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

This year, we, iGEM15_HUST-China, constructed a light control system basing on yeast two hybrid, which was used to drive the expression of downstream viscous protein.
We totally constructed, sequenced and submitted 26 parts.

Following are all of our parts.

Basic Parts

1.Light Control

Part Number	Description	Abbreviation
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 the Yeast-Two-Hybrid system. Cryptochrome 2 (CRY2) is a blue light stimulated photoreceptor, when exposed to blue light, it would interact with CIB1. A Gal4 DNA sequence was fused to their C terminus, thus the interaction between two proteins would activate the downstream expression.
We measured β-galactosidase activity as the validation test of our Light-control system.

2.Viscous Protein

Mcfp

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 foot protein secreted from Mytilus californianus. The protein is of significance to the formation of byssus to help mussels permanently or temporarily tether to the surface of solid surface of reef or ship-body.
LIP2 and XPR2 are signal peptides. Signal peptide is added to the behind of Mcfp3 sequence, thus Mcfp can be secreted out of cell.

Si-tag Collection

Part Number	Description	Abbreviation
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 Si-tag proteins. Si-tag is 50S ribosomal protein L2 in the genome of E.coli, which was found to bind tightly to silicon particles. The Si-tag consists of three domains showing different binding strength. We combined the single domain, and tested their final binding strength.

Besides, we added LIP prepro and YLcwp3(see below) to the terminals of Si-tag, thus Si-tag can be secreted then surface displayed on the cell wall.

Click HERE or part number to see more details.

3.Secrete and Surface display

Part Number	Description	Abbreviation
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 interest protein, the expression products will be secreted out of cell.

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

4.Others

Part Number	Description	Abbreviation
BBa_K1592004	promoter hp4d	Php4d

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

Improved part

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

Ptrp is a promoter of trp operon. It will be repressed by trpR and LovTAP.
We improved BBa_K191007, removed the illegal sites without affecting its function by site-directed mutagenesis for meeting the requirements of RFC10. Finally we successfully constructed three ptrp mutant, called Ptrp mutant1, Ptrp mutant2, and Ptrp mutant3.

(These promoters didn’t really applied to our project, for they were our reserve choices)

Composite Part

Part Number	Description
BBa_K1592018	Pgal1+rox1+cyc1_terminator
BBa_K1592019	Panb1+XPR2 pre-Mcfp3
BBa_K1592023	Ptrp mutant1+RBS+GFP
BBa_K1592024	Ptrp mutant2+RBS+GFP
BBa_K1592025	Ptrp mutant3+RBS+GFP