Bacterium biofactory

Enhancing B. subtilis' productivity, along with synchronizing its population under a single regulatory loop through genetic modifications, to increase synthesizing level of phosphotriesterase enzyme utilized in pesticide-related bioremediation.

Through the use of bioremediation, the harm done towards habitats exposed to agricultural pesticides such as organophosphates can be backtracked. This is done by utilizing the phosphotriesterase enzyme, which naturally degrades said pesticide. For this to work, its synthetization must become highly efficient, so as to meet demands. This project is divided into two stages:

Stage 1 focuses on modifying the bacteria’s chromosomes. When growing bacteria within a controlled environment, certain genes become unnecessary. Under natural conditions, they are intended to keep the organism ready for environmental changes. In an in vitro setting, energy is continuously directed towards these non-required metabolic routes, although such waste means additional production costs.  By determining and silencing unnecessary protein-coding genes, “wasteful” energy consumption can be relocated towards the production of the enzyme, for an increment in its production levels. The bacteria is able to produce more of the phosphotriesterase enzyme, ensuring a quicker degradation of pollutants. 

Stage 2 seeks to synchronize the entire bacteria population under a single clock. A genetic circuit is placed inside a plasmid, which has a toggle-switch effect on gene expresion, allowing for the enzyme's sythetization to become time-related.

Agricultural pesticides are one of the biggest contaminants affecting the environment. Their large-scale discharge threatens the existence of many habitats. Such contaminants are organophosphate compounds (OP). These synthetic chemicals are extremely toxic due to their long degradation process. Although banned in multiple countries, they are still used by many farmers, especially in third world countries because of its low production costs.

One of the biggest setbacks when using enzymes for bioremediation is the difficulties associated with manufacturing, as the production of said enzymes becomes insufficient when compared to the high demands in which they are needed. Humongous amounts of bacteria would need to be produced, in an unscalable way. However, through the implementation of this “super-bacteria”, a standard batch of B. subtilis with the phosphotriesterase insert would be able to achieve higher production rates in less time. Additionally, it's production would be underway in a cyclical fashion, delivered in organized lots. 

It becomes eminent to note how this new, modified super-bacteria can be used on endless projects and industries, for a higher production of whatever compound is required. This benefits: financially and socially. The former, as this bacteria is intended to produce higher amounts of the recombinant protein while maintaining other factors the same as before. The latter allows a larger outreach for medical trials, bioremediation initiatives, nutritional substitutes for those in need, etc. Overall, the project is of high scalability and reach, with staggering growth potential due to its vast application possibilities.

This issue worsens when analyzed in a third world country setting: poverty and impunity towards the norms in place pave the way towards staggering levels of contamination by pesticides. Despite the ever-growing harmful effects of pesticides on the environment and human health, their use continues to increase during the last decades; in Mexico alone, the use of pesticides has reached 95,000 tons annually (2014). These substances represent a risk to human health, as well as the environment because they contaminate soil, water, sediments and air.

In the last decades, the use of OPs as pesticides for virtually any kind of agricultural need has skyrocketed its popularity, becoming the preferred method among farmers. While this has benefited society, its harmful consequences must also be taken into account; these being the immense amount of toxic components that end up in the environment, resulting in severe contaminations in aquaculture areas. 

Taking this challenge head-on through the use of biotechnology allows for a remediation of the past. Simply banning certain pesticides, although an important step in bettering the issue, is not enough, as the already-contaminated habitats must be cleansed. Solventing this issue translates to lower risks of cancer, lung problems, and a better overall health for those living near polluted sites. It also involves a bettering of the involved habitats; the extension and survival of organisms living in or near contaminated waters as well as an overall minimization of environmental hazards such as water shortages and loss of species.

In order to understand the needs of the mexican population, it is important to analyze statistics: In Mexico over 140 pesticides that are currently authorized by the authorities are simultaneously banned in other countries for their slow degradation and toxicity. In addition, 183 active ingredients classified as dangerous by various international organizations are still very much used in said county, although many of these pesticides are causes of cancer in humans, such as the organophosphate compounds. This image becomes jarringly real after visiting polluted sites and agricultural centers in the state of Jalisco, where many of its inhabitants suffer respiratory issues, as well as skin diseases. Not to mention the damage all around, in bodies of water and polluted soils with dying organisms. 

Because of this, an idea on how to help these peoples began to form. After analyzing all issues and setbacks involved in bioremediation through the use of enzymes, the first version of today’s project was born. Because of this, an idea on how to help these peoples began to form. After analyzing all issues and setbacks involved in bioremediation through the use of enzymes, the first version of today’s project was born. The in vitro proposal was designed through multiple engagements with professionals and experts, who helped bring to light the hardships experienced by the affected communities, as well as ways to fix at least some of the harm done unto them by OP contaminants. 

After a complex investigation on bacteria's metabolic routes, specifically,  B. subtilis' gene functionality and proteome expression, an in silico design of the new, enhanced bacteria was designed. This model of a "super-bacteria" is able to produce higher amounts of the enzyme insert, and its population is able to synchronize production under a specific time lapse. The bacteria will be able to increment the enzyme’s production, meaning higher  revenues for any industry that uses it. It also vastly expands the possibilities of habitats beginning bioremediation without additional costs. Finally, it means a quicker degradation of the OP pollutants, inducing healing much faster. 

Additionally, through this process, a web of connections and professional consultations was generated, which allows access into laboratories so as to implement the design. Although this project is of high scalability and reach, with staggering growth potential due to its vast application possibilities, it reaches a breaking point, where its implementation relies on financial investment.

This project is an implementation of biotechnologies, as its purpose is the modification of genetics in order to give rise to a new product, able to sustainably bioremedy the organophosphate compounds’ hazardous contamination. It includes the following biology-related technologies:

Gene editing uses up and coming technologies such as CRISPR-Cas in order to manipulate and modify the genetic makeup of a given organism. Said  system consists of two key molecules, an enzyme called Cas9 and an RNA guide, that introduce mutations into the DNA through cutting in specific sites.

Recombinant protein is a protein encoded by a gene — recombinant DNA — that has been cloned in a system that supports its expression. In other words, its production is based on the use of bacterium or yeasts to carry out their synthesis as a host organism. This technology allows distinct organisms with specific characteristics with optimize production, to synthesis an outside gene 

The genetic circuits function much as toggles and switches in electrical engineering, and are used to manipulate bacteria so as to synchronize cell separation, lysis, among other basic functions. They specifically allow for a certain communication among a given population, which can be useful for many applications.

This project is currently in an in vitro stage. Therefore, its application into a prototype is still pending. It also becomes difficult to name a specific number of those potentially benefited, as laboratory investigations must be realized beforehand. However, the fact remains that the bioremediation of the environment benefits all of society, not only those suffering first hand pollutant-related issues.

The intended impact for the next year is, first and foremost, creating a prototype which is authorized for implementation inside harmed habitats. The prototype is to be a highly efficient bacteria population in terms of energy waste and consumption, which is able to synthesize the enzyme insert in higher rates, therefore removing pesticide-related pollutants from contaminated sites in shorter periods of time. This bacteria culture is to be synchronized, sequencing the enzyme’s gene in a synthetically regulated way, so as to produce even more enzymes in series. In terms of application, a site is to be selected and authorized by the authorities to bioremediate with this new product, previously tested inside a laboratorium. Its overall impact is to be measured by the reduction of OP contaminants from said sites after having passed through the enzyme-producing bacteria.

The main goal for this year is to generate a prototype of the modified bacteria that is able to produce higher quantities of the enzyme insert in an cyