Skip to main content
This website uses cookies to ensure you get the best experience. By continuing to use this site, you agree to the use of cookies.

Please note: Your browser does not support the features used on Addgene's website. You may not be able to create an account or request plasmids through this website until you upgrade your browser. Learn more

Please note: Your browser does not fully support some of the features used on Addgene's website. If you run into any problems registering, depositing, or ordering please contact us at [email protected] Learn more

Plasmid kit icon

UbiGate Collection
(Kit # 1000000144, 1000000145 )

Depositing Lab: Marco Trujillo

The UbiGate collection enables the straightforward reconstitution of the ubiquitination cascades of different organisms in Escherichia coli by 'Golden Gate' cloning. The UbiGate collection contains two plasmid kits:

Kit #1000000144

The UbiGate Components (set1) contains plasmids that allow generation of polycistronic expression cassettes for coexpression in bacteria of up to six ORFs simultaneously (contains ubiquitin, the E1 UBA1, a complete collection of E2s, a selection of E3s and their cognate substrates, to reconstitute ubiquitination assays).

Kit #1000000145

The UbiGate E2 activity screen (set2) contains constructs for E3 autoubiquitination assay (includes expression cassettes containing ubiquitin, the E1 UBA1 in combination with all Arabidopsis thaliana E2's).


Both kits are shipped as bacterial glycerol stocks in a 96-well plate format.

Ordering

Item Catalog # Description Quantity Price (USD)
Kit 1000000144 UbiGate Components (set1) 1 $350 Add to Cart
Kit 1000000145 UbiGate E2 activity screen (set2) 1 $350 Add to Cart

Description

Kit #1000000144: UbiGate Components (set1)

This set allows generation of polycistronic expression cassettes for coexpression in bacteria of up to six ORFs simultaneously. In addition, the kit contains ubiquitin, the E1 UBA1, a complete collection of E2s, a selection of E3s and their cognate substrates, to reconstitute the ubiquitination assay. Genes of interest can be cloned in the standard Level 0 cloning vectors pUG-41308 to generate tag-less and N-terminally tagged versions, as well as in pUG-1287 for C-terminal tagging. All vectors contain standardized genetic modules that can be used for hierarchical assembly based on the Golden Gate cloning technique. Six control constructs to test in bacteria ubiquitination, purification, and detection procedures are also included (please see information on control plasmids at bottom of page).


Set1 UbiGate assembly of polycistronic operons for ubiquitination analysis.
  • Figure Set1: UbiGate assembly of polycistronic operons for ubiquitination analysis.

    (a) Open reading frames (ORFs) amplified by PCR with flanking BpiI recognition sites are cloned by restriction-ligation into level 0 vectors (only the resulting level 0 constructs containing an insert are shown). Level 0 vectors containing an ORF, and optionally a tag, are cloned into level 1 positional vectors using BsaI. The four nucleotide unique cleavage overhangs (UCOs) resulting from type IIS restriction are depicted as coloured boxes over BpiI or BsaI recognition sites. Note that UCOs are generated in distinct orientations in dependence of recognition site orientation. (b) Two sets of six level 1 positional vectors allow cloning of ORFs with N- and C-terminal tags for six defined positions. (c) The destination vector pET-28 Golden-Gate (pET-28GG) was created based on the pET-28b vector containing a LacZ flanked by BpiI sites for blue/white selection. (d) Level 1 vectors are assembled into the pET-28GG by BpiI restriction-ligation, resulting in the desired level 2 polycistronic operon. (e) Inducibility of the new pET-28GG vector containing His–Ub (pUG-205) was tested by applying 0.5mM IPTG and analysing accumulation at the indicated time points. His–Ub was purified by immobilized metal ion chromatography (IMAC) and resolved by SDS-PAGE using 12% tricine gels and analysed by immunoblot (IB) using anti-Ub and coomassie brilliant blue (CBB).


Kit #1000000145: UbiGate E2 activity screen (set2)

This collection includes expression cassettes containing ubiquitin, the E1 UBA1 in combination with all Arabidopsis thaliana E2s (ubiquitin-conjugating enzymes). Constructs can be employed to assay the auto-ubiquitination activity of an E3 ubiquitin ligase of interest in the presence of different E2s, by coexpression in bacteria. Six control constructs to test in bacteria ubiquitination, purification, and detection procedures are also included (please see information on control plasmids at bottom of page).


Set 2. E3 autoubiquitination assay by co-transformation with UbiGate constructs.
  • Figure Set2: E3 autoubiquitination assay by co-transformation with UbiGate constructs.

