MoClo Pichia Toolkit
(Kit #
1000000108
)
Depositing Lab: Volker Sieber
This collection of control elements and fluorescent reporters can be used in combination with the MoClo-YTK toolkit for protein expression and secretion in Pichia pastoris.
This kit will be sent as bacterial glycerol stocks in 96-well plate format.
Original Publication
A Modular Toolkit for Generating Pichia pastoris Secretion Libraries. Obst U, Lu TK, Sieber V. ACS Synth Biol. 2017 May 2;6(6):1016-1025. doi: 10.1021/acssynbio.6b00337. PubMed (Link opens in a new window) Article (Link opens in a new window)
Description
This collection of control elements and fluorescent reporters can be used in combination with the MoClo-YTK toolkit for protein expression and secretion in P. pastoris.
The Sieber Lab developed the MoClo Pichia Toolkit as a set of genetic parts to control transcription, translation, and protein secretion in P. pastoris. This toolkit will allow for generation of large and diverse secretion libraries in order to explore a wide range of secretion constructs, achieve successful secretion, and better understand the regulatory factors that play a role in protein production.
This toolkit contains 17 new control elements specific for P. pastoris. Combining these parts with existing S. cerevisiae parts allows for a gene of interest to be expressed in over 4000 different ways.
* Indicates 7 synthetic hybrid tags designed to have variable signal peptides, but still ensure full cleavage of the tag during secretion
Figure 1. Pichia pastoris protein secretion platform.
Proteins produced in P. pastoris can be expressed intracellularly or secreted extracellularly. The secretion pathway allows the possibility for protein modifications (e.g. proteolytic maturation, glycosylation or disulfide bonds).
After P. pastoris transformation, high-throughput screening of expression construct libraries can be performed in 96-well plates to find optimal expression constructs.
Standardized and combinatorial assembly strategy for designing expression vectors. Parts are defined by flanking overhangs and are flexible and interchangeable. For intracellular protein expression the coding sequence must be designed as a part with type 3 to ensure the correct overhang to the previous part type 2. For protein secretion, the secretion signal (part type 3a) and the coding sequence (part type 3b) are needed. White boxes correspond to new Pichia toolkit elements developed in this study; gray shaded boxes are taken from the existing MoClo-YTK toolkit.
(Image with permission from ACS Synthetic Biology. Copyright 2017 American Chemical Society).
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 MoClo Pichia Toolkit was a gift from Volker Sieber (Addgene kit #1000000108).”
For your Reference section:
A Modular Toolkit for Generating Pichia pastoris Secretion Libraries Obst U, Lu TK, Sieber V. ACS Synthetic Biology. 2017 May 2; doi: 10.1021/acssynbio.6b00337. PubMed PMID: 28252957.
MoClo Pichia toolkit - #1000000108
- 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
| Chloramphenicol |
Inventory
| Well | Plasmid | Resistance |
|---|---|---|
| A / 1 | pPTK003-2-pENO1 | Chloramphenicol |
| A / 2 | pPTK002-2-pGAP | Chloramphenicol |
| A / 3 | pPTK001-2-pAOX1 | Chloramphenicol |
| A / 4 | pPTK004-2-pTPI1 | Chloramphenicol |
| A / 5 | pPTK005-3a-αMF | Chloramphenicol |
| A / 6 | pPTK006-3a-αMF no EAEA | Chloramphenicol |
| A / 7 | pPTK007-3a-αMFΔ | Chloramphenicol |
| A / 8 | pPTK008-3a-αMFΔ no Kex | Chloramphenicol |
| A / 9 | pPTK009-3a-αAmylase-αMFΔ | Chloramphenicol |
| A / 10 | pPTK010-3a-Glucoamylase-αMFΔ | Chloramphenicol |
| A / 11 | pPTK011-3a-SA-αMFΔ | Chloramphenicol |
| A / 12 | pPTK012-3a-Inulinase-αMFΔ | Chloramphenicol |
| B / 1 | pPTK013-3a-Invertase-αMFΔ | Chloramphenicol |
| B / 2 | pPTK014-3a-Killer-αMFΔ | Chloramphenicol |
| B / 3 | pPTK015-3-yEGFP_Intra | Chloramphenicol |
| B / 4 | pPTK016-3-RFP_Intra | Chloramphenicol |
| B / 5 | pPTK017-3b-yEGFP_Sec | Chloramphenicol |
| B / 6 | pPTK018-3b-RFP_Sec | Chloramphenicol |
| B / 7 | pPTK019-4-tAOX1 | Chloramphenicol |
| B / 8 | pPTK020-7-attB | Chloramphenicol |
| B / 9 | pPTK021-7-PARS | Chloramphenicol |