Editing Editing Team:HUST-China/Modeling on Cellular Level
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With the benefit of synthetic biology, we built up our network with some characterized parts to carry out the mission of sand solidification. However, whether our circuit will be able to achieve its goal depends on the key to the following questions.
- Will the darkness induction system be able to switch efficiently to control the expression of target proteins?
- How much Si-tag and Mcfp-3 our strains produce in the end?
- What strategy should we take in practice to make full use of our product?
The answer to these questions will be shown in our modeling as well as the guidance for future wet-lab experiments.
To ensure that our circuit is capable, we built a DDEs (Delay Differential Equations) model based on Michaelis-Menten equation and Chemical reaction rate equation, which includes three parts, to get the insight of how each part works cooperatively.
The code of our DDEs model can be downloaded here.
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_K159209 | 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_K159212 | 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 with trp operator. It will be repressed by trpR and LovTAP.
We improved BBa_K191007, remove the illegal sites without affecting its function by site-directed mutagenesis for meeting the requirements of RFC10. Finally we 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 |
BBa_K1592018
BBa_K1592018
BBa_K1592018
