MultiGreen Kit
(Kit #
1000000250
)
Depositing Lab: Wayne Parrott
MultiGreen enables expedient, multi-gene construct assembly for plant transformation using a backward compatible design for the GreenGate cloning system. MultiGreen enables users to assemble multiplexed plant transformation vectors in series, in parallel, or a combination of the two. Each MultiGreen intermediary assembly is made in a binary vector backbone, permitting testing of individual expression units or even co-transformation of unique assemblies prior to producing the final multiplexed vector.
This kit will be sent as bacterial glycerol stocks in 96-well plate format.
Original Publication
MultiGreen: A multiplexing architecture for GreenGate cloning. Pennetti VJ, LaFayette PR, Parrott WA. PLoS ONE. 2024 Sep 18;19(9):e0306008. doi: 10.1371/journal.pone.0306008. PubMed (Link opens in a new window) Article (Link opens in a new window)
Description
This kit contains 48 plasmids:
- 12 intermediate destination vectors corresponding to each entry position with either kanamycin or spectinomycin resistance
- 3 final destination vectors with either kanamycin or spectinomycin resistance, one containing a visual reporter in place of ccdB/CmR
- 7 MultiGreen 1.0 multiplexing in series expanded entry vectors
- 20 linker modules spanning overhang combinations for assemblies lacking all overhangs
- 6 empty entry vectors with backbones domesticated for Esp3I sites
These plasmids can be used to take existing GreenGate-compatible entry vectors and produce multigene constructs in a similar manner to conventional GreenGate cloning, with the added Type IIS enzyme, Esp3I. Note that in some cases, components containing internal Esp3I sites can be used to produce multigene constructs with MultiGreen, but it is recommended to domesticate any internal cut sites whenever possible — particularly when a cut site produces an undesirable GreenGate-compatible overhang.
Figure 1: MultiGreen 1.0 — multiplexing in series. A) Schematic overview of a standard GreenGate reaction condensing six entry vectors, AB Promoter, BC N-tag, CD gene of interest, DE C-tag, EF terminator, and FG resistance, into a destination vector using a one-pot BsaI mediated restriction ligation reaction. B) MultiGreen 1.0 expansion modules introduce the H overhang as a split of two new Level 0 entry vectors, the FH module is reserved for transcription blockers and the HG module is reserved for the multiplexer. The HG multiplexer module contains a pair of Esp3I sites internal to the BsaI sites for conventional GreenGate assembly, but external to the chromoprotein reporter. Each assembly that incorporates the level 0 HG and FH modules for multiplexing can be visually selected for integration of the multiplexer module. Subsequent MultiGreen 1.0 reactions should alternate visual reporters to efficiently screen one assembly round from the next, as the chemical selection is set by the initial destination vector. The assembly for the final transcriptional unit should either include the FH transcription blocker and HG filler modules to terminate the multiplexing, or alternatively a level 0 FG plant selectable marker module can be included. C) Detailed view of how the multiplexer operates by incorporating an external set of Esp3I sites flanking a visual reporter. Inclusion of the FH and HG modules in lieu of the FG followed by selection for colonies expressing the visual reporter enables iterative stacking in series. Image reused from Pennetti et al. 2024, under Creative Commons Attribution License (Link opens in a new window).
Figure 2: A schematic overview of multiplexing in parallel with MultiGreen 2.0. MultiGreen 2.0 uses a suite of level 1 acceptor vectors to produce initial concatenations of entry vectors. The level 1 acceptor vectors are not ampicillin resistant, allowing for chemical counterselection from level 0 entry vectors. Level 1 acceptor vectors are binary vectors themselves and can be used directly in Agrobacterium for (co)infection. Up to six level 1 assemblies can be combined per reaction. By alternating selection choice of level 1 acceptor vectors, assemblies can be iteratively combined in multiples of six. Note that alternating selection of level 1 acceptor vectors is only necessary for assemblies with more than six transcriptional units. Conventional GreenGate level 0 entry modules can be incorporated in any level 2 assembly with compatible overhangs, provided both Type IIS enzymes are included in the one-pot reaction cocktail. Image reused from Pennetti et al. 2024, under Creative Commons Attribution License (Link opens in a new window).
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 MultiGreen Kit was a gift from Wayne Parrott (Addgene kit #1000000250)."
For your Reference section:
MultiGreen: A multiplexing architecture for GreenGate cloning. Pennetti VJ, LaFayette PR, Parrott WA. PLoS ONE. 2024 Sep 18;19(9):e0306008. doi: 10.1371/journal.pone.0306008. PubMed (Link opens in a new window) Article (Link opens in a new window)
MultiGreen - #1000000250
- 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 | |
| Chloramphenicol and Spectinomycin |
Inventory
| Well | Plasmid | Resistance |
|---|---|---|
| A / 1 | pVP084 | Ampicillin |
| A / 2 | pVP085 | Ampicillin |
| A / 3 | pVP086 | Ampicillin |
| A / 4 | pVP088 | Ampicillin |
| A / 5 | pVP090 | Ampicillin |
| A / 6 | pVP333 | Ampicillin |
| A / 7 | pVP334 | Ampicillin |
| A / 8 | pVP120 | Ampicillin |
| A / 9 | pVP121 | Ampicillin |
| A / 10 | pVP122 | Ampicillin |
| A / 11 | pVP123 | Ampicillin |
| A / 12 | pVP183 | Ampicillin |
| B / 1 | pVP184 | Ampicillin |
| B / 2 | pVP185 | Ampicillin |
| B / 3 | pVP186 | Ampicillin |
| B / 4 | pVP224 | Ampicillin |
| B / 5 | pVP225 | Ampicillin |
| B / 6 | pVP226 | Ampicillin |
| B / 7 | pVP227 | Ampicillin |
| B / 8 | pVP228 | Ampicillin |
| B / 9 | pVP229 | Ampicillin |
| B / 10 | pVP230 | Ampicillin |
| B / 11 | pVP231 | Ampicillin |
| B / 12 | pVP293 | Ampicillin |
| C / 1 | pVP294 | Ampicillin |
| C / 2 | pVP096 | Kanamycin |
| C / 3 | pVP091 | Chloramphenicol and Ampicillin |
| C / 4 | pVP089 | Chloramphenicol and Ampicillin |
| C / 5 | pVP295 | Chloramphenicol and Ampicillin |
| C / 6 | pVP296 | Chloramphenicol and Ampicillin |
| C / 7 | pVP297 | Chloramphenicol and Ampicillin |
| C / 8 | pVP298 | Chloramphenicol and Ampicillin |
| C / 9 | pVP299 | Chloramphenicol and Ampicillin |
| C / 10 | pVP300 | Chloramphenicol and Ampicillin |
| C / 11 | pVP311 | Chloramphenicol and Kanamycin |
| C / 12 | pVP312 | Chloramphenicol and Kanamycin |
| D / 1 | pVP313 | Chloramphenicol and Kanamycin |
| D / 2 | pVP314 | Chloramphenicol and Kanamycin |
| D / 3 | pVP315 | Chloramphenicol and Kanamycin |
| D / 4 | pVP316 | Chloramphenicol and Kanamycin |
| D / 5 | pVP077 | Chloramphenicol and Kanamycin |
| D / 6 | pVP076 | Chloramphenicol and Spectinomycin |
| D / 7 | pVP078 | Chloramphenicol and Spectinomycin |
| D / 8 | pVP079 | Chloramphenicol and Spectinomycin |
| D / 9 | pVP080 | Chloramphenicol and Spectinomycin |
| D / 10 | pVP081 | Chloramphenicol and Spectinomycin |
| D / 11 | pVP082 | Chloramphenicol and Spectinomycin |
| D / 12 | pVP083 | Chloramphenicol and Spectinomycin |