T2A Split/Link TurboID Kit
(Kit #
1000000249
)
Depositing Lab: Sven Eyckerman
The T2A Split/Link TurboID Kit can be used to assemble mammalian expression vectors for proximity labeling experiments. These vectors can be assembled in PiggyBac or lentiviral backbones, enabling researchers to choose their preferred cellular engineering method with the same basic cloning modules.
This kit will be sent as bacterial glycerol stocks in 96-well plate format.
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.
- Figure 1: T2A Split/Link TurboID Kit system. Plasmids are arranged in a hierarchy depending on their content. Level-0 are basic parts flanked by inward-cutting BsaI restriction sites. BsaI-based Golden Gate cloning of six Level-0 plasmids (with each position present) and an A-G backbone causes unidirectional assembly of each of these parts in the backbone plasmid. These Level-1 plasmids express transcriptional units, and can similarly be assembled in a higher-order Level-2 plasmid containing two Level-1 transcriptional units by BsmBI-based Golden Gate assembly. Image used with permission from Delhaye et al., bioRxiv. 2023.
![A two part schematic of the assembly vectors used in the T2A split/link TurboID kit. Part A is a table listing the makeup of the Level-0 plasmids. The column headings are Overhang, 5’ Overhang sequence, Module content, 3’ Overhang sequence, and Overhang. Row one is A, ACCT-, Promoter, -AACA, B. Row two is B [will not repeat overhang sequence], Kozak + ATG + N-terminal tag, -GGCT, C. Row three is C, POI, -TCAG, D. Row four is D, Linker, -CTGC, E. Row five is E, C-terminal tag + STOP, -ACTA, F. The final row is F-, Transcriptional terminator or post-transcriptional element, -GTAT, G. The Level-0 modules (all positions) and the Level-1 backbone, in a one-pot BsaI assembly, form the Level-1 plasmid containing the six modules. Part B contains a table with the same headings. Row one is W, TGCC-, Transcriptional unit (TU)1 (e.g. POI-TurboID), -GCAA, X. The final row is X, Transcriptional unit 2 (e.g. selection marker), -CAGA, Z. The Level-1 TUs and the Level-2 backbone, in a one-pot BsmBI assembly, form the Level 2 plasmid containing the two modules.](https://media.addgene.org/data/easy-thumbnails/filer_public/cms/filer_public/5b/49/5b4959c1-1f17-4ccd-99da-d003e43a4096/eyckerman_figure_2.png__900x751_subsampling-2_upscale.png)
- Figure 2: Assembly vectors used in the T2A Split/Link TurboID Kit. (A) Overhangs and contents of the different positions within the Level-0 hierarchy. (B) Overhangs and contents of the different positions within the Level-1 hierarchy.
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).
T2A Split/Link TurboID Kit - #1000000249
- 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
| Ampicillin | |
| Kanamycin | |
| Chloramphenicol and Ampicillin | |
| Chloramphenicol and Kanamycin |