yeast Tunable Expression Systems Toolkit (yTEST)
(Kit #
1000000266
)
Depositing Lab: Jeff Hasty
The yeast Tunable Expression Systems Toolkit (yTEST) contains a library of MoClo-compatible parts and plasmids for assembling five distinct small-molecule inducible promoter systems in Saccharomyces cerevisiae. Users can select from a variety of highly characterized inducible activation systems responsive to doxycycline (Dox), abscisic acid (ABA), danoprevir (DNV), 1-naphthaleneacetic acid (NAA), or 5-phenyl-indole-3-acetic acid (5-Ph-IAA), enabling highly versatile and tunable control of gene expression.
This kit will be sent as bacterial glycerol stocks in 96-well plate format.
Original Publication
A Standardized Set of MoClo-Compatible Inducible Promoter Systems for Tunable Gene Expression in Yeast. O'Laughlin R, Tran Q, Lezia A, Ngamkanjanarat W, Emmanuele P, Hao N, Hasty J. ACS Synth Biol. 2024 Jan 19;13(1):85-102. doi: 10.1021/acssynbio.3c00184. PubMed (Link opens in a new window) Article (Link opens in a new window)
Description
The yeast Tunable Expression Systems Toolkit (yTEST) was developed to provide researchers with a broader collection of small-molecule inducible promoter systems for use in S. cerevisiae. This MoClo-compatible toolkit includes 25 part plasmids and over 30 cassette plasmids for designing, constructing, and utilizing five distinct classes of inducible promoter systems regulated by doxycycline (Dox), abscisic acid (ABA), danoprevir (DNV), 1-naphthaleneacetic acid (NAA), or 5-phenyl-indole-3-acetic acid (5-Ph-IAA). For each system, we built and extensively characterized multiple variants, each with distinct induction properties, giving researchers the flexibility to choose the most appropriate system for their experimental needs.
The Dox-inducible systems feature three rtTA variants (rtTA3, rtTA-v10, and rtTA-v16) whose properties can be further tuned by selecting different upstream promoters. The ABA-inducible systems use ABI and PYL1 for chemically induced proximity (CIP) and include variants with three distinct activation domains. The DNV-inducible systems comprise six variants based on the viral NS3 protein, with the NS3a-DNCR2 CIP pair containing a linker sequence providing the strongest induction. The NAA-inducible system is based on TIR1-U1 and AID∆34 CIP. TIR1-U1 includes three mutations that block its interaction with the SCF (Skp1, Cullin, and an F-box protein) complex, converting a system typically used for protein degradation into an inducible activation system. The 5-Ph-IAA-inducible system uses a modified version of TIR1-U1, called TIR1-U2, that enables 5-Ph-IAA, instead of NAA, to induce binding with AID∆34. Finally, in addition to part and cassette plasmids for each inducible promoter system variant described above, yTEST includes four multiple cloning site (MCS) plasmids that allow genes of interest to be placed under the control of a Dox-, ABA-, or DNV-inducible promoter using simple restriction enzyme cloning methods.
Figure 1: Summary of yeast Tunable Expression Systems Toolkit (yTEST) contents and advantages.
Additional Information
The yTEST kit is meant to be used alongside and in conjunction with other yeast MoClo toolkits. For assembly and characterization of inducible promoters in yTEST, parts from the MoClo-YTK kit and the Yeast GPCR-sensor Toolkit were used. We recommend yTEST users acquire these kits as well in order to get full and complete use from yTEST.
How to Cite this Kit
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which they were created, and include Addgene in the Materials and Methods of your future publications.
For your Materials and Methods section:
"The yeast Tunable Expression Systems Toolkit (yTEST) was a gift from Jeff Hasty (Addgene kit #1000000266)."
For your Reference section:
A Standardized Set of MoClo-Compatible Inducible Promoter Systems for Tunable Gene Expression in Yeast. O'Laughlin R, Tran Q, Lezia A, Ngamkanjanarat W, Emmanuele P, Hao N, Hasty J. ACS Synth Biol. 2024 Jan 19;13(1):85-102. doi: 10.1021/acssynbio.3c00184. PubMed (Link opens in a new window) Article (Link opens in a new window)
yeast Tunable Expression Systems Toolkit (yTEST) - #1000000266
- Resistance Color Key
Each circle corresponds to a specific antibiotic resistance in the kit plate map wells.