    (a) GST-AvrPtoB in pGEX-4T-1 and an operon containing His-ubiquitin, UBA1, HA-UBC8 in pET-28GG were co-transformed in BL21(DE) cells. Protein expression was induced with 0.5mM IPTG and cells were harvested after 3h. Proteins were purified with glutathione sepharose, eluted with 15 mM reduced glutathione (GSH) and resolved in a 7% PAGE (glycine) and analysed by IB using the indicated anti-bodies. Blue triangles indicate the unmodified E3, red triangles and lines E3-Ub(n) conjugates. (b) GST-Mib (aa 738-1030) from zebrafish (Danio rerio) in pGEX-1T and an operon containing His-ubiquitin, UBA1, HA-UBC8 in pET-28GG were co-transformed in BL21(DE) cells. Protein expression was induced with 0.5mM IPTG and cells were harvested after 3h. Proteins were purified with glutathione sepharose, eluted with 15 mM reduced glutathione (GSH) and resolved in a 7% PAGE (glycine) and analysed by IB using the indicated anti-bodies. Blue triangles indicate the unmodified E3, red triangles and lines E3-Ub(n) conjugates.


Control Plasmids

The 6 control plasmids listed below are included in both Kit #1000000144: UbiGate Components (set1) and Kit #1000000145: UbiGate E2 activity screen (set2).

Addgene ID Plasmid Name
104635 pUG-124
104636 pUG-125
104637 pUG-153
104638 pUG-412
104639 pUG-413
104640 pUG-417

Depositor Information

Protocols available in original publication listed below.


Original Publication

UbiGate: a synthetic biology toolbox to analyse ubiquitinatio. Kowarschik K, Hoehenwarter W, Marillonnet S, Trujillo M.New Phytol.2018. Mar;217(4):1749-1763. doi: 10.1111/nph.14900. PubMed PMID 29194629

How to Cite these Kits

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 UbiGate Components (set1) was a gift from Marco Trujillo (Addgene Kit #1000000144).”

“The UbiGate E2 activity screen (set2) was a gift from Marco Trujillo (Addgene Kit #1000000145).”

UbiGate Components (set1) - #1000000144

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

Spectinomycin
Ampicillin
Kanamycin

Inventory

Well Plasmid Resistance
A / 1 pUG-41308
Spectinomycin
A / 2 pUG-1287
Spectinomycin
A / 3 pUG-P1
Ampicillin
A / 4 pUG-P2
Ampicillin
A / 5 pUG-P3
Ampicillin
A / 6 pUG-P4
Ampicillin
A / 7 pUG-P5
Ampicillin
A / 8 pUG-P6
Ampicillin
A / 9 pUG-TP1
Ampicillin
A / 10 pUG-TP2
Ampicillin
A / 11 pUG-TP3
Ampicillin
A / 12 pUG-TP4
Ampicillin
B / 1 pUG-TP5
Ampicillin
B / 2 pUG-TP6
Ampicillin
B / 3 pET-28GG
Kanamycin
B / 4 pUG-225
Spectinomycin
B / 5 pAGM18012
Spectinomycin
B / 6 pAGT861
Spectinomycin
B / 7 pUG41722
Spectinomycin
B / 8 pUG41744
Spectinomycin
B / 9 pUG41766
Spectinomycin
B / 10 pUG41780
Spectinomycin
B / 11 pUG41800
Spectinomycin
B / 12 pUG41822
Spectinomycin
C / 1 pUG-UBA1
Spectinomycin
C / 2 pUG-UBC1
Spectinomycin
C / 3 pUG-UBC2
Spectinomycin
C / 4 pUG-UBC4
Spectinomycin
C / 5 pUG-UBC3
Spectinomycin
C / 6 pUG-UBC5
Spectinomycin
C / 7 pUG-UBC6
Spectinomycin
C / 8 pUG-UBC7
Spectinomycin
C / 9 pUG-UBC8
Spectinomycin
C / 10 pUG-UBC9
Spectinomycin
C / 11 pUG-UBC10
Spectinomycin
C / 12 pUG-UBC11
Spectinomycin
D / 1 pUG-UBC12
Spectinomycin
D / 2 pUG-UBC13
Spectinomycin
D / 3 pUG-UBC14
Spectinomycin
D / 4 pUG-UBC15
Spectinomycin
D / 5 pUG-UBC16
Spectinomycin
D / 6 pUG-UBC17
Spectinomycin
D / 7 pUG-UBC18
Spectinomycin
D / 8 pUG-UBC19
Spectinomycin
D / 9 pUG-UBC20
Spectinomycin
D / 10 pUG-UBC21
Spectinomycin
D / 11 pUG-UBC22
Spectinomycin
D / 12 pUG-UBC23
Spectinomycin
E / 1 pUG-UBC24
Spectinomycin
E / 2 pUG-UBC25
Spectinomycin
E / 3 pUG-UBC26
Spectinomycin
E / 4 pUG-UBC27
Spectinomycin
E / 5 pUG-UBC28
Spectinomycin
E / 6 pUG-UBC29
Spectinomycin
E / 7 pUG-UBC30
Spectinomycin
E / 8 pUG-UBC31
Spectinomycin
E / 9 pUG-UBC32
Spectinomycin
E / 10 pUG-UBC33
Spectinomycin
E / 11 pUG-UBC34
Spectinomycin
E / 12 pUG-UBC35
Spectinomycin
F / 1 pUG-UBC36
Spectinomycin
F / 2 pUG-UBC37
Spectinomycin
F / 3 pUG-135
Spectinomycin
F / 4 pUG-136
Spectinomycin
F / 5 pUG-137
Spectinomycin
F / 6 pUG-138
Spectinomycin
F / 7 pUG-21
Spectinomycin
F / 8 pUG-22
Spectinomycin
F / 9 pUG-248
Spectinomycin
F / 10 pUG-233
Spectinomycin
F / 11 pUG-234
Spectinomycin
F / 12 pUG-146
Spectinomycin
G / 1 pUG-131
Spectinomycin
G / 2 pUG-57
Spectinomycin
G / 3 pUG-489
Spectinomycin
G / 4 pUG-65
Spectinomycin
G / 5 pUG-349
Spectinomycin
G / 6 pUG-350
Spectinomycin
G / 7 pUG-351
Spectinomycin
G / 8 pUG-419
Spectinomycin
G / 9 pUG-124
Kanamycin
G / 10 pUG-125
Kanamycin
G / 11 pUG-153
Kanamycin
G / 12 pUG-412
Kanamycin
H / 1 pUG-413
Kanamycin
H / 2 pUG-417
Kanamycin
Data calculated @ 2019-11-16

Kit Plate Map - #1000000144

UbiGate E2 activity screen (set2) - #1000000145

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

Kanamycin

Inventory

Well Plasmid Resistance
A / 1 pUG-293
Kanamycin
A / 2 pUG-294
Kanamycin
A / 3 pUG-296
Kanamycin
A / 4 pUG-295
Kanamycin
A / 5 pUG-297
Kanamycin
A / 6 pUG-298
Kanamycin
A / 7 pUG-299
Kanamycin
A / 8 pUG-300
Kanamycin
A / 9 pUG-301
Kanamycin
A / 10 pUG-302
Kanamycin
A / 11 pUG-303
Kanamycin
A / 12 pUG-304
Kanamycin
B / 1 pUG-305
Kanamycin
B / 2 pUG-306
Kanamycin
B / 3 pUG-307
Kanamycin
B / 4 pUG-308
Kanamycin
B / 5 pUG-309
Kanamycin
B / 6 pUG-310
Kanamycin
B / 7 pUG-311
Kanamycin
B / 8 pUG-312
Kanamycin
B / 9 pUG-313
Kanamycin
B / 10 pUG-314
Kanamycin
B / 11 pUG-315
Kanamycin
B / 12 pUG-316
Kanamycin
C / 1 pUG-317
Kanamycin
C / 2 pUG-318
Kanamycin
C / 3 pUG-319
Kanamycin
C / 4 pUG-320
Kanamycin
C / 5 pUG-321
Kanamycin
C / 6 pUG-322
Kanamycin
C / 7 pUG-323
Kanamycin
C / 8 pUG-324
Kanamycin
C / 9 pUG-325
Kanamycin
C / 10 pUG-326
Kanamycin
C / 11 pUG-331
Kanamycin
C / 12 pUG-332
Kanamycin
D / 1 pUG-333
Kanamycin
D / 2 pUG-334
Kanamycin
D / 3 pUG-335
Kanamycin
D / 4 pUG-336
Kanamycin
D / 5 pUG-337
Kanamycin
D / 6 pUG-338
Kanamycin
D / 7 pUG-352
Kanamycin
D / 8 pUG-124
Kanamycin
D / 9 pUG-125
Kanamycin
D / 10 pUG-153
Kanamycin
D / 11 pUG-412
Kanamycin
D / 12 pUG-413
Kanamycin
E / 1 pUG-417
Kanamycin
Data calculated @ 2019-11-16

Kit Plate Map - #1000000145

Content blocked, you may need to disable your ad-blocker.