Periodic Reporting for period 1 - Colour cotton (Bioengineer novel eco-friendly natural colour fibre through genetic manipulation of the phenylpropanoid and betalain pathways in cotton)
Reporting period: 2022-03-28 to 2024-03-27
The project was trying to develop a more versatile and broadly applicable biological toolbox with coloured pigments as the report signals, as outlined in the objectives below:
1) functionally split DODA⍺ for protein complementation assay;
2) use split DODA⍺ in testing protein-protein interaction as an alternative to the widely used split fluorescent protein (BiFC) or luciferase (FLCA) systems;
3) characterize the new protein complementation system;
4) explore alternative applications of using betalain as a signal reporter through manipulating DODA⍺.
The project allowed me to explore a plant pigment-based report system in multiple molecular biological assays. With its special characteristics in signalling presenting and observation, the new system provides academic research and industry with a novel toolbox for testing protein-protein interaction and developing novel biosensors.
1) functionally split DODA⍺ for protein complementation assay;
2) use split DODA⍺ in testing protein-protein interaction as an alternative to the widely used split fluorescent protein (BiFC) or luciferase (FLCA) systems;
3) characterize the new protein complementation system;
4) explore alternative applications of using betalain as a signal reporter through manipulating DODA⍺.
The project allowed me to explore a plant pigment-based report system in multiple molecular biological assays. With its special characteristics in signalling presenting and observation, the new system provides academic research and industry with a novel toolbox for testing protein-protein interaction and developing novel biosensors.
The main results achieved so far are:
1, functionally split the key enzyme in betalain biosynthesis.
2, the split enzyme enables visualizing protein-protein interaction with betalains.
3, the split enzyme can be used in visualizing chemicals-induced protein-protein interaction.
4, the split enzyme can visualize protein affinity in an interaction complex.
5, the betalain biosynthesis enzyme is dysfunctional when tethered to the membranes.
6, visualize protease with betalains
The main toolbox I developed from the project is a novel bimolecular complementation assay to test protein-protein interaction. The other part of the work could be used to develop novel biosensors for visualizing protease. It shows several special properties that make it competitive or outstanding to other widely used toolboxes.
a, it applies the betalain pigments as a report signal. The signal is stable, visible, measurable and eco-friendly.
b, it is genetically or partially encoded and supplied with a substrate.
c, it shows a low background (nonself-assembly) and protein conformation-dependent manner.
d, it can be used to visualize protein affinity.
e, it is low-cost and feasible.
1, functionally split the key enzyme in betalain biosynthesis.
2, the split enzyme enables visualizing protein-protein interaction with betalains.
3, the split enzyme can be used in visualizing chemicals-induced protein-protein interaction.
4, the split enzyme can visualize protein affinity in an interaction complex.
5, the betalain biosynthesis enzyme is dysfunctional when tethered to the membranes.
6, visualize protease with betalains
The main toolbox I developed from the project is a novel bimolecular complementation assay to test protein-protein interaction. The other part of the work could be used to develop novel biosensors for visualizing protease. It shows several special properties that make it competitive or outstanding to other widely used toolboxes.
a, it applies the betalain pigments as a report signal. The signal is stable, visible, measurable and eco-friendly.
b, it is genetically or partially encoded and supplied with a substrate.
c, it shows a low background (nonself-assembly) and protein conformation-dependent manner.
d, it can be used to visualize protein affinity.
e, it is low-cost and feasible.
The new methods developed from this project would facilitate research, especially in protein-protein interaction, and biosensor development. I have most of the work done in plants, but it should be easy to transfer to other research organisms. The basic advantage of these outputs is making the relative area more feasible and approachable while maintaining the same or even higher quality. For applying in a qualitative study, it doesn’t need any expensive or specialized devices and chemicals. For applying in a quantitative study, it offers several ways to measure the report signals including digital quantification on images, primary quantification through spectrum absorbent analysis or precise analysis by HPLC. The stability of the report signal makes it less affected by environmental changes.
With the development of AlphaFold, protein research will be more feasible than before. More and more biological research will benefit from an insight into protein structure. The bimolecular complementation assay developed from this project will be useful in verifying protein interaction with confident structural information. It can be useful in verifying protein structure that is predicted by AlphaFold. The split protein method benefits from the structural information acquired but it can be used to explore protein structure and folding.
With the development of AlphaFold, protein research will be more feasible than before. More and more biological research will benefit from an insight into protein structure. The bimolecular complementation assay developed from this project will be useful in verifying protein interaction with confident structural information. It can be useful in verifying protein structure that is predicted by AlphaFold. The split protein method benefits from the structural information acquired but it can be used to explore protein structure and folding.