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Saccharomyces cerevisiae

S. cerevisiae testing devices

Escherichia coli

Parts Table

Code	Name	Type	Description
BBa_K5466001	IGG6	Other	9-bp sequence that enables polycistronic gene expression in yeasts and other filamentous fungi. Identified and characterized by Yue et al. (2023), it has been proven functional for bicistronic expression in the yeasts Saccharomyces cerevisiae, Pichia pastoris and Yarrowia lipolytica, as well as the filamentous fungus Aspergillus nidulans. IGG6 has shown polycistronic expression up to 4 CDSs in Saccharomyces cerevisiae.
BBa_K5466002	AGA2P Signal Peptide	Tag	Signal peptide consisting on the 18 first amino acids from Saccharomyces cerevisiae AGA2 protein. Attachment to the N-terminal end of a protein can efficiently drive translocation across the plasma membrane. Additional fusion of AGA2P to the C-terminal can be used for protein display. AG was added to the C-terminus of the signal peptide based on the discovery by Wang et al. (2005) regarding the importance of specific amino acids for proper peptide cleavage.
BBa_K5466003	Flexible linker 3x (GGGGS) Saccharomyces cerevisiae codon optimized	Protein_Domain	A flexible 15 aa long linker, designed for protein fusion.
BBa_K5466004	AntiAFB1- Nb28-S102D	Protein_Domain	Single-domain antibody (sdAb) with improved binding activity to AFB1, without a start codon or stop codon, prepared for yeast surface display.
BBa_K5466005	AGA2P for yeast surface display, without N-terminal signal peptide	Protein_Domain	AGA2P enables yeast surface display at both the N- and C-terminus. AGA2P is an extracellularly-secreted protein that It is part of the cell wall and is linked to anchorage subunit Aga1p via two disulfide bonds.
BBa_K5466006	SUC2 Signal Peptide. Saccharomyces cerevisiae codon optimized	Tag	Signal peptide consisting on the 19 first amino acids from Saccharomyces cerevisiae SUC2 invertase. SUC2 is an extracellularly-secreted enzyme that hydrolyzes sucrose into glucose and fructose. Attachment to the N-terminal end of a protein can efficiently drive translocation across the plasma membrane.
BBa_K5466007	EpoR-D2-TM	Protein_Domain	D2 and transmembrane domain from mammalian erythropoietin receptor (residues 118-272). The scaffold of the GEMS sensing platform. Also, for yeasts the scaffold of a receptor inspired in GEMS platform: Patrol Yeast, using a split-ubiquitin yeast two hybrid as signaling module. In the N-terminal there is a short flexible peptide (SGEF), allowing movement on the ligand binding domain (LBD).
BBa_K5466008	NubG	Protein_Domain	N-terminal part of the split ubiquitin (residues 1-34) with a flexible linker. It can re-associate with Cub (BBa_K5466009) in vivo and form active ubiquitin. Use in the assessment of membrane protein intaraction, through the split ubiquitin system, a membrane based Yeast-Two Hybrid. The intracellular and signaling region of a receptor inspired in GEMS platform: Patrol Yeast. Spontaneous reconstitution of the split protein is prevented through mutation I13G, rendering NubG. Only when NubG and Cub are brought into close proximity upon receptor dimerization, can the ubiquitin be reconstituted and recognized by cellular deubiquitinases, leading to hydrolysis and release of a transcription factor fused to the C-terminal end of Cub.
BBa_K5466009	Cub	Protein_Domain	C-terminal part of the split ubiquitin (residues 35-76) with a flexible linker. It can re-associate with NubG (BBa_K5466008)in vivo and form active ubiquitin. Use in the assessment of membrane protein intaraction, through the split ubiquitin system, a membrane based Yeast-Two Hybrid. The intracellular and signaling region of a receptor inspired in GEMS platform: Patrol Yeast. Only when NubG and Cub are brought into close proximity upon receptor dimerization, can the ubiquitin be reconstituted and recognized by cellular deubiquitinases, leading to hydrolysis and release of a transcription factor fused to the C-terminal end of Cub.
BBa_K5466010	LexA-VP16	Coding	LexA-VP16 is a synthetic transcription factor formed by the fusion of LexA DNA binding domain (BD) and Herpesviurs VP16 activation domain (AD). Due to its structure, it can exert potent transcriptional activation on DNA sequences harboring LexA operators followed by native core promoter elements. SV40 NLS was attached to the C-terminal.
BBa_K5466011	LexA-VP16-activated promoter	Regulatory	Synthetic promoter composed of the core promoter region from Saccharomyces cerevisiae TDH3 promoter, preceded by 4 lexA operators. This part allows for powerful activation by transcription factors harboring LexA DBD fused to an activation domain such as herpes simplex VP16 AD, for example, BBa_K5466010.
BBa_K5466012	CadC	Protein_Domain	Intracellular domain of the EMeRALD sensing platform, consist of CadC DNA binding domain and Juxtamembrane region and an artificial Leu (16) transmembrane region. Upon dimerization active pCadBA (BBa_K3425101). Without stop codon for fusion with LBD for use in EMeRALD sensing platform.
BBa_K5466013	AntiAFB1-scFv1. Saccharomyces cerevisiae codon optimized without STOP codon	Protein_Domain	AntiAFB1-scFv1 (BBa_K2247006) from iGEM17_Tsinghua, without a STOP codon to be used as a ligand binding domain for AFB1 with other protein domains. For example, in our project, it is the LBD of a composite part, a Patrol Yeast receptor, fused with EpoR and NubG (BBa_K5466018), to detect AFB1.
BBa_K5466014	AntiAFB1-scFv2. Saccharomyces cerevisiae codon optimized without STOP codon	Protein_Domain	AntiAFB1-scFv2 (BBa_K2247007) from iGEM17_Tsinghua, without a STOP codon to be used as a ligand binding domain for AFB1 with other protein domains. For example, in our project, it is the LBD of a composite part, a Patrol Yeast receptor, fused with EpoR and Cub (BBa_K5466019), to detect AFB1.
BBa_K5466015	AntiAFB1-scFv1. Escherichia coli codon optimized	Coding	AntiAFB1-scFv1 (BBa_K2247006) from iGEM17_Tsinghua, with E. coli codon optimization. Can be used as a ligand binding domain for AFB1 with other protein domains. For example, in our project, it is the LBD of a composite part, as extracellular region of the EMeRALD platform, with CadC intracellular domain (BBa_K5466012), to detect AFB1.
BBa_K5466016	AntiAFB1-scFv2. Escherichia coli codon optimized	Coding	AntiAFB1-scFv2 (BBa_K2247007) from iGEM17_Tsinghua, with E. coli codon optimization. Can be used as a ligand binding domain for AFB1 with other protein domains. For example, in our project, it is the LBD of a composite part, as extracellular region of the EMeRALD platform, with CadC intracellular domain (BBa_K5466012), to detect AFB1.
BBa_K5466017	Nb28-S102D Aga2P	Composite: Coding	Yeast surface display of Nb28-S102D with Aga2P. The localization of the VHH on the cell wall allows the capture of AFB1 from the environment.
BBa_K5466018	antiAFB1-scFv1 NubG	Composite: Coding	Component of the signaling platform Patrol Yeast. The NubG component of the split-ubiquitin system with scFv1 to detect AFB1 in yeast. Use with antiAFB1-scFv2 Cub (BBa_K5466019). Dimerization of Cub and NubG occurs only when both scFvs bind to aflatoxin, this reconstitutes the ubiquitin, leading to hydrolysis and release of the transcription factor, activating the expression of (BBa_K5466011).
BBa_K5466019	antiAFB1-scFv2 Cub	Composite: Coding	Component of the signaling platform Patrol Yeast. The Cub component of the split-ubiquitin system with scFv1 to detect AFB1 in yeast, fused with the LexA-VP16 (BBa_K5466010) transcriptional factor. Use with antiAFB1-scFv1 NubG (BBa_K5466018). Dimerization of Cub and NubG occurs only when both scFvs bind to aflatoxin, this reconstitutes the ubiquitin, leading to hydrolysis and release of the transcription factor, activating the expression of (BBa_K5466011).
BBa_K5466020	Policistronic response to detection of AFB1 by split-ubiquin system	Composite: Device	This device elicit three response upon AFB1 detection, activated by (BBa_K5466018) and (BBa_K5466019): Expression of Nb28-S102D Aga2P, a VHH with high affinity to AFB1 anchored to the cell wall, capturing the AFB1 present in the environment. A positive feedback loop amplifies the response, ensuring that upon the first detection of aflatoxin, the maximum response is triggered. This allows the yeast cell wall to become saturated with Nband ensuring that the response is sustained over time.This positive feedback occurs because the transcription factor, that activates the expression of this device, is synthesized. A reporter RFP that allows us to visualize the presence of aflatoxin in the environment and confirms that the response is occurring.
BBa_K5466021	Constitutive expression of receptor antiAFB1-scFv1 NubG	Composite: Device	The expression of receptor antiAFB1-scFv1 NubG (BBa_K5466018) is controlled by the constitutive promoter TDH3 (BBa_K124002) to ensure it is always available for the detection of AFB1. Device the express a component of the signaling platform Patrol Yeast. The NubG component of the split-ubiquitin system with scFv1 to detect AFB1 in yeast. Use with constitutive expression of receptor antiAFB1-scFv2 Cub (BBa_K5466022). Dimerization of Cub and NubG occurs only when both scFvs bind to aflatoxin, this reconstitutes the ubiquitin, leading to hydrolysis and release of the transcription factor, activating the expression of (BBa_K5466011).
BBa_K5466022	Constitutive expression of receptor antiAFB1-scFv2 Cub	Composite: Device	The expression of receptor antiAFB1-scFv2 Cub (BBa_K5466019) is controlled by the constitutive promoter TDH3 (BBa_K124002) to ensure it is always available for the detection of AFB1. Device the express a component of the signaling platform Patrol Yeast. The Cub component of the split-ubiquitin system with scFv2 to detect AFB1 in yeast. Use with constitutive expression of receptor antiAFB1-scFv1 Nub (BBa_K5466021). Dimerization of Cub and NubG occurs only when both scFvs bind to aflatoxin, this reconstitutes the ubiquitin, leading to hydrolysis and release of the transcription factor, activating the expression of (BBa_K5466011).
BBa_K5466023	Constitutive expression of AFB1 detection platform	Composite: Device	The expression of signaling platform Patrol Yeast to detect AFB1. IGG6 (BBa_K5466001) allow co-transcription of antiAFB1-scFv1 NubG (BBa_K5466018) and antiAFB1-scFv2 Cub (BBa_K5466019) reducing the number of base pairs required for the expression of both receptors. By utilizing IGG6, the total construct size is reduced by approximately 1000 bp compared to the combined 5558 bp of (BBa_K5466021) and (BBa_K5466022) required for the same function. This optimize the size of the genetic construct. The expression is controlled by the constitutive promoter TDH3 (BBa_K124002) to ensure it is always available for the detection of AFB1.
BBa_K5466024	antiAFB1-scFv1 CadC	Composite: Coding	Component of the EMeRALD platform to detect AFB1, with antiAFB1-scFv2 CadC (BBa_K5466025). Upon dimerization with antiAFB1-scFv2 CadC (BBa_K5466025), active pCadBA (BBa_K3425101).
BBa_K5466025	antiAFB1-scFv2 CadC	Composite: Coding	Component of the EMeRALD platform to detect AFB1, with antiAFB1-scFv1 CadC (BBa_K5466024). Upon dimerization with antiAFB1-scFv1 CadC (BBa_K5466024) , active pCadBA (BBa_K3425101).
BBa_K5466026	Autoregulated antiAFB1-scFv1 CadC receptor	Composite: Device	The promoter leakage maintains low expression of the receptor. Since it is regulated by the promoter which is activated by the receptors, this allows for an increase in expression upon detection of aflatoxin, creating a positive feedback loop (until all AFB1 is captured), thereby saturating the receptor membrane and facilitating the capture of AFB1. Use with Autoregulated antiAFB1-scFv2 CadC receptor (BBa_K5466027).
BBa_K5466027	Autoregulated antiAFB1-scFv2 CadC receptor	Composite: Device	The promoter leakage maintains low expression of the receptor. Since it is regulated by the promoter which is activated by the receptors, this allows for an increase in expression upon detection of aflatoxin, creating a positive feedback loop (until all AFB1 is captured), thereby saturating the receptor membrane and facilitating the capture of AFB1. Use with Autoregulated antiAFB1-scFv1 CadC receptor (BBa_K5466026).
BBa_K5466028	RFP expression controlled by CadC	Composite: Device	Expression of RFP (BBa_B1010) upon dimerization of CadC.
BBa_K5466029	Split mCerulean N-terminal. Saccharomyces cerevisiae codon optimized	Protein_Domain	N-terminal part of the split mCerulean (residues 1-172) with a flexible linker. It can re-associate with split mCerulean C-terminal (BBa_K5466030) in vivo and reconstitute mCerulean that can emit fluorescence. Use to study protein–protein interactions. Can be fused with EpoR-D2-TM (BBa_K5466007) to verify that ligand-induced dimerization is produced on the cytoplasmic membrane by the two ligand-binding domains targeting the same ligand to test if they are good candidates as LBD on the GEMS platform or Yeast Patrol.
BBa_K5466030	Split mCerulean C-terminal. Saccharomyces cerevisiae codon optimized	Protein_Domain	C-terminal part of the split mCerulean (residues 155-238) with a flexible linker. It can re-associate with split mCerulean N-terminal (BBa_K5466029) in vivo and form mCerulean that can emit fluorescence. Use to study protein–protein interactions. Can be fused with EpoR-D2-TM (BBa_K5466007) to verify that ligand-induced dimerization is produced on the cytoplasmic membrane by the two ligand-binding domains targeting the same ligand to test if they are good candidates as LBD on the GEMS platform or Yeast Patrol.
BBa_K5466031	AntiAFB1-scFv1 EpoR Split-N-mCerulean	Composite: Coding	Using this part along with antiAFB1-scFv2 EpoR Split-C-mCerulean (BBa_K5466032) allows us to observe if, in the presence of aflatoxin, dimerization occurs and mCerulean is reconstituted.
BBa_K5466032	AntiAFB1-scFv2 EpoR Split-C-mCerulean	Composite: Coding	Using this part along with antiAFB1-scFv1 EpoR Split-N-mCerulean (BBa_K5466031) allows us to observe if, in the presence of aflatoxin, dimerization occurs and mCerulean is reconstituted.
BBa_K5466033	Triple fluorescence tricistronic device	Composite: Device	A device that constitutively produces three fluorescent proteins—mCherry, spGFP, and mCerulean3—under a single promoter to test the behavior of IGG6.
BBa_K5466034	mCerulean3 Saccharomyces cerevisiae codon optimized	Coding	mCerulean3 is cyan fluorescent protein derived from Aequorea victoria.
BBa_K5466035	Constitutive expression of Nb28-S102D Aga2P	Composite: Device	The expression of surface sdAb to capture AFB1 from the environment. Controlled by the constitutive promoter TDH3 (BBa_K124002) to ensure that there is always sdAb on the surface to capture AFB1.
BBa_K5466036	Constitutive expression of AntiAFB1-scFv1 EpoR Split-N-mCerulean	Composite: Device	The expression of surface AntiAFB1-scFv1 EpoR Split-N-mCerulean to observe if, in the presence of aflatoxin, dimerization occurs and mCerulean is reconstituted. Use with constitutive expression of AntiAFB1-scFv2 EpoR Split-C-mCerulean (BBa_K5466031).
BBa_K5466037	Constitutive expression of AntiAFB1-scFv2 EpoR Split-C-mCerulean	Composite: Device	The expression of surface AntiAFB1-scFv2 EpoR Split-C-mCerulean to observe if, in the presence of aflatoxin, dimerization occurs and mCerulean is reconstituted. Use with constitutive expression of AntiAFB1-scFv1 EpoR Split-N-mCerulean (BBa_K5466030)