AspFlex Kit
(Kit #
1000000217
)
Depositing Lab: Veronica Godoy-Carter
This collection is a set of Modular Cloning (MoClo) plasmids for Acinetobacter spp. and Acinetobacter baumannii for protein expression analyses and CRISPRi experiments.
This kit will be sent as bacterial glycerol stocks in 96-well plate format.
Original Publication
AspFlex: Molecular Tools to Study Gene Expression and Regulation in Acinetobacter baumannii. Brychcy M, Kokodynski A, Lloyd D, Godoy VG. ACS Synth Biol. 2023 Aug 16. doi: 10.1021/acssynbio.3c00167. PubMed (Link opens in a new window) Article (Link opens in a new window).
Description
This collection is a set of MoClo plasmids for both Acinetobacter spp. and Acinetobacter baumannii. The plasmids support easy Golden Gate cloning for protein expression analyses in Acinetobacter spp., including the construction of transcriptional reporters or fusion proteins. The collection is an expansion pack for the EcoFlex collection for E. coli (Addgene Kit#: #1000000080). The kit introduces a new plasmid based CRISPRi Golden Gate system to A. baumannii, with plasmids for cloning sgRNAs and plasmids containing a catalytically deficient Cas9 (dCas9).
Several accessory plasmids are also available, which contain an additional Tellurite resistance cassette for multi-drug resistance strains. These plasmids are not part of the kit but can be ordered separately (see the Accessory Plasmids tab).
Assembly mechanism of molecular elements in AspFlex. (A) A DNA recombinant approach to clone any transcriptional unit (for example for protein expression, transcriptional reporters, etc.). The system is organized in the standardized modular level based MoClo fashion. Here, a Level 0 plasmid consists of the elements necessary to form a transcriptional unit, a Level 1 plasmid places the elements from Level 0 plasmids to create a single transcriptional unit, while a Level 2 plasmid combines multiple transcriptional units from Level 1 plasmids to create a plasmid with two to five independent transcriptional units. Lastly, a Level 3 plasmid allows for the combination of six or more independent transcriptional units. (B) Workflow to assemble a CRISPRi unit. Here, a Level 0 allows the possibility to create a custom sgRNA, which can then be transferred into a shuttle vector in a Level 1 plasmid containing a (inducible) dCas9 provided with the kit. A Level 0 plasmid containing dCas9, which can be moved into different level plasmids is also provided. Up to four independent sgRNAs can be used in Level 2 plasmids. Level 3 then allows for five or more sgRNAs.
Kit Documentation
AspFlex Kit Protocols (PDF, 263 KB)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 AspFlex Kit was a gift from Veronica Godoy-Carter (Addgene kit #1000000217).”
For your Reference section:
AspFlex: Molecular Tools to Study Gene Expression and Regulation in Acinetobacter baumannii. Brychcy M, Kokodynski A, Lloyd D, Godoy VG. ACS Synth Biol. 2023 Aug 16. doi: 10.1021/acssynbio.3c00167. PMID: 37587063 (Link opens in a new window)
AspFlex Kit - #1000000217
- 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 |
Inventory
| Well | Plasmid | Resistance |
|---|---|---|
| A / 1 | pMB1-A | Ampicillin |
| A / 2 | pMB1-B | Ampicillin |
| A / 3 | pMB1-C | Ampicillin |
| A / 4 | pMB1-D | Ampicillin |
| A / 5 | pMB1-D1 | Ampicillin |
| A / 6 | pMB1-E | Ampicillin |
| A / 7 | pMB3-A | Ampicillin |
| A / 8 | pMB3-B | Ampicillin |
| A / 9 | pMB1-A-ptet-dCas9 | Ampicillin |
| A / 10 | pMB1-A-ptet-FLAG-dCas9 | Ampicillin |
| A / 11 | pMB1-A-pBAD-dCas9 | Ampicillin |
| A / 12 | pMB2a | Kanamycin |
| B / 1 | pMB2b | Kanamycin |
| B / 2 | pMB2-A | Kanamycin |
| B / 3 | pMB2-B | Kanamycin |
| B / 4 | pMB2-C | Kanamycin |
| B / 5 | pMB2-D | Kanamycin |
| B / 6 | pBP_sgRNA | Chloramphenicol |
| B / 7 | pBP_dCas9 | Chloramphenicol |
Kit Plate Map - #1000000217
Accessory Plasmids
The following related plasmids are also available with additional Tellurite resistance cassette for multi-drug resistance strains. These plasmids are not included in the kit, but can be ordered separately: