Difference between revisions of "Team:HUST-China/Basic part"

This year, we iGEM_HUST-China team constructed a darkness induction system based on yeast two hybrid, which is used to promote the expression of downstream working proteins: Si-tag and Mcfp-3.
With the basic or composite parts we used in our Euk.cement project, together with some former parts in pools we improved by mutating correction of some mistakes, this year we totally constructed, sequenced, fully documented and submitted 26 parts.

Following documented our parts submitted.

Parts in Our Euk.Cement Project

1. Basic Parts : Light Control Parts of Darkness Induction System

Part Number	Description	Nickname
BBa_K1592005	GalBD-CRY2 Fusion for Yeast-Two-Hybrid	BD-CRY2
BBa_K1592006	GalAD-CIB1 Fusion for Yeast-Two-Hybrid	AD-CIB1
BBa_K1592015	photoreceptor cryptochrome 2	CRY2
BBa_K1592016	a basic helix-loop-helix protein	CIB1

Our light-control system is based on a Yeast-Two-Hybrid system. Cryptochrome 2 (CRY2) is a blue light stimulated photoreceptor, when exposed to blue light, CRY2 fused with BD would interact with CIB1 fused with AD ,this CRY2-CIB1 interaction will bring AD and BD of Gal4 gene together to activate the downstream negative control parts.
We verified our Light-control system by measuring β-galactosidase activity that induced by Gal4.

Composite part

Part Number	Description
BBa_K1592018	Pgal1+rox1+cyc1_terminator

This composite part BBa_K1592018 consists of a Pgal1 promoter, a ROX1 inhibitor gene that is controlled by Pgal1 promoter and a terminator, The Pgal1 promoter can be activated by CRY2-CIB1 light-control parts through UAS. Expressed ROX1 protein will inhibit following working parts from expression.

2. Basic Parts: Secrete and Surface Display

Part Number	Description	Nickname
BBa_K1592000	LIP2 prepro(signal peptide)	LIP2 prepro
BBa_K1592002	Yarrowia lipolytica cell wall protein 3	YLcwp3

LIP2 prepro is a signal peptide. When fused to the N-terminal of target protein, the expression products will be secreted out of cell.

YLcwp3, also known as an anchor domain, is a yeast cell wall protein. When fused to the C-terminal of target protein, the expression products will be displayed on the cell wall.

3. Basic Parts: Silica binding proteins (Si-tag) Collection

Part Number	Description	Nickname
BBa_K1592007	LIP prepro + E. coli ribosomal protein L2 (1-60) + YLcwp3 Fusion	Si-tag1-his
BBa_K1592008	LIP prepro + E. coli ribosomal protein L2 (61-202) + YLcwp3 Fusion	Si-tag2-his
BBa_K1592009	LIP prepro + E. coli ribosomal protein L2 (203-273) + YLcwp3 Fusion	Si-tag3-his
BBa_K1592010	LIP2 prepro + E. coli ribosomal protein L2 (1-202)+ YLcwp3 Fusion	ST12
BBa_K1592011	LIP prepro + E. coli ribosomal protein L2 (61-273) + YLcwp3 Fusion	ST23
BBa_K1592012	LIP prepro + E. coli ribosomal protein L2 (1-60,203-273) + YLcwp3 Fusion	ST13
BBa_K1592013	LIP prepro + E. coli ribosomal protein L2 (1-60,GS linker,202-273) + YLcwp3	ST1L3-his
BBa_K1592014	LIP prepro + E. coli ribosomal protein L2 (1-273) + YLcwp3	ST123

This collection consists of several silica binding proteins (Si-tag) that can be surface displayed. Si-tag is 50S ribosomal protein L2 in the genome of E.coli, which was found to bind tightly to silica particles. The full length 50S ribosomal protein L2 protein consists of three domains showing different silica binding binding intensity. We constructed 8 different Si-tag isoforms with different truncations and domain combinations of this 50S ribosomal protein L2. We further added LIP prepro and YLcwp3(see below) to the terminals of Si-tag, thus Si-tag can be secreted and surface displayed on the cell wall.Finally we tested their silica binding intensity in situ.

Click HERE or part number to see more details.

4. Basic Parts: Flocculating Protein Mcfp3

Part Number	Description	Abbreviation
BBa_K1592001	Mytilus californianus foot protein 3(Mcfp3) variant 3	Mcfp3
BBa_K1592003	Mcfp3 with LIP2 prepro	LIP-Mcfp
BBa_K1592017	Mcfp3 with XPR2 pre	XPR2-Mcfp

Mcfp-3 is a flocculating protein secreted by Mytilus californianus (mussels). The protein is of significance to the formation of filopodia to help mussels permanently or temporarily tether to the surface of solid surface of reef or ships.
LIP2 and XPR2 are signal peptides. Signal peptide is added to the top of Mcfp3 sequence, thus Mcfp-3 can be secreted out of cell.

Composite Part

Part Number	Description
BBa_K1592019	Panb1+XPR2 pre-Mcfp3

This composite part BBa_K1592019 consists of a Panb1 promoter, and a XPR2 pre signal peptide initiated Mcfp3 gene. With this part, the Mcfp-3 gene can be expressed and The product flocculating protein will finally secreted outside cell.

5.Basic Parts: Promoter hp4d

Part Number	Description	Nickname
BBa_K1592004	promoter hp4d	Php4d

Promoter hp4d is a recombinant promoter which can strongly promote gene expression in any culture medium. The gene promoted by Php4d usually expresses at the early stage of stabilization.

Parts Improvement

Basic Parts: Improved Ptrp Promoter

Part Number	Description	Nickname
BBa_K1592020	Ptrp mutant1	Ptrp1
BBa_K1592021	Ptrp mutant2	Ptrp2
BBa_K1592022	Ptrp mutant3	Ptrp3

Ptrp is a promoter of trp operon exists in parts pools with cat. Number BBa_K191007. It will be repressed by trpR and LovTAP. This year, We improved this BBa_K191007 biobrick. We removed the illegal restriction sites without affecting its function by site-directed mutagenesis to fulfill the requirement of RFC10. Finally we constructed three ptrp mutants, named Ptrp mutant1, Ptrp mutant2, and Ptrp mutant3.

Composite Part

Part Number	Description
BBa_K1592023	Ptrp mutant1+RBS+GFP
BBa_K1592024	Ptrp mutant2+RBS+GFP
BBa_K1592025	Ptrp mutant3+RBS+GFP

These composite parts consist of a improved Ptrp promoter, a RBS and a GFP fluorescent report gene. With this parts, the promoting function of Ptrp promoter can be evaluated.

These Ptrp promoters were not really applied in our project. However, we successfully found the defects of existed biobrick in pools and improved it to fulfill the basic requirement of biobrick property. This parts improvement work is a required achievement in judging