- Inventory
Searchable and sortable table of all plasmids in kit. The Well column lists the plasmid well location in its plate. The Plasmid column links to a plasmid's individual web page.
- Kit Plate Map
96-well plate map for plasmid layout. Hovering over a well reveals the plasmid name, while clicking on a well opens the plasmid page.
Resistance Color Key
| Chloramphenicol | |
| Ampicillin | |
| Kanamycin |
Inventory
| Well | Plasmid | Resistance |
|---|---|---|
| A / 1 | pRO129 | Chloramphenicol |
| A / 2 | pRO112 | Chloramphenicol |
| A / 3 | pRO217 | Chloramphenicol |
| A / 4 | pRO232 | Chloramphenicol |
| A / 5 | pRO174 | Chloramphenicol |
| A / 6 | pRO175 | Chloramphenicol |
| A / 7 | pRO177 | Chloramphenicol |
| A / 8 | pRO286 | Chloramphenicol |
| A / 9 | pRO271 | Chloramphenicol |
| A / 10 | pWN022 | Chloramphenicol |
| A / 11 | pWN050 | Chloramphenicol |
| A / 12 | pWN083 | Chloramphenicol |
| B / 1 | pAL009 | Chloramphenicol |
| B / 2 | pWN018 | Chloramphenicol |
| B / 3 | pWN079 | Chloramphenicol |
| B / 4 | pWN080 | Chloramphenicol |
| B / 5 | pWN043 | Chloramphenicol |
| B / 6 | pWN062 | Chloramphenicol |
| B / 7 | pRO117 | Chloramphenicol |
| B / 8 | pRO176 | Chloramphenicol |
| B / 9 | pRO275 | Chloramphenicol |
| B / 10 | pRO276 | Chloramphenicol |
| B / 11 | pRO288 | Chloramphenicol |
| B / 12 | pWN102 | Chloramphenicol |
| C / 1 | pAL002 | Chloramphenicol |
| C / 2 | pRO211 | Ampicillin |
| C / 3 | pRO261 | Ampicillin |
| C / 4 | pRO262 | Ampicillin |
| C / 5 | pRO263 | Ampicillin |
| C / 6 | pRO264 | Ampicillin |
| C / 7 | pRO227 | Ampicillin |
| C / 8 | pRO229 | Ampicillin |
| C / 9 | pRO230 | Ampicillin |
| C / 10 | pRO231 | Ampicillin |
| C / 11 | pRO246 | Ampicillin |
| C / 12 | pRO247 | Ampicillin |
| D / 1 | pRO253 | Ampicillin |
| D / 2 | pRO254 | Ampicillin |
| D / 3 | pRO204 | Ampicillin |
| D / 4 | pRO241 | Ampicillin |
| D / 5 | pRO279 | Ampicillin |
| D / 6 | pRO280 | Ampicillin |
| D / 7 | pRO236 | Ampicillin |
| D / 8 | pAL001 | Ampicillin |
| D / 9 | pAL008 | Ampicillin |
| D / 10 | pWN020 | Ampicillin |
| D / 11 | pWN021 | Ampicillin |
| D / 12 | pWN057 | Ampicillin |
| E / 1 | pWN059 | Ampicillin |
| E / 2 | pWN061 | Ampicillin |
| E / 3 | pWN069 | Ampicillin |
| E / 4 | pWN075 | Ampicillin |
| E / 5 | pWN076 | Ampicillin |
| E / 6 | pWN081 | Ampicillin |
| E / 7 | pWN086 | Ampicillin |
| E / 8 | pWN103 | Ampicillin |
| E / 9 | pWN104 | Ampicillin |
| E / 10 | pWN113 | Ampicillin |
| E / 11 | pAL004 | Kanamycin |
| E / 12 | pAL003 | Kanamycin |
| F / 1 | pAL005 | Kanamycin |
| F / 2 | pAL007 | Kanamycin |