Sensing Module (Quorum Sensing-Based Population Detection)

The Sensing Module detects bacterial population size using a quorum sensing mechanism. As bacteria grow, they produce an autoinducer molecule called AHL (N-acyl homoserine lactone) via the enzyme LuxI. The AHL concentration rises with the bacterial population. When it hits a critical level, AHL binds to the LuxR transcription factor inside the bacteria.

This binding causes AHL-LuxR complexes to dimerize, and these dimers pair up to form tetramers. These tetramers then bind to specific DNA sequences known as lux boxes, located upstream of the plux promoter. This binding activates the plux promoter, leading to the expression of genes in the Execution and Regulation modules. Essentially, this module links bacterial population size directly to gene expression. More bacteria mean more AHL, and once the threshold is reached, the gene expression cascade kicks in.

Execution Module (Cell Cycle Arrest for Population Control)

The Execution Module controls bacterial growth once the population hits a preset threshold. This is achieved through the expression of a cell cycle arrest gene (cca) located downstream of the plux promoter. When enough AHL is produced, the plux promoter activates, leading to the expression of cca.

The cca gene causes a halt in cell division across the bacterial population, preventing further growth. This is crucial because unchecked growth could lead to nutrient depletion and the buildup of toxic byproducts, reducing linalool production efficiency.

Introducing our cell cycle arrest gene

• Antisense against dnaA

Why antisense?

Steric Blocking Antisense Technology involves using antisense RNA to inhibit gene expression by binding to target mRNA, preventing translation. This technique gained traction in the 1990s, paving the way for therapeutic applications in genetic disorders.

Longer antisense RNAs may be more effective at silencing DnaA compared to shorter oligonucleotides for several reasons. Their increased length allows for tighter and more stable binding to the target mRNA, enhancing the silencing effect. Additionally, longer RNAs can cover more regions of the mRNA, potentially blocking multiple functional sites and more effectively preventing translation. They can also be designed to specifically target the desired mRNA, which reduces the chances of interacting with unintended transcripts—an issue often seen with shorter oligonucleotides.

Furthermore, longer antisense RNAs tend to be more stable within the cell, allowing for prolonged gene silencing. Finally, they can engage different pathways for RNA degradation, providing multiple mechanisms for effective silencing. These advantages collectively make longer antisense RNAs a powerful tool for targeting DnaA and other genes.

The silencing effect of longer antisense RNAs is reversible due to several factors. First, once the antisense RNA is degraded or removed, the target mRNA can be free to resume translation and expression. This is particularly important because the cellular mechanisms involved in RNA degradation do not permanently alter the mRNA itself. Additionally, the binding of longer antisense RNAs is non-covalent, meaning that it doesn’t create permanent changes in the target or the cell’s genetic material.

This allows for a dynamic regulation of gene expression, where the effects can be turned on or off as needed, enabling researchers to study gene function and potentially tailor therapies without long-lasting impacts.

Why dnaA?
In our project, we aim to control bacterial population growth in a reversible manner without permanently affecting cell viability. To achieve this, we targeted DnaA, a key regulator of replication initiation. DnaA reaches its peak concentration and plays a vital role by binding to the origin of replication (oriC) in its ATP-bound form (DnaA-ATP). This binding triggers DNA unwinding and initiates replication.

DnaA utilizes the energy from ATP hydrolysis to introduce a bend or kink in the DNA, creating tension in the double helix. This tension facilitates the unwinding of DNA at the AT-rich regions of oriC, allowing the replication process to commence effectively.

Complete Arrest and Population Recovery

Our approach focuses on the reversible regulation of DnaA expression through the use of antisense RNA, which binds to DnaA mRNA, blocking its translation and temporarily halting DNA replication. This mechanism stops cell division without permanently compromising cell viability. The regulation of antisense RNA production is dependent on a quorum-sensing module that responds to population density. When the bacterial population reaches a certain threshold, the quorum-sensing system activates antisense RNA production, inhibiting DnaA expression and halting replication. As the population declines, the system reduces antisense RNA production, allowing replication to resume.

Complete arrest isn’t ideal for long-term functionality since cells naturally die over time. To keep the population sustainable, we need periodic recovery. As old or damaged cells die, new ones should replace them, keeping the population functional. This is where the Regulation Module comes in, managing population recovery.

Regulation Module (Recovery and Modulation of Population Growth)

The Regulation Module adds reversibility and fine control over the bacterial population. After triggering cell cycle arrest, the system allows periodic recovery to maintain a healthy population.

This module features two negative feedback loops to manage AHL levels and modulate population control:

TetR Control of AHL Production

To prevent continuous AHL production and indefinite population arrest, we regulate the luxI gene (responsible for AHL production) under the ptet promoter. TetR, a repressor protein, binds to the ptet promoter, blocking luxI expression and halting further AHL production. This keeps AHL levels from rising and prolonging arrest.

AHL Degradation by AiiA

We also need to remove existing AHL to allow recovery. The enzyme AiiA degrades AHL in the media. We’ve added a degradation tag to AiiA to ensure it breaks down AHL only as long as needed. This way, AHL concentrations decrease once the population is arrested.

Both TetR and AiiA are expressed under the plux promoter, so they’re produced only when AHL levels are high. As bacteria die and AHL is degraded, its concentration drops. When AHL falls below the critical threshold, LuxR dissociates from the plux promoter, stopping the expression of cca, TetR, and AiiA. This ends cell cycle arrest and allows the population to recover and grow.

Cyclic Control and Population Oscillations

Together, these modules create a dynamic system that oscillates between growth and arrest phases, keeping the bacterial population stable. Negative feedback loops ensure that when the population becomes too large, cell cycle arrest is triggered. As cells die and AHL levels drop, the population can recover and resume growth.

This cyclical behavior keeps the population healthy and functional without depleting resources.The frequency and amplitude of these oscillations depend on parameters like AHL production and degradation rates, as well as the effectiveness of the arrest and recovery mechanisms. By fine-tuning these parameters, we can maintain a stable bacterial density that supports continuous linalool production while avoiding uncontrolled growth.

MEV SYSTEM DESIGN

Our project revolves around production of Linalool, a monoterpene, efficiently to battle against bad odor and foot microbial infections, with a genetically modified organism, placed in the vicinity of the foot but not in its direct contact. An integral part of E.solei the production of Linalool in our experimental chassis (E.coli), which we categorize as our Module 1. For this we delve deeper into the origin and pathways through which monoterpenes like Linalool as produced in other organisms.

The Mevalonate Pathway (MEV) in Plant Species: The BluePrint

The mevalonate pathway in plants is a crucial metabolic route responsible for synthesising isoprenoids, which include a wide variety of essential compounds such as terpenes, sterols, and hormones. This pathway begins with the condensation of acetyl-CoA to form HMG-CoA, which is then converted to mevalonate by the enzyme HMG-CoA reductase. Mevalonate is subsequently phosphorylated and decarboxylated to produce isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), the fundamental building blocks of isoprenoids.

The MEV pathway governs productions of isoprenoids which contribute to the formation of secondary metabolites, having ecological functions such as attracting pollinators or deterring herbivores.

The production of Linalool, needed us to direct the MEV pathway from the compounds DMAPP to GPP (geranyl diphosphate) and finally Linalool. For this we needed to put in two genes of our own : namely GPPS (geranyl diphosphate synthase) and LIS (Linalool synthase).

Initially the team had believed in trying to place the entirety of the pathway into our chassis E.coli for the production of linalool, just like other research teams have done before. However, this required us to place a plasmid (of approx. length 15 kb) with the full gene circuit, inside our chassis. Due to time constraints this made us wonder if we could come up with something new.

The MEP Pathway in E. coli: The Link

Unlike some other organisms, E. coli primarily utilizes the non-mevalonate pathway (the MEP pathway) for isoprenoid synthesis. It is intrinsic to the organism and vital for various cellular functions, including membrane composition and the production of signaling molecules. The pathway begins with the condensation of pyruvate and glyceraldehyde-3-phosphate but culminates in the production of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are essential for synthesizing various isoprenoids.

This was the link we were searching for. Due to further time constraints, the team decided to follow the path less traveled. We decided to exploit the MEP pathway due to the formation of a common substrate between MEV and MEP pathway : DMAPP. For our further reactions we planned on housing the two new genes with a pSB1C3 backbone and transforming that plasmid into the chassis.

Our perspective: The two new genes placed inside would act on DMAPP substrate present in the cytosol of the chassis, and act on at least a part of it to synthesise Linalool. We agree that it might lead to less production of Linalool because of flux division of DMAPP between this pathway and other existing pathways and also due to the MEP pathway being a low metabolic flux pathway.

However, even if unsuccessful it would provide valuable insight on the preference of Linalool production and other types of terpene productions like FPP (farnesyl pyrophosphate), and also enzyme reactivity comparison.

Future Prospects: Optimisation of Linalool Production

The team did extensive research on ways to improve the mevalonate pathway but due to time constraints could not carry out experiments on the same.

Choice of Media

Research showed that using different compositions of growth media can alter the amount of the compound of interest being produced. MEP pathway begins with the condensation of pyruvate which hints at the fact that there must be a carbon source like glucose further up the pathway which fuels the availability of pyruvate substrate. This is the logic behind using various carbon sources as growth media.

Several research experiments were done in the past on Lycopene production in E. coli wherein, the MEV pathway was exploited along with the MEP pathway. When glucose, fructose, glycerol, or arabinose were supplied as an auxiliary carbon source, respectively, to the LB medium for lycopene production, fructose exhibited the highest lycopene yield.

This approach remains largely uncultivated and the team wants to delve deeper into the same approach for optimizing production of Linalool