TTTT: Carbon nanodot-based tracked, transformation, translation, and trans-regulation system

Why TTTT?

Genetic transformation stands as a pivotal realm within the domain of functional genomics research and molecular breeding within the realm of plant science. The focal point of genetic engineering, molecular biology, and genetic breeding revolves around the creation of secure, efficient, and innovative genetic transformation techniques. Conventional approaches for transforming plants encompass the use of Agrobacterium tumefaciens infection, particle bombardment, and viral infection. These techniques have been extensively applied in various model plants, such as Arabidopsis thaliana and tobacco, as well as annual or biennial herbs. In recent years, with the swift progress of nanoscience and nanotechnology during the latter part of the 20th century, nanomaterials have gained widespread adoption in nanobiology and gene therapy. Their appeal lies in their diminutive size, expansive surface area, biocompatibility, biodegradability, low toxicity, and reduced immunogenic properties.

TTTT 101

In the past three years, our team members (Main contributors: Lei Yingjie (UIUC-USA); Wu Jixiao (Imperial College London-UK); Xu Leyi (University of Toronoto-CA); Tao Yujie(Soochow University-China)) with Jianhuang's lab at Soochow university focused on the research of PEI-modified CDs (CDP). A type of carbon quantum dot with a positive charge can be implemented through transfection. The CDP could easily penetrate and transfer plasmid DNA into different plant cells by smearing plant leaves or soaking roots resulting in high protein expression levels. Therefore, we not only used this tool in our iGEM project this year but also listed some future plans that we are going to implement in the next few years.

TTTT VS Traditional solution

Agrobacterium tumefaciens infection, particle bombardment, and viral infection. These techniques have been extensively applied in various model plants. Although these techniques have well-established protocols or even commercial kits on sale, as well as multiple useful features. Our TTTT system still has several advantages.

1. TTTT VS Agrobacterium tumefaciens infection

Agrobacterium, a Gram-negative bacterium and plant pathogen, causes crown gall disease in over 140 plant species. Agrobacterium can insert T-DNA, a portion of its tumor-inducing plasmid DNA (pDNA), into plant cells with the aid of virulence proteins. Virulence proteins assist the transport of T-DNA from Agrobacterium to the plant cell wall and plasma membrane and promote the integration of T-DNA into the plant nuclear genome at a random location.

Although Agrobacterium tumefaciens infection technique has been used for more than 50 years in plant biology, with lots of protocols and commercial kits on sale, Our CDP still have some advantage when compare with it.

Major advantages contain:
- Species dependent and higher efficiency: The CDP transform technique do not require pathogen infection, therefore, it can be applied to more species. Since some species will have natural immunity to Agrobacterium and will be hard to infect.
- Faster: Time taken for transient expression using CDP only take 3-5 days

1. TTTT VS Gene Gun In 1987, Sanford et al. developed the technique of biolistic delivery, also known as particle bombardment or gene gun. In this approach, high-pressure helium pulses are used to accelerate gold or tungsten particles coated with DNA. These particles gain sufficient momentum to pierce recipient cells at high speed. However, several experimental parameters affect plants' penetration efficiency and genetic transformation levels, such as helium gas pressure, net particle size, and dosing frequency. This method also considered as the time taken was too long(5-7 weeks) as the transformed leaf tab needed to be tissue cultured and regrow into a whole plant.

Key features of our solution:

Applications for the TTTT system:

Hence, we tried several applications based on this system.
1. Transient expression Bt toxin: Biopesticide 2021 SZ-SHD | Part:BBa_K3686010

2. Low phosphate phytosensor (SZ-SHD 2021)
Plants usually have severe responses under the pressure of low phosphate. Some genes will be turned on to allow plants to better adapt to this situation. Therefore, in order to build a low-cost, real-time soil phosphate sensor, we first found a low phosphate response promoter in Maize which was reported by Jianrong Bai in 2018...... Engineering

Conclusion...till 10/11/2023

Since we are still improving and developing potential applications of CDP, we might find more interesting uses for it. We are eagerly looking forward to discussing with researchers working in similar fields and bringing more interesting plant synbio iGEM projects the next year! Or change our design into business? Nothing is impossible.