MoClo Yeast Secretion Biosensor (YSB) Toolkit
(Kit # 1000000268 )

Depositing Lab:   Joseph Brock

The MoClo YSB Toolkit contains a library of plasmids for use in protein secretion optimization in yeast. This kit contains 46 signal peptides (Type 3a*), two yeast episomal expression vectors with GFP or RFP dropout sites (Type ‘234’), eight C-terminal tags (Type 4a), including C-myc negative control and various alpha-mating factor tag designs, and a human serum albumin (HSA) coding sequence (Type 3b*). The components can be assembled via Golden Gate assembly (standard or combinatorial), along with parts from the MoClo Yeast Toolkit (YTK), to create protein expression cassettes.

This kit will be sent as bacterial glycerol stocks in 96-well plate format.

Original Publication

High-throughput optimisation of protein secretion in yeast via an engineered biosensor. Cleaver A, Luo R, Smith OB, Murphy L, Schwessinger B, Brock J. Trends Biotechnol. 2025 Apr;43(4):838-867. doi: 10.1016/j.tibtech.2024.11.010. PubMed Article

Please visit https://doi.org/10.1101/2024.05.15.594099 for bioRxiv preprint.

Description

The MoClo Yeast Secretion Biosensor (YSB) toolkit is designed for optimizing secretion of recombinant proteins in Saccharomyces cerevisiae. The kit's components are best used with the protein secretion biosensor strain detailed in the original publication (Cleaver et al., 2025), but can be applied in other yeast strains. The protein secretion biosensor assay is intended as an initial optimization tool, after which the ideal protein of interest (POI) expression cassette can be inserted into an industrial or high protein production yeast strain.

Successful secretion of a given POI relies on the use of an optimal secretion signal or signal peptide (SP). The kit contains 46 SPs (Type 3a*), 24 from S. cerevisiae and 22 from Homo sapiens. They originate from experimentally characterized secreted proteins found on UniProt and were selected for maximal sequence diversity. The SP components are compatible with other species like Komagataella phaffii (formerly Pichia pastoris).

The kit also contains two yeast episomal expression vectors with either GFP or RFP dropout sites (Type ‘234’). These include a CEN6/ARS4 yeast origin of replication, as well as an ampicillin resistance gene, and a pBR322 origin of replication for Escherichia coli. Expression cassettes can be assembled into these vectors via Golden Gate assembly, using BsaI.

As the Type 3a-3b cloning junction between the SP and POI introduces two amino acids, we redesigned the junction to encode an alanine-proline (Ala-Pro) rather than the glycine-serine (Gly-Ser) defined by the MoClo assembly standard. These altered junctions are denoted by the symbol ‘*’. This reuses the MoClo YTK Type 8-1 overhang (CCCT), which should be taken into account when assembling expression cassettes in alternative vectors.

The seven C-terminal alpha-mating factor tags (Type 4a) contain the S. cerevisiae 13-amino-acid alpha-mating factor (aMF). This peptide is detected by the G protein-coupled receptor Ste13, which in the protein secretion biosensor strain is coupled to a sfGFP reporter gene. When the POI is successfully secreted, the aMF tag is secreted alongside and triggers activation of the biosensor pathway, resulting in a green fluorescent read-out. The Type 4a C-terminal c-Myc tag is intended as a negative control for the secretion biosensor assay.

Recombinant HSA was used as the test protein for the secretion biosensor assay. The HSA coding sequence (Type 3b*) can be used as a positive control, as well as a template for designing custom POI coding sequences (Type 3b*).

Figure 1: Yeast protein secretion biosensor assay schematic. (A) MoClo Combinatorial Golden gate assembly of 23 promoters (from MoClo YTK, not included in this kit), 46 signal peptides, a protein of interest (POI), a C-terminal linker with alpha-mating factor (aMF) tag, and six terminators (from MoClo YTK, not included in this kit), into the RFP (Type ‘234’) dropout site of a yeast episomal expression vector (6,348 total expression cassette combinations). Assembled plasmids are then transformed into the secretion assay yeast strain, containing a G protein-coupled receptor biosensor for aMF with GFP output. (B) 1. POI represented in blue, along with aMF tag represented in red, is expressed and translocated to the endoplasmic reticulum. 2. POI with aMF tag is translocated to the Golgi apparatus. 3. In the Golgi apparatus, proteases cleave the tag from the POI (dependent on tag design used). 4. POI and cleaved aMF tag are translocated to a secretory vesicle. 5. POI and cleaved aMF tag are secreted from the cell. 6. aMF tag interacts with biosensor cell surface receptor triggering GFP expression. (C) Cells are plated out on -URA auxotrophic selective media, cell fluorescence indicates successful POI secretion. The most fluorescent colonies can be plasmid sequenced to determine successful expression cassette combinations for POI secretion.

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 Yeast Secretion Biosensor (YSB) Toolkit was a gift from Joseph Brock (Addgene kit #1000000268)."

For your Reference section:

High-throughput optimisation of protein secretion in yeast via an engineered biosensor. Cleaver A, Luo R, Smith OB, Murphy L, Schwessinger B, Brock J. Trends Biotechnol. 2025 Apr;43(4):838-867. doi: 10.1016/j.tibtech.2024.11.010. PubMed Article

MoClo Yeast Secretion Biosensor (YSB) Toolkit - #1000000268

Inventory

Well	Plasmid	Resistance
A / 1	YEE_GFP	Ampicillin
A / 2	YEE_RFP	Ampicillin
A / 3	SP_A1	Chloramphenicol
A / 4	SP_A2	Chloramphenicol
A / 5	SP_A3	Chloramphenicol
A / 6	SP_A4	Chloramphenicol
A / 7	SP_A5	Chloramphenicol
A / 8	SP_A6	Chloramphenicol
A / 9	SP_B1	Chloramphenicol
A / 10	SP_B2	Chloramphenicol
A / 11	SP_B3	Chloramphenicol
A / 12	SP_B4	Chloramphenicol
B / 1	SP_B5	Chloramphenicol
B / 2	SP_B6	Chloramphenicol
B / 3	SP_C1	Chloramphenicol
B / 4	SP_C2	Chloramphenicol
B / 5	SP_C3	Chloramphenicol
B / 6	SP_C4	Chloramphenicol
B / 7	SP_C5	Chloramphenicol
B / 8	SP_C6	Chloramphenicol
B / 9	SP_D1	Chloramphenicol
B / 10	SP_D2	Chloramphenicol
B / 11	SP_D3	Chloramphenicol
B / 12	SP_D4	Chloramphenicol
C / 1	SP_D5	Chloramphenicol
C / 2	SP_D6	Chloramphenicol
C / 3	SP_E1	Chloramphenicol
C / 4	SP_E2	Chloramphenicol
C / 5	SP_E3	Chloramphenicol
C / 6	SP_E4	Chloramphenicol
C / 7	SP_E5	Chloramphenicol
C / 8	SP_E6	Chloramphenicol
C / 9	SP_F1	Chloramphenicol
C / 10	SP_F2	Chloramphenicol
C / 11	SP_F3	Chloramphenicol
C / 12	SP_F4	Chloramphenicol
D / 1	SP_F5	Chloramphenicol
D / 2	SP_F6	Chloramphenicol
D / 3	SP_G1	Chloramphenicol
D / 4	SP_G2	Chloramphenicol
D / 5	SP_G3	Chloramphenicol
D / 6	SP_G6	Chloramphenicol
D / 7	SP_H1	Chloramphenicol
D / 8	SP_H2	Chloramphenicol
D / 9	SP_H3	Chloramphenicol
D / 10	SP_H4	Chloramphenicol
D / 11	SP_H5	Chloramphenicol
D / 12	SP_H6	Chloramphenicol
E / 1	HSA_3b*_AP	Chloramphenicol
E / 2	C-term_aMFx1 (4a)	Chloramphenicol
E / 3	C-term_aMFx1_noKREAEA (4a)	Chloramphenicol
E / 4	C-term_aMFx4 (4a)	Chloramphenicol
E / 5	C-term_TEV_aMFx1 (4a)	Chloramphenicol
E / 6	twinStrep_FLAG_aMFx1 (4a)	Chloramphenicol
E / 7	twinStrep_HA_aMFx1 (4a)	Chloramphenicol
E / 8	twinStrep_aMFx1 (4a)	Chloramphenicol
E / 9	C-term_C-myc (4a)	Chloramphenicol

Data calculated @ 2025-09-17