Easy-MISE Toolkit
(Kit #
1000000230
)
Depositing Lab: Paola Branduardi
The Easy-MISE toolkit, which stands for Easy-Modular Integrative fuSion-ready Expression toolkit, is a complete vector set leading to fast engineering procedures for the improvement of heterologous production in S. cerevisiae. It is characterized by a reduced design complexity thanks to a single-step assembly of the integration-ready expression cassette.
The final device obtained is a flexible double transcription unit cassette. Each transcription unit is transcribed in the opposite direction. Each integration cassette contains an upstream and a downstream homology sequence for targeted genome insertion. This is obtained by exploiting the CRISPR-Cas9 system and already available gRNA vectors from the EasyClone-MarkerFree Vector Set, targeting 11 previously well-characterized genome loci.
The toolkit allows an easy in-frame tag of every ORF of interest. This is achieved by the presence of a DNA spacer between each ORF and terminator that can be substituted with the desired sequence, for example with a GFP tag to study cell localization of the protein of interest, or with a specific ligand targeting the protein to a specific subcellular localization.
This kit will be sent as bacterial glycerol stocks in 96-well plate format.
Original Publication
Easy Modular Integrative fuSion-ready Expression (Easy-MISE) Toolkit for Fast Engineering of Heterologous Productions in Saccharomyces cerevisiae. Maestroni L, Butti P, Milanesi R, Pagliari S, Campone L, Serra I, Branduardi P. ACS Synth Biol. 2023 May 19;12(5):1508-1519. doi: 10.1021/acssynbio.3c00015. PubMed (Link opens in a new window) Article (Link opens in a new window).
Description
The Easy-MISE toolkit is modular and based on the use of chromoprotein-based screening. Indeed, it comprises two new acceptor vectors for Golden Gate Assembly.
The level 0 acceptor vector, called pGA-blue, carries the blue amilCP chromoprotein that generates blue colonies once expressed in E. coli. pGA-blue is used to build the library of parts needed to obtain the final device and has a final blue/white screening to select positive clones.
A similar logic stands behind the second acceptor vector, pGA-red, built as a level 1 acceptor vector. pGA-red plasmids carry the red-fluorescence protein (mRFP1) chromophore. After the Golden Gate Assembly reaction, it is possible to assemble the final device with up to 10 different parts. In this case, red clones are considered negative, while the positive clones will appear white.
Once the final device is ready, the yeast cells can be transformed to obtain the final engineered strains.
The kit comes with the three novel acceptor vectors (pGA-Blue, pGA-Red-mini, and pGA-Red-maxi) and a library of level 0 parts (pEM plasmids) made of promoters, terminators, adaptors, fluorescent tags, and homology regions in order to build the final device, speeding up the time to develop engineered strains.
- Figure 1. Easy Modular Integrative fuSion-ready Expression (Easy-MISE) Toolkit for fast engineering of heterologous production in Saccharomyces cerevisiae. Steps 1-4 show the process of generating the final device used for engineering strains.
Kit Documentation
Easy-MISE toolkit protocol (PDF, 390 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 Easy-MISE Toolkit was a gift from Paola Branduardi (Addgene kit #1000000230).”
For your Reference section:
Easy Modular Integrative fuSion-ready Expression (Easy-MISE) Toolkit for Fast Engineering of Heterologous Productions in Saccharomyces cerevisiae. Maestroni L, Butti P, Milanesi R, Pagliari S, Campone L, Serra I, Branduardi P. ACS Synth Biol. 2023 May 19;12(5):1508-1519. doi: 10.1021/acssynbio.3c00015. PubMed (Link opens in a new window) Article (Link opens in a new window).
Easy-MISE toolkit - #1000000230
- 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 |
Inventory
| Well | Plasmid | Resistance |
|---|---|---|
| A / 1 | pGA-red-maxi | Ampicillin |
| A / 2 | pGA-red-mini | Ampicillin |
| A / 3 | pGA-blue | Kanamycin |
| A / 4 | pEM.P01L | Kanamycin |
| A / 5 | pEM.P01R | Kanamycin |
| A / 6 | pEM.P02L | Kanamycin |
| A / 7 | pEM.P02R | Kanamycin |
| A / 8 | pEM.P03L | Kanamycin |
| A / 9 | pEM.P03R | Kanamycin |
| A / 10 | pEM.P04L | Kanamycin |
| A / 11 | pEM.P04R | Kanamycin |
| A / 12 | pEM.P05L | Kanamycin |
| B / 1 | pEM.P05R | Kanamycin |
| B / 2 | pEM.P06L | Kanamycin |
| B / 3 | pEM.P06R | Kanamycin |
| B / 4 | pEM.T01L | Kanamycin |
| B / 5 | pEM.T02R | Kanamycin |
| B / 6 | pEM.A01L | Kanamycin |
| B / 7 | pEM.A01R | Kanamycin |
| B / 8 | pEM.A02L | Kanamycin |
| B / 9 | pEM.A02R | Kanamycin |
| B / 10 | pEM.F01L | Kanamycin |
| B / 11 | pEM.F01R | Kanamycin |
| B / 12 | pEM.F02L | Kanamycin |
| C / 1 | pEM.F02R | Kanamycin |
| C / 2 | pEM.F03L | Kanamycin |
| C / 3 | pEM.F03R | Kanamycin |
| C / 4 | pEM.H01L | Kanamycin |
| C / 5 | pEM.H01R | Kanamycin |
| C / 6 | pEM.H02L | Kanamycin |
| C / 7 | pEM.H02R | Kanamycin |
| C / 8 | pEM.H03L | Kanamycin |
| C / 9 | pEM.H03R | Kanamycin |
| C / 10 | pEM.H04L | Kanamycin |
| C / 11 | pEM.H04R | Kanamycin |
| C / 12 | pEM.H05L | Kanamycin |
| D / 1 | pEM.H05R | Kanamycin |
| D / 2 | pEM.H06L | Kanamycin |
| D / 3 | pEM.H06R | Kanamycin |
| D / 4 | pEM.H07L | Kanamycin |
| D / 5 | pEM.H07R | Kanamycin |
| D / 6 | pEM.H08L | Kanamycin |
| D / 7 | pEM.H08R | Kanamycin |
| D / 8 | pEM.H09R | Kanamycin |
| D / 9 | pEM.H09L | Kanamycin |
| D / 10 | pEM.H10L | Kanamycin |
| D / 11 | pEM.H10R | Kanamycin |
| D / 12 | pEM.H11L | Kanamycin |
| E / 1 | pEM.H11R | Kanamycin |