Team:HUST-China/Basic part
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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.
BBa_K1592018
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 |
| BBa_K1592008 | LIP prepro + E. coli ribosomal protein L2 (61-202) + YLcwp3 Fusion | Si-tag2 |
| BBa_K1592009 | LIP prepro + E. coli ribosomal protein L2 (203-273) + YLcwp3 Fusion | Si-tag3 |
| BBa_K1592010 | LIP2 prepro + E. coli ribosomal protein L2 (1-202)+ YLcwp3 Fusion | Si-tag12 |
| BBa_K1592011 | LIP prepro + E. coli ribosomal protein L2 (61-273) + YLcwp3 Fusion | Si-tag23 |
| BBa_K1592012 | LIP prepro + E. coli ribosomal protein L2 (1-60,203-273) + YLcwp3 Fusion | Si-tag13 |
| BBa_K1592013 | LIP prepro + E. coli ribosomal protein L2 (1-60,GS linker,202-273) + YLcwp3 | Si-tag1L3 |
| BBa_K1592014 | LIP prepro + E. coli ribosomal protein L2 (1-273) + YLcwp3 | Si-tag123 |
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 | Nickname |
|---|---|---|
| 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.
| 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.
BBa_K1592018
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.
BBa_K1592018
