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Contribution

Contribution in Synthetic Biology

Our iGEM project brings several groundbreaking contributions, including a novel genetic circuit that integrates population control, safety mechanisms, and targeted therapeutic applications.

Innovative Genetic Circuit

A key innovation in our design is the use of antisense mRNA to target dnaA, a gene essential for initiating DNA replication in bacteria. This approach, which has not been widely explored, offers a new and novel method for controlling bacterial growth by specifically downregulating dnaA expression. By reducing dnaA protein levels, we can achieve reversible cell cycle arrest, adding precision to our quorum sensing-based population regulation system. This unique targeting of dnaA through antisense technology, combined with our linalool-producing circuit, enhances our system's ability to maintain stable bacterial populations while ensuring biosafety through additional features like auxotrophy and a kill switch.

Part Contributions

The main part contributed by us is Part:BBa_K5425004 This is the complete circuit responsible for regulating reversible cell growth arrest. It is designed to control bacterial population dynamics through a quorum sensing mechanism. Other parts contributed are:

Part: Part:BBa_K5425004

Part name: Population Control Module

The population control module is a carefully engineered circuit that regulates cell density through quorum sensing.

Type: COMPOSITE

Length: 3933 bp

Part: Part:BBa_K5425002

Part name: Antisense dnaA

This part encodes an antisense mRNA that is complementary to approximately 400 bp of the primary transcript of dnaA (a replication initiator protein) in Escherichia coli.

Type: BASIC

Length: 437 bp

Part: Part:BBa_K5425005

Part name: Oscillation characterization circuit

This circuit is designed to fine-tune the parameters of various components of part BBa_K5425004.

Type: COMPOSITE

Length: 4240 bp

Part: Part:BBa_K5425007

Part name: Threshold characterization circuit

This circuit was designed to determine the threshold population at which the pLux promoter becomes active due to the binding of TetR and AHL.

Type: COMPOSITE

Length: 2555 bp

Applications of Population Control Module for the Future

The bacterial population control circuit we have developed offers exciting future extensions and possibilities, making it a powerful tool in synthetic biology. Here are several ways it could be applied in the future and come handy to synthetic biologist in the following listed ways.

Bioproduction and Metabolic Engineering:

In industrial contexts, bacterial populations are often used to produce valuable compounds like biofuels, pharmaceuticals, fragrances, and more. Our circuit can optimize these production processes by maintaining a healthy, stable bacterial population, preventing overgrowth that could lead to resource depletion and the accumulation of toxic byproducts. This control would enable more efficient and continuous bioproduction, enhancing both productivity and sustainability.

Bioremediation:

The system could also be adapted for environmental applications such as bioremediation, where bacteria are employed to break down contaminants. By controlling bacterial growth, we could design self-sustaining systems that effectively clean polluted environments like soil and water without overwhelming the ecosystem. This would allow for more targeted and efficient treatment of contaminated sites.

Therapeutic Applications:

In medicine, genetically engineered bacteria are being explored as vehicles for drug delivery to specific tissues, such as targeting cancer cells or modifying the gut microbiome. Our circuit could be used to regulate the population of therapeutic bacteria, ensuring that they deliver drugs at the right time and location while preventing overgrowth that could lead to complications. This population control would help optimize the safety and efficacy of bacterial therapies in clinical settings.


By incorporating population control into various fields, this circuit could revolutionize how we use bacteria in both industrial and medical applications, offering a more controlled and sustainable approach to harnessing microbial functions.

DryLab Contribution

Population Equation

We developed a new growth equation taking reference from the original logistic growth model, which helps in measuring the onset of the death phase of the bacterial population as a function of toxic secondary metabolite accumulation and substrate concentration in the medium.

The resulting equation is given below:

        dN/dt  =  r0/((1+(m*[CCA])^r3))N –  r0/K  N² –  (aNt⁴)/((t⁴+ b⁴)(1+(c*S)^n))
                    

We introduced this new term, (aNt⁴)/((t⁴+ b⁴)(1+(c*S)^n)), which governs the above-stated death phase onset.

AntiRNA Software

We developed AntiRNA, which can predict the best antisense mRNA length for binding to the sense mRNA of DnaA to obtain the best desired output from our quorum sensing circuit. This is done by integrating our developed math model for quorum sensing with the IntaRNA docking software and DegScore deep learning algorithm.

Our algorithm just takes the sense mRNA sequence as input, and by following the directions generated by the code, it produces the best antisense mRNA length.

The repository link of our software is here.

WetLab Contribution

We conducted a fungal survival assay using various concentrations of linalool, a naturally fragrant monoterpenoid, to observe its effects on fungal growth. Linalool was found to ilinaloolungal growth at a minimum inhibitory concentration (MIC) of 256 µg/ml without being toxic to the cells. This is the first time linalool has been applied in this context in iGEM, successfully demonstrating its potential to inhibit fungal growth. we further approach the development of the protocols developed to grow the fungus and the survival assay which can be very well used by future teams.

Link to fungal assay here.

Human Practices Contribution

As part of our lasting impact, we created educational posters for each stall, which were key in engaging students during the event. These posters covered topics like bacterial diversity, chemical safety in cosmetics, and synthetic biology innovations. Now archived, they serve as resources for future iGEM teams to build on our outreach efforts. By contributing these materials, we aim to ensure that public awareness of synthetic biology continues to grow, fostering greater accessibility and engagement for future generations.