Boost the engineering of cyanobacteria

• As we discussed in the Human Practices, one of the reasons that restricts other iGEM teams from working with cyanobacteria is the lack of suitable lighting equipment. Therefore, we attempted to explore whether cyanobacteria could be normally cultured without the use of a professional lighting incubator.
  
  We selected four conditions: placing the culture by the window, at the entrance of the laboratory, under fluorescent lights, and in a professional lighting incubator. And test the feasibility of culturing Synechococcus elongatus PCC 7942 without the presence of light incubator.





We found that when cyanobacteria were directly placed under fluorescent lights, they also exhibited a good growth condition. In less than 120 hours, the cyanobacteria under the fluorescent lights were able to reach an OD600 of greater than 0.4, which is the biomass level sufficient for genetic transformation. In comparison, it took over 72 hours for cyanobacteria in the professional lighting incubator to reach this level (note that the initial OD600 of the two groups was not consistent), and the time difference between the two conditions was not as large as we initially anticipated.




Next, we aimed to further accelerate the growth rate of cyanobacteria to shorten the DBTL (Design-Build-Test-Learn) cycle. We expressed the katG gene in cyanobacteria in hopes of promoting faster growth. We introduced the constructed KatG plasmid and an empty control plasmid into cyanobacteria, then tested their growth under fluorescent lights. We found that cyanobacteria expressing the KatG plasmid indeed grew faster, reducing the time required to reach OD600 = 0.4 by approximately 12 hours, while also achieving a higher final biomass.



Additionally, we increased the amount of sucrose released into the environment by cyanobacteria, which could help other teams interested in co-culturing with cyanobacteria.

Complementary choice of model plant

• In plant biology research, while model plants like tobacco and Arabidopsis have been specifically designed to facilitate experimental studies, their cultivation remains both time-consuming and technically demanding. These plants often require precise growth conditions, extended life cycles, and careful maintenance, which can slow down the pace of research. Moreover, the expensive equipment and specialized facilities needed for plant-related projects, such as controlled growth chambers and advanced imaging systems, pose significant barriers to entry, especially for smaller labs or research groups with limited resources. These challenges highlight the need for more accessible and efficient model systems to accelerate discoveries in plant science.



Therefore, we would like to introduce the potential of using Wisconsin Fast Plants, a rapid-cycling relative of Arabidopsis developed by Professor Emeritus Paul H. Williams at the University of Wisconsin-Madison. These plants offer a much simpler operation and boast a significantly faster lifecycle of just 35 days, compared to traditional model plants like tobacco and Arabidopsis. Additionally, Wisconsin Fast Plants are fully compatible with our TTTT system and exhibit superior performance in leaf spray transfection, thanks to their smooth leaf surface, which lacks the villi or waxy layer that can hinder efficient transfection in other plants. This makes them an ideal alternative for accelerating research and experimentation in plant biology.

Accelerated plant synbio research

• 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.



In the past four years, our team members 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. In iGEM 2023, our team built a project based on it and gave the system a name- Carbon nanodot-based tracked, transformation, translation, and trans-regulation(TTTT) system.



This year, by enhancing the preparation process of CDP, the latest version, known as Smart-CD (patent pending), can effectively transfect plants through leaf spray for transient expression and seed soaking for genomic insertion. This method has been tested on several common crops.

TTTT VS Conventional solutions

• Agrobacterium, a Gram-negative bacterium and plant pathogen, causes crown gall disease in over 140 plant species. It can insert T-DNA, a portion of its tumor-inducing plasmid DNA, into plant cells with the help of virulence proteins. These proteins facilitate the transport of T-DNA from Agrobacterium to the plant cell wall and plasma membrane, promoting its integration into the plant nuclear genome at random locations.
  In 1987, Sanford et al. developed biolistic delivery, also known as particle bombardment or the gene gun. This method uses high-pressure helium to accelerate gold or tungsten particles coated with DNA, allowing them to penetrate recipient cells at high speed. However, factors such as helium pressure, particle size, and dosing frequency affect penetration efficiency and transformation levels.
  Although these conventional techniques have been used for over 50 years in plant biology, with numerous protocols and commercial kits available, our CDP method offers several advantages:
  
  • Species Independence and Higher Efficiency: Unlike Agrobacterium, the CDP technique doesn't rely on pathogen infection, making it applicable to a wider range of species, especially those naturally immune to Agrobacterium.
  • Faster Process: The time required can be reduced by up to 50% compared to conventional methods.
  • Versatile Applications: More scenarios can be designed around the TTTT system, which will be discussed in the following sections.

TTTT mediated plug-in stacking crops

• Evolution of resistance in pests can reduce the effectiveness of insecticidal proteins such as Bacillus thuringiensis (Bt) produced by transgenic crops. Introduction of more than one gene into crop plants simultaneously or sequentially, called transgene stacking, has been a more effective strategy for conferring higher and durable insect and disease resistance in transgenic plants than single-gene technology. It was thought to be "creating crops for the future."



Carbon dot-mediated transformation (CDP) holds significant potential for building stacking crops faster and more efficiently due to its species independence and higher transformation efficiency compared to conventional methods like Agrobacterium. Since CDP bypasses the need for pathogen-based infection, it can be applied to a broader range of plant species, including those that are resistant to Agrobacterium. This versatility accelerates the introduction and stacking of multiple transgenes into plants, enhancing resistance to pests and diseases. Furthermore, CDP's faster process allows for quicker generation of transgenic crops different gene parts, which are customized with the needs, therefore potentially improving agricultural productivity and sustainability.

TTTT mediated smarter fertilizer applying

• One of the pivotal issues that plagues phosphate utilization in agriculture is the highly inefficient uptake of this essential nutrient by plants. Since phosphate is very reactive, and most of the phosphate in the fertilizers went back to insoluble forms when they meet calcium and other earth elements in the soil before they are absorbed in plant. As a result, to enhance the crop production, we continue to dig out and extract resources from our planet, at a rate that the known mineral phosphate reserves are estimated to deplete within the next 50 to 100 years.



This inefficiency not only results in substantial resource wastage but also contributes to a plethora of environmental problems. Phosphate runoff from agricultural fields often leads to eutrophication in water bodies, causing harmful algal blooms, dead zones, and severe damage to aquatic ecosystems.
Therefore, in the year 2023 , our team introducing groundbreaking "phytosensor" revolutionizes the way we monitor phosphate levels within plants. This ingenious gene circuit comprises a low-phosphate-responsive promoter, an advanced low-noise expression amplifier part, and a highly luminous GFP as a reporter. The incorporation of this technology enables real-time monitoring of a plant's phosphorus status, thus providing valuable insights into its nutrient health. The utilization of a bright GFP protein as a reporter allows plant caretakers to visually assess the plant's phosphate status with ease, as it fluoresces brightly under UV light.
The product empowers farmers and plant scientists to make informed decisions about phosphate supplementation, ensuring that plants receive precisely what they need without the risk of overapplying fertilizers, without those expensive analytical devices. When entering the plant cells, the sensor detects the low-phosphate stress with its low-phosphate-responsive promoter. This signal then enters the amplifier system, through an artificial pathway with super-cool artificial transcription factors, that eventually yields a large quantity of super bright GFP protein that can be seen under UV light with the naked eye!

TTTT mediated allel tracking

• Together with local chromatin structure, gene accessibility, and the presence of transcription factors, gene positioning is implicated in gene expression regulation. Fluorescence in situ hybridization (FISH) approaches, such as padlock-FISH, enable to detect a single-copy locus using the fixed plant material. However, imaging techniques using non-living organisms are insufficient to track spatial and temporal dynamics of loci.
  The ANCHOR method is a DNA-labeling tool derived and optimized from chromosome partitioning complex of bacteria. A single-copy of parS−1-kb-long fragment—serves as a binding platform for ParB proteins.



Therefore, the TTTT system can combined with ANCHOR method. By transforming plants using TTTT rather than agrobacterium which orginally use in ANCHOR, the experiment time can be much shorter and it can be preformed on more species without the limitation.



The project already initiated and was tested by our team and imperial-college team. Although we are unable to share more results at this time. But we are looking forward to the update in next year.

TTTT mediated system biology research

• Using the TTTT system to study gene networks in the field of system biology was also one of our proposals. With the recently added genomic insertion capability for TTTT system, it can be use to study the gene network in more kind of tissue type. Such as stem or root which are hard to be transfect by straying. Although the project is still on going, we are looking forward to presenting more updates in 2025.

TTTT planned update in 2025

To better understand the performance, we also set a series of experiments after the competition. And we are looking forward to presenting them in iGEM 2025.
1. Validate the effectiveness of genomic insertion on sorghum.

• Soak the sorghum seed in transformation mixture during germination > grow the sorghum plant to vegetative stage > validate the genomic insertion (TAIL-PCR/southern blot/nanopore sequencing) /Validate the Protein expression(western blot/ Elisa/ fluorescent imaging) > Assess if the genomic insertion is heritable in the offspring.

2. Validate the effectiveness of transfection and transient expression on Sorghum leaf.

• spray the transformation mixture onto the sorghum leaf > Validate the Protein expression(western blot/ ELISA/ fluorescent imaging)

3. Increase the genomic insertion event by add parts on the transformation vector to increase the change of homology-directed repair or combine with CRISPR

• - Construct different transformation vector by adding parts to increase the HDR efficiency >Soak the sorghum seed in transformation mixture during germination > grow the sorghum > measure the effectiveness of genomic insertion (LM-PCR/southern blot/nanopore sequencing) /measure the strength of Protein expression(western blot/ ELISA/ fluorescent imaging)

This series of work can provide new ideas for the iGEM community and subsequent teams, so that the subsequent iGEM teams are more daring to try to conceive the topic of carbon sequestration and negative carbon, space-related microbial co-culture and plant modification, which can greatly enrich the research community related to iGEM and even synthetic biology. The collaboration with imperial-college team (IC HP(Collaboration)) was just a begining. But we are looking forward to a larger plant synbio community in iGEM later.
We are looking forward to showing more updates soon! Please refer to: SZ-SHD2025 Plant

References

[1]Tabashnik, Bruce E., Thierry Brévault, and Yves Carrière. "Insect resistance to Bt crops: lessons from the first billion acres." Nature biotechnology 31.6 (2013): 510-521.
[2]Improving Seed Shattering Resistance in Wild O. alta Rice with Mesoporous Silica Nanoparticle Delivery Systems Zhujiang Liu, Jingkun Zhang, Yao Cai, Hang Wang, Mingjie Luo, Jiayang Li, Hong Yu, Xiangbing Meng, and Yuhong CaoNano Letters 2024 24 (38), 11823-11830DOI: 10.1021/acs.nanolett.4c02297
[3]Meschichi, Anis, et al. "ANCHOR: a technical approach to monitor single-copy locus localization in planta." Frontiers in Plant Science 12 (2021): 677849.