T2A Split/Link TurboID Kit
(Kit # 1000000249 )

Original Publication

Leveraging a self-cleaving peptide for tailored control in proximity labeling proteomics. Delhaye L, Moschonas GD, Fijalkowska D, Verhee A, De Sutter D, Van de Steene T, De Meyer M, Grzesik H, Van Moortel L, De Bosscher K, Jacobs T, Eyckerman S. Cell Reports Methods. 2024 Jul 9, 4, 100818. doi: 10.1016/j.crmeth.2024.100818. PubMed (Link opens in a new window) Article (Link opens in a new window).

Description

The T2A Split/Link TurboID Kit is a sequential Golden Gate assembly method originally based on the GreenGate and Golden GATEway assembly kits. The T2A Split/Link TurboID Kit system was adjusted to contain two sequential type IIS restriction reactions.

The T2A Split/Link TurboID Kit contains a vector hierarchy of three levels (Figure 1). First, six modules (Level-0) are cloned in a one-pot BsaI-based Golden Gate assembly (Figure 2) into transcriptional units (TU; Level-1). Depending on the insert module, Level-0 modules are flanked by BsaI recognition sites that generate unique overhangs (A to G) to allow unidirectional assembly of the modules in Level-1 vectors. TUs can be used as such for transient mammalian expression experiments or can be assembled with another TU in Level-2 vectors in a one-pot BsmBI-based Golden Gate assembly. Similar to the Level-0 vectors, Level-1 vectors are flanked by BsmBI-recognition sites that generate unique overhangs (W, X, and Z). Level-2 vectors expressing two TUs can be generated with a PiggyBac or lentiviral backbone, enabling both cellular engineering methods with the same basic cloning modules. For ease of use, a number of mammalian antibiotic selection markers (puromycin, blasticidin, neomycin, and hygromycin resistance genes) are already provided as Level-1 TUs for both PiggyBac and lentiviral vectors.

Level-0 (provided by the GreenGate kit), Level-1, and Level-2 backbones encode a ccdB toxin gene between the BsaI or BsmBI sites driven by a constitutive lacUV5 bacterial promoter to provide negative selection of backbone transformants. Level-0 and Level-2 vectors confer carbenicillin or ampicillin resistance, while Level-1 vectors confer kanamycin resistance to prevent selection of transformants containing lower-ordered plasmids within the hierarchy.

Note for plasmids containing the TRE promoter: The TRE promoter requires cells engineered with a doxycycline-inducible transcriptional transactivator (rtTA) or silencer (tTS) for dose-responsive expression of the downstream ORF.

Please visit https://doi.org/10.1101/2023.11.03.565112 (Link opens in a new window) for bioRxiv preprint.

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 T2A Split/Link TurboID Kit was a gift from Sven Eyckerman (Addgene kit #1000000249).”

For your Reference section:

Leveraging a self-cleaving peptide for tailored control in proximity labeling proteomics. Delhaye L, Moschonas GD, Fijalkowska D, Verhee A, De Sutter D, Van de Steene T, De Meyer M, Grzesik H, Van Moortel L, De Bosscher K, Jacobs T, Eyckerman S. Cell Reports Methods. 2024 Jul 9, 4, 100818. doi: 10.1016/j.crmeth.2024.100818. PubMed (Link opens in a new window) Article (Link opens in a new window).