Yeast Secrete and Detect
(Kit #
1000000166
)
Depositing Lab: Sylvestre Marillonnet
This kit contains a collection of 42 plasmids to enable a Golden Gate cloning-based one step-one pot assembly of transcription units for protein secretion in the yeast organisms Saccharomyces cerevisiae and Pichia pastoris. It contains a variety of methanol utilization (MUT) promoters, signal peptides and C-terminal tags as well as matching episomal and integrative acceptor plasmids for target gene expression.
This kit will be sent as bacterial glycerol stocks in 96-well plate format.
Original Publications
A modular two yeast species secretion system for the production and preparative application of unspecific peroxygenases. Püllmann P, Knorrscheidt A, Münch J, Palme PR, Hoehenwarter W, Marillonnet S, Alcalde M, Westermann B, Weissenborn MJ. Commun Biol. 2021 May 12;4(1):562. doi: 10.1038/s42003-021-02076-3. PubMed PMID: 33980981.
Improving the Heterologous Production of Fungal Peroxygenases through an Episomal Pichia pastoris Promoter and Signal Peptide Shuffling System. Püllmann P, Weissenborn MJ. ACS Synth Biol. 2021 Jun 2. doi: 10.1021/acssynbio.0c00641. PubMed PMID: 34075757.
A modular cloning system for standardized assembly of multigene constructs Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S. PLoS One. 2011 Feb 18;6(2):e16765. doi: 10.1371/journal.pone.0016765. PubMed PMID: 21364738.
Description
Basic modules (1) consisting of 17 unique signal peptides, 11 MUT promoters, and 7 C-terminal tags for protein detection and/or protein purification are cloned into pAGM9121. In combination with the gene of a protein of interest (POI) (1), transcription units are assembled within the acceptor expression plasmids. The assembly occurs in a standard Golden Gate one step-one pot digestion-ligation reaction (2). Based on the chosen setup (3), signal peptide (up to 17 unique combinations) or promoter/signal peptide shuffling libraries (up to 187 unique combinations) can be constructed. After construction, the resulting libraries can be transformed into the respective organisms and subsequently screened for POI secretion in a high throughput manner (e.g. 96 well plate format) (4). In addition to classical enzyme activity-based measurements, the presence of a C-terminal GFP11-Protein tag (27 aa) further allows target protein quantification based on a split-GFP Assay. By omitting the respective modules and correct design of the gene module overhangs, the kit can be used for intracellular and/or non-tagged protein production as well.
- Schematic overview of the Yeast Secrete and Detect system.
Protocols
A modular two yeast species secretion system for the production and preparative application of unspecific peroxygenases. Püllmann P, Knorrscheidt A, Münch J, Palme PR, Hoehenwarter W, Marillonnet S, Alcalde M, Westermann B, Weissenborn MJ. Commun Biol. 2021 May 12;4(1):562. doi: 10.1038/s42003-021-02076-3. PubMed PMID: 33980981.
Improving the Heterologous Production of Fungal Peroxygenases through an Episomal Pichia pastoris Promoter and Signal Peptide Shuffling System. Püllmann P, Weissenborn MJ. ACS Synth Biol. 2021 Jun 2. doi: 10.1021/acssynbio.0c00641. PubMed PMID: 34075757.
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 Yeast Secrete and Detect kit was a gift from Sylvestre Marillonnet (Addgene kit #1000000166).”
For your Reference section:
A modular two yeast species secretion system for the production and preparative application of unspecific peroxygenases. Püllmann P, Knorrscheidt A, Münch J, Palme PR, Hoehenwarter W, Marillonnet S, Alcalde M, Westermann B, Weissenborn MJ. Commun Biol. 2021 May 12;4(1):562. doi: 10.1038/s42003-021-02076-3. PubMed PMID: 33980981.
Improving the Heterologous Production of Fungal Peroxygenases through an Episomal Pichia pastoris Promoter and Signal Peptide Shuffling System. Püllmann P, Weissenborn MJ. ACS Synth Biol. 2021 Jun 2. doi: 10.1021/acssynbio.0c00641. PubMed PMID: 34075757.
A modular cloning system for standardized assembly of multigene constructs Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S. PLoS One. 2011 Feb 18;6(2):e16765. doi: 10.1371/journal.pone.0016765. PubMed PMID: 21364738.
Yeast Secrete and Detect - #1000000166
- 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 | |
| Spectinomycin |
Inventory
| Well | Plasmid | Resistance |
|---|---|---|
| A / 1 | pAGT572_Nemo | Ampicillin |
| A / 2 | pPAP001 | Ampicillin |
| A / 3 | pPAP002 | Ampicillin |
| A / 4 | pPAP004 | Ampicillin |
| A / 5 | pPAP003 | Kanamycin |
| A / 6 | pAGM22082_sfGFP1-10 | Kanamycin |
| A / 7 | pAGM9121_Sce-Prepro SP | Spectinomycin |
| A / 8 | pAGM9121_Kma-Inulinase SP | Spectinomycin |
| A / 9 | pAGM9121_Sce-Invertase 2 SP | Spectinomycin |
| A / 10 | pAGM9121_Sce-Acid Phosphatase SP | Spectinomycin |
| A / 11 | pAGM9121_Gma-UPO SP | Spectinomycin |
| A / 12 | pAGM9121_Hsa-Serum Albumin SP | Spectinomycin |
| B / 1 | pAGM9121_Aaw-Glucoamylase SP | Spectinomycin |
| B / 2 | pAGM9121_Sce-Killer Protein SP | Spectinomycin |
| B / 3 | pAGM9121_Mro-UPO SP | Spectinomycin |
| B / 4 | pAGM9121_Cfo-CPO SP | Spectinomycin |
| B / 5 | pAGM9121_Cgl-UPO SP | Spectinomycin |
| B / 6 | pAGM9121_Cci-UPO SP | Spectinomycin |
| B / 7 | pAGM9121_Ani- α Amylase SP | Spectinomycin |
| B / 8 | pAGM9121_Sce- α Galactosidase SP | Spectinomycin |
| B / 9 | pAGM9121_Gga-Lysozym SP | Spectinomycin |
| B / 10 | pAGM9121_Aae-UPO* SP | Spectinomycin |
| B / 11 | pAGM9121_Mth-UPO SP | Spectinomycin |
| B / 12 | pAGM9121_GFP11 | Spectinomycin |
| C / 1 | pAGM9121_OctaHis-GFP11 | Spectinomycin |
| C / 2 | pAGM9121_TEV-His-GFP11 | Spectinomycin |
| C / 3 | pAGM9121_His2-GFP11 | Spectinomycin |
| C / 4 | pAGM9121_Strep II | Spectinomycin |
| C / 5 | pAGM9121_TwinStrep | Spectinomycin |
| C / 6 | pAGM9121_TwinStrep-GFP11 | Spectinomycin |
| C / 7 | pAGM9121_pAOX1 | Spectinomycin |
| C / 8 | pAGM9121_pFDH1 | Spectinomycin |
| C / 9 | pAGM9121_pFLD1 | Spectinomycin |
| C / 10 | pAGM9121_pPMP20 | Spectinomycin |
| C / 11 | pAGM9121_pDAS1 | Spectinomycin |
| C / 12 | pAGM9121_pDAS2 | Spectinomycin |
| D / 1 | pAGM9121_pCAT1 | Spectinomycin |
| D / 2 | pAGM9121_pALD4 | Spectinomycin |
| D / 3 | pAGM9121_pDHAS | Spectinomycin |
| D / 4 | pAGM9121_pFMD | Spectinomycin |
| D / 5 | pAGM9121_pMOX | Spectinomycin |
| D / 6 | pAGM9121 | Spectinomycin |