Overview

The NKU-China-2025 team consists of 19 basic parts and 2 composite parts. They are used to sense early AD biomarkers in the gut and activate the butyrate synthesis pathway after "And-gate" judgement. Butyrate is then used to alleviate neuroinflammation and to prevent and treat the diseases.

These parts embody our synthetic biology approach: modular, standardized gene expression to achieve predictable outcomes across organisms. And the feasibility of building multifunctional biological machines. Most of the parts are compliant with RFC[10] or RFC[1000] (except one), two sets of composite parts have been functionally tested, and the "And-gate" logic has been successfully constructed.All parts are available for public use.

Basic Parts

Number	Short description	Long description
BBa_2549Z28B	Hbd: 3-hydroxybutyryl-CoA dehydrogenase	This enzyme catalyzes the reduction of acetoacetyl-coa to 3-hydroxybutoxyl-coa and is an element in the butyrate synthesis pathway.
BBa_25MON07J	Crt: Crotonase	This enzyme catalyzes the dehydration of 3-hydroxybutoxyl-coa to crotonyl-coa and is an element in the butyrate synthesis pathway.
BBa_25Q8O7D1	Ter: Trans-2-enoyl-CoA reductase	This enzyme is responsible for the reduction of crotonyl-coa to butoyl-coa.
BBa_25GUL9GW	BCoAT: Butyryl-CoA:acetate CoA-transferase	BCoAT catalyzes butyryl-coa to yield the final product butyric acid.
BBa_25RNTN9I	Radical response promoter pTRR1	This element can respond to the stimulation signal of free radicals and initiate subsequent gene expression. The promoter region of pTRR1 contains specific Oxidative Stress Response Elements (OSREs), which are recognized and bound by redox-sensitive transcription factors (Yap1 transcription factors).
BBa_25EJSV4B	GPA1/Gα1 fusion protein	The five amino acids of the C terminus of yeast GPA1 protein were replaced by the C terminus of human Gα1 protein. This genetic element can respond to human GPCR receptor binding, transmitting signals downstream.
BBa_251M1Q8U	GPCR receptor for human TAAR5 protein (N terminus with membrane homing peptide)	This element has yeast membrane-directed peptides that can be expressed on the cell membrane of yeast, bind to TMA and activate yeast GPCR receptor downstream signaling pathways
BBa_25XWUN58	pFUS1 promotor	The pFUS1 promoter is a promoter that specifically responds to the yeast pheromone mating pathway (MAPK signaling pathway), which can be activated to drive downstream gene expression (such as the Hbd enzyme or reporter gene EGFP) after the TAAR5 receptor is activated by TMA and signals through the chimeric G protein Gpa1.
BBa_257KIJW9	Codon-optimized human Bax protein	Bax protein translocates to the yeast mitochondrial outer membrane after expression and is inserted into the mitochondrial outer membrane by a C-terminal hydrophobic anchor sequence, where it undergoes oligomerization at the membrane. Bax oligomers form pores or channels in the outer mitochondrial membrane, disrupting mitochondrial membrane integrity and increasing mitochondrial membrane permeability. Cytochrome c is released from yeast mitochondria and induces apoptosis.
BBa_25T2KDPN	TETR promoter （responses to rtTA3）	Based on the yeast constitutive promoter CYC1, this element can bind to rtTA3 protein and initiate subsequent gene expression by adding seven tet operon sequence repeats.
BBa_25DVG514	rtTA3 protein (containing VP16 sequence + yeast NLS)	The optimized codon-optimized rtTA3 protein, with a VP16 sequence derived from Herpes Simplex Virus, is a very potent transcriptional activation domain that recruits the RNA polymerase II complex. The yeast NLS sequence can import the rtTA3 protein into the nucleus. The rtTA3 protein binds to tetracycline and can enter the nucleus to bind to the TETR promoter to initiate subsequent gene transcription.
BBa_25TX0FU3	common insulator in yeast	The insulator element contains the yeast tRNA gene and blocks the potential activation of the system by transcriptional activation domains or other synchronous transcriptional activation elements (such as enhancers) on the yeast genome.
BBa_252GI6E4	pPcha promoter recognized by the Lrp protein	Part of the pPcha promoter feedback shutdown system. The system consists of elements such as Lrp, pPcha, TetR system and TETR promoter. When butyrate synthesized by the engineered yeast reaches a certain concentration, Lrp binds to butyrate, activates the TetR system and expresses the TetR protein to repress the TETR promoter.
BBa_25MIZ9C7	Butyrate binding protein Lrp	Lrp proteins are part of the butyrate negative feedback shutdown system. The system consists of elements such as Lrp, pPcha, TetR system and TETR promoter. When butyrate synthesized by the engineered yeast reaches a certain concentration, Lrp binds to butyrate, activates the TetR system and expresses the TetR protein to repress the TETR promoter.
BBa_25OZXWPX	TetR protein Transcription factors binding to the TETR promoter	The TetR protein is part of the butyrate negative feedback shutdown system. The system consists of elements such as Irp, pPcha, TetR system and TETR promoter. When butyrate synthesized by engineered yeast reaches a certain concentration, it binds to Irp, activates the TetR system and expresses the TetR protein to repress the TETR promoter.
BBa_25MM73P7	EGFP protein	EGFP was as report gene used in our project, the element USES GenSript codon optimization tool codon optimization (https://www.genscript.com.cn/tools/gensmart-codon-optimization), Thus, it is adapted to yeast expression system
BBa_251X4W66	LcGC protein	LcGC can adhere yeast to colon cells after expression.
BBa_25ZVKXJ3	pADH1: promoter	Strong constitutive promoter from Saccharomyces cerevisiae driving high-level gene expression. Optimized to meet the requirements of RFC1000
BBa_250YD0ZH	pTEF1: promoter	Robust constitutive promoter derived from the TEF1 gene for reliable expression in yeast（Saccharomyces cerevisiae).

Composite Part

Number	Short description	Long description
BBa_25L5UP5N	TMA detection and reporting complex system in yeast	pRS416-TAAR5-GPA1-pFUS1-EGFP : The system consists of a human TAAR5 protein with an N-terminal membrane guidance peptide under the control of a yeast ( Saccharomyces cerevisiae ) constitutive promoter, a GPA1 (Gα1) fusion protein with a strong promoter, and an EGFP reporter gene under the control of a pFUS1 promoter. This system enables yeast to detect TMA, signal transduction and finally express a reporter gene, which is an important part of the "and gate" judgment in our project. In addition, it can be extended into a biological switch targeting TMA molecules in the future. Since it is a fully synthetic DNA element, there are no sequences between the elements and no restriction sites as well.
BBa_25VXKMA5	ROS detection and reporting complex systems in yeast	pGBKT7-pTRR1-EGFP: The system consists of a free radical responsive promoter, pTRR1, and a reporter gene, EGFP, which can be expressed in yeast upon free radical stimulation.