and 100 μg/ml in the transformation efficiency comparisons with the pRS series (Figs 3 and 4). The concentration of ClonNat used for pLS408 and pLS418 selection is 25 μg/ml for both the transformation efficiency comparisons (Figs 3 and 4) and Fig. 2. The ability of the pLS minimal shuttle vectors to rescue the auxotrophic mutations in the haploid W303 yeast strain background confirmed their functionality.
Figure 2.
Functional validation of the integrating pLS minimal shuttle vectors in Saccharomyces cerevisiae.
Figure 3.
Integration efficiency of the integrating pLS minimal shuttle vectors compared to the pRS series.
Figure 4.
Transformation efficiency of the centromeric pLS minimal shuttle vectors compared to pRS416.
Results
Components of minimal shuttle vectors
We took the DNA sequences for the prototrophic biosynthetic markers HIS3, TRP1, LEU2, and URA3 from the S228C laboratory strain sequence published in the Saccharomyces Genome Database [34–37]. For the drug resistance markers KanMX and natMX6, which confer resistance to kanamycin and nourseothricin, respectively, the DNA sequences were obtained from the SnapGene website [38, 39]. For KanMX and natMX6, we adopted the commonly used promoter and terminator of the TEF1 gene from Ashbya gossypii. These yeast selection markers, ranging from 1051 to 1905 bp, represent the largest elements in our plasmids.
For the bacterial selection marker AmpR, we used the sequence in the minimal cloning vector pUCmu, where a deletion enabled the resistance marker to utilize a shortened terminator sequence, reducing the size from 1095 to 947 bp [27]. Next, we incorporated the ORI, which is essential for plasmid propagation in bacterial hosts. The ORI sequence was derived from the minimal cloning vector pUCmini, where a random deletion mutation of the pUC variant of the pMB1 ORI was identified to reduce the size of the plasmid, resulting in an ORI of just 589 bp compared to 750 bp [27, 40].
The MCS spans 56 bp and includes 10 unique restriction sites for the widely used 6-cutter enzymes BssHII, Eco53kI/SacI, KpnI/Acc65I, SmaI/XmaI, BamHI, XbaI, SalI, PstI (except in pLS400, pLS406, and pLS408 due to its presence in the promoter of the yeast selection marker), SphI, and AvrII. This MCS is similar to that of pICOz [27]. To facilitate seamless sequencing and verification of insert integration, we positioned the M13 forward and reverse sequences, each 17 bp in length, to flank the MCS.
Introduction of mutations to eliminate restriction sites outside multiple cloning site
We removed nearly all restriction enzyme recognition sites outside the MCS (Tables 1 and 2). Because our DNA sequences were synthesized de novo, we could easily introduce multiple mutations in the open reading frames (ORFs) and terminators of the selection markers AmpR, KanMX, HIS3, TRP1, LEU2, URA3, and natMX6 at the same time. The changes in the ORF DNA sequences exclusively switched synonymous codons. Hence, we removed all 6-bp and 8-bp restriction enzyme recognition sites (unless the restriction site was also present in a promoter) without altering the encoded amino acid sequences [41]. Furthermore, we introduced mutations in the gene terminators, substituting guanine and cytosine with adenine and thymine, to recover additional restriction sites within the selection markers. However, to prevent potential disruptions in gene expression, we did not modify promoters. Similarly, the ORI was left unchanged to ensure that plasmid replication in the bacterial host remained unaffected. On average, 68 mutations were introduced per minimal shuttle vector, effectively removing unnecessary restriction sites while preserving the original functionality. Since some of the recovered sites are still present in the regions of the plasmid backbone we did not change (promoters, ORI), the final number of recovered restriction sites is reduced. Information on the number of mutations introduced in each selection marker and recovered restriction sites per minimal shuttle vector is provided in Table 1. Detailed information on the restriction enzyme recognition sites recovered for each selectable marker is provided in Table 2. As shown in Fig. 1, nearly all restriction sites were removed from the ORFs and terminators of the bacterial and yeast selection markers, leaving only a few restriction sites in the promoters and ORI regions. The final plasmid sequences have been validated by whole-plasmid sequencing, and the DNA sequences of the pLS minimal shuttle vectors are provided in Supplementary Notes 1–6 and as annotated sequence maps in gbk format in Supplementary Files.
Table 1.
Overview of mutations introduced and recovered restriction sites.
| AmpR | KanMX | HIS3 | TRP1 | LEU2 | URA3 | natMX6 | |
|---|---|---|---|---|---|---|---|
| Number of mutations introduced | 36 | 31 | 28 | 20 | 43 | 34 | 37 |
| Number of recovered cut sites in gene | 33 | 17 | 24 | 15 | 27 | 26 | 29 |
| Recovered cut sites in plasmid |
pLS400
27 |
pLS403
33 |
pLS404
24 |
pLS405
31 |
pLS406
34 |
pLS408
33 |
Some recoded restriction sites remain present in the unchanged regions of the plasmid backbone, i.e. the promoters and ORI, reducing the number of overall recovered restriction sites.
Table 2.
Overview of restriction sites in genes in the pLS minimal shuttle vectors.
| AmpR | KanMX | HIS3 | TRP1 | LEU2 | URA3 | natMX6 | |
|---|---|---|---|---|---|---|---|
| Acc65I | X | X | X | ||||
| AccI | X | X | X | ||||
| AclI | X | O | O | O | O | X | O |
| AcuI | X | X | |||||
| AflII | O | O | O | O | X | O | O |
| AflIII | O | X | O | X | X | ||
| AgeI | O | O | O | O | X | O | X |
| AhdI | X | ||||||
| AleI | O | O | X | O | O | O | O |
| ApaI | O | O | O | O | O | X | O |
| ApaLI | X | ||||||
| ApoI | O | X | O | O | |||
| AseI | X | X | O | X | X | X | O |
| AsiSI | O | X | O | O | O | O | O |
| AvaI | X | X | |||||
| AvrII | X | ||||||
| BaeGI | X | ||||||
| BanI | X | X | X | X | |||
| BanII | X | X | X | X | |||
| BbsI | O | O | X | O | X | X | X |
| BclI | O | O | X | O | O | O | O |
| BfuAI | X | X | |||||
| BglI | X | O | X | O | O | O | X |
| BglII | O | O | X | X | O | O | O |
| Bme1580I | X | ||||||
| BmrI | X | O | O | X | O | O | |
| BmtI | O | O | X | O | O | O | O |
| BpmI | X | O | O | O | X | X | X |
| BpuEI | X | ||||||
| BsaAI | O | O | X | O | O | O | |
| BsaBI | O | O | X | O | O | O | O |
| BsaHI | X | O | O | O | O | X | |
| BsaI | X | O | O | O | O | X | O |
| BsaWI | X | X | |||||
| BseRI | O | O | O | X | O | ||
| BsgI | O | O | O | X | O | O | |
| BsiHKAI | X | ||||||
| BsiWI | O | O | X | O | O | O | X |
| BsmBI | O | X | O | O | O | X | O |
| BsmI | O | X | X | O | O | X | X |
| BsoBI | X | X | |||||
| Bsp1286I | X | ||||||
| BspDI | O | X | O | O | X | O | O |
| BspMI | X | X | |||||
| BspQI | O | O | O | O | O | X | O |
| BsrDI | X | O | O | O | X | X | O |
| BsrFI | X | X | O | O | O | X | |
| BsrGI | O | O | O | O | X | O | O |
| BssHII | X | X | |||||
| BssSI | X | ||||||
| BstAPI | O | O | O | X | O | O | X |
| BstBI | O | O | O | X | X | O | |
| BstEII | O | O | O | O | O | X | |
| BstXI | O | O | X | X | X | O | O |
| BstYI | X | ||||||
| BstZ17I | O | O | X | O | X | O | |
| Bsu36I | O | O | O | X | O | O | O |
| BtgI | O | O | O | X | X | ||
| BtsI | X | X | O | O | O | O | O |
| ClaI | O | X | O | O | X | O | O |
| DraI | X | O | O | O | |||
| EaeI | X | ||||||
| EciI | X | X | |||||
| EcoO109I | O | O | X | O | X | X | |
| EcoNI | O | X | O | O | X | O | O |
| EcoP15I | X | ||||||
| EcoRI | O | O | O | X | O | O | |
| EcoRV | O | O | O | X | X | X | O |
| Esp3I | O | X | O | O | O | X | O |
| FspI | X | O | O | O | O | ||
| HincII | X | X | |||||
| HindIII | O | X | X | X | O | X | O |
| KasI | O | O | O | O | O | O | X |
| KpnI | X | X | X | ||||
| MfeI | O | O | O | X | X | O | O |
| MflI | X | ||||||
| MreI | O | O | O | O | O | O | X |
| MscI | O | O | X | O | O | O | O |
| MspA1I | X | ||||||
| NaeI | O | O | O | O | O | O | X |
| NarI | O | O | O | O | O | O | X |
| NcoI | O | O | O | O | X | ||
| NdeI | O | O | X | O | O | O | |
| NgoMIV | O | O | O | O | O | O | X |
| NheI | O | O | X | O | O | O | O |
| NmeAIII | X | O | O | O | O | O | X |
| NruI | O | X | O | O | O | O | X |
| NsiI | O | X | X | O | O | X | O |
| NspI | X | X | X | X | |||
| PciI | O | O | X | O | X | X | O |
| PflFI | O | O | O | O | O | O | X |
| PflMI | O | X | O | O | O | O | |
| PfoI | O | O | O | O | O | O | X |
| PluTI | O | O | O | O | O | O | X |
| PpuMI | O | O | O | O | X | X | |
| PshAI | O | O | O | O | O | O | X |
| PsiI | O | O | O | O | X | O | |
| PspOMI | O | O | O | O | O | X | O |
| PstI | X | X | |||||
| PvuI | X | X | O | O | O | O | O |
| SapI | O | O | O | O | O | X | O |
| SbfI | X | ||||||
| ScaI | X | X | O | O | O | X | X |
| SfcI | X | X | |||||
| SfiI | O | O | X | O | O | O | O |
| SfoI | O | O | O | O | O | O | X |
| SmaI | X | X | |||||
| SmlI | X | ||||||
| SphI | X | ||||||
| StuI | O | O | O | X | O | X | O |
| StyI | X | X | X | ||||
| TaqII | X | X | X | O | O | O | |
| TatI | X | X | X | X | X | ||
| TsoI | X | X | O | O | O | O | |
| TspMI | X | X | |||||
| Tth111I | O | O | O | O | O | O | X |
| XbaI | X | ||||||
| XcmI | O | O | O | O | X | X | X |
| XmaI | X | X | |||||
| XmnI | X | O | O | O | O |
X indicates restriction sites that have been recovered; O indicates restriction sites that were already absent. The table includes restriction enzymes with degenerate recognition sequences, which may result in redundancies among the recovered cut sites recognized by multiple enzymes.
Functional validation of the pLS minimal shuttle vectors in S. cerevisiae
We validated the functionality of the pLS minimal shuttle vectors for genetic manipulation in both E. coli and S. cerevisiae. The bacterial selection marker AmpR and the compact ORI were confirmed to be functional, as evidenced by the successful propagation of all plasmids in XL10 E. coli cells. We observed robust bacterial growth with transformation plates exhibiting a high density of colonies and efficient DNA recovery after miniprep purification (data not shown). To validate the functionality of the recoded yeast selection markers and the utility of the minimal shuttle vectors for genetic manipulation of S. cerevisiae, we introduced sequences from the non-essential gene YCR051W into the MCS of pLS400, pLS403, pLS404, pLS405, pLS406, and pLS408 for single-copy genomic integration (Supplementary Fig. S1, Supplementary Table S1). The ability of the pLS minimal shuttle vectors to rescue auxotrophic mutations in the haploid W303 yeast strain background confirmed their functionality (Fig. 2). Compared to their respective negative controls, the pLS vectors successfully complemented the auxotrophic deficiencies, as demonstrated by high-density colony growth on drop-out plates, validating the functionality of the recoded yeast selection markers. Transformed strains were screened for single-copy integration using polymerase chain reaction (PCR) with primer sets designed to differentiate between single and multiple construct copies in the genome (Supplementary Fig. S1, Supplementary Table S1, Supplementary Note 19). The DNA sequences of the single-copy integration fragment YCR051W and the primers used for its amplification are provided in Supplementary Notes 20 and 21. To assess whether the reduced size of the pLS vectors confers an advantage for yeast transformation, we compared their integration efficiencies to those of the widely used pRS vectors. The results indicate that the pLS minimal shuttle vectors exhibit higher integration efficiencies per microgram of plasmid DNA across all selection markers (Fig. 3). To evaluate the selection capacity of the pLS400 and pLS408 minimal shuttle vectors, we performed a dose–response assay by transforming yeast with linearized pLS400 and pLS408 at increasing concentrations of G418 (0–600 μg/ml) and ClonNat (0–300 μg/ml), respectively. As expected, the number of colony-forming units (CFUs) decreased with higher G418 and ClonNat concentrations, reflecting the increasing stringency of selection (Supplementary Fig. S2). This analysis provides insight into the range of G418 and ClonNat concentrations suitable for selecting pLS400 and pLS408 transformants, respectively. Collectively, these results demonstrate the successful design and functional validation of the pLS minimal shuttle vectors, showcasing their potential to aid genetic engineering in yeast.
Development and functional validation of centromeric pLS minimal shuttle vectors
To expand the versatility of the pLS series, we developed CEN/ARS-based versions of each pLS minimal shuttle vector. The incorporation of the CEN/ARS sequence into the pLS vector backbone allows for stable maintenance and replication in yeast cells without integration into the genome. We named the centromeric pLS plasmids in accordance with the well-known pRS400 series: pLS410 (KanMX), pLS413 (HIS3), pLS414 (TRP1), pLS415 (LEU2), pLS416 (URA3), and pLS418 (natMX6). The DNA sequence of the CEN/ARS sequence [13] and the primers used for its amplification and cloning are provided in Supplementary Notes 22 and 23. The sequence contains 8 restriction sites recognized by Type IIP and IIS restriction endonucleases, which are thus no longer available for gene engineering. We validated the functionality of the centromeric pLS minimal shuttle vectors for genetic manipulation in both E. coli and S. cerevisiae. The results demonstrate that the CEN/ARS-containing pLS vectors exhibit high transformation efficiency in S. cerevisiae, with successful propagation and maintenance of the plasmids (Fig. 4). The final plasmid sequences have been validated through whole-plasmid sequencing, and the DNA sequences of the centromeric pLS minimal shuttle vectors are provided in Supplementary Notes 13–18 and as annotated sequence maps in gbk format in Supplementary Files.
Discussion
The design of the pLS series simplifies a range of potential applications. For example, the small size of the vectors allows the entire backbone to be amplified more easily by PCR. This simplifies mutagenesis, allowing specific mutations to be introduced into target inserts using divergent primers. (The small size facilitated the construction of the other minimal shuttle vectors starting with pLS405, as described in Materials and methods).
Moreover, the lack of restriction sites in the pLS backbone enhances the vectors’ utility for genetic manipulation of inserts. For instance, it allows the insertion of regulatory domains at various unique restriction sites in the target gene. Examples of such applications are in the work by Azoitei et al. and Reynolds et al. [42, 43], who screened for grafting sites within the target to identify clones with optimal functional activity. Lee et al. [20] constructed a light-sensitive TurboID protein by testing 31 different sites for inserting AsLOV2. The construction of complex genetic backgrounds in budding yeast [44, 45] is generally simplified by greater flexibility in plasmid design.
Depleting this large number of restriction sites in the pLS plasmids was only practical with de novo DNA synthesis. Further optimization of the pLS vectors may be possible by replacing the selection markers with shorter alternatives, performing deletion engineering of the promoters and terminators, or by utilizing size-reduced origins of replication. For example, the minimal ORI from the low-copy plasmid pSC101 is only 220 bp in length but requires an additional initiator protein [46]. With regard to the selection markers, replacing AmpR with the dfrB10 gene, which is only 237 bp long [25], or nano-antibiotics [47, 48] may be considered.
In conclusion, our study introduces a novel set of minimal shuttle vectors tailored for yeast applications, harbouring the marker genes KanMX (pLS400 and pLS410), HIS3 (pLS403 and pLS413), TRP1 (pLS404 and pLS414), LEU2 (pLS405 and pLS415), URA3 (pLS406 and pLS416), and natMX6 (pLS408 and pLS418). By removing non-essential elements and strategically eliminating restriction sites, these vectors provide valuable tools for synthetic biology in S. cerevisiae.
Supplementary Material
Contributor Information
Lorenzo Scutteri, Laboratory of the Physics of Biological Systems, École polytechnique fédérale de Lausanne (EPFL), Lausanne, CH-1015, Switzerland; Interfaculty Institute of Bioengineering, École polytechnique fédérale de Lausanne (EPFL), Lausanne, CH-1015, Switzerland; Ludwig Institute for Cancer Research, Agora Research Center, Lausanne, CH-1005, Switzerland.
Patrick Barth, Interfaculty Institute of Bioengineering, École polytechnique fédérale de Lausanne (EPFL), Lausanne, CH-1015, Switzerland; Ludwig Institute for Cancer Research, Agora Research Center, Lausanne, CH-1005, Switzerland.
Sahand Jamal Rahi, Laboratory of the Physics of Biological Systems, École polytechnique fédérale de Lausanne (EPFL), Lausanne, CH-1015, Switzerland.
Author contributions
L.S. constructed the plasmids, created the bacterial and yeast strains, performed the functional validation, and analysed the data. L.S. and S.R. conceptualized the project and wrote the manuscript. S.R. and P.B. supervised the project and acquired funding.
Conflict of interest
No potential conflict of interest was reported by the authors.
Funding
L.S. is supported by the EPFLglobaLeaders doctoral fellowship; an EPFL Science Seed Fund awarded to P.B. and S.J.R.; and SNSF grants CRSK-3_190526, 310030_204938, and CRSK-3_221036 awarded to S.J.R. This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska–Curie grant agreement No 945363.
Data availability
The data generated in the paper are available at SYNBIO online.
All plasmids and maps have been deposited with Addgene.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The data generated in the paper are available at SYNBIO online.
All plasmids and maps have been deposited with Addgene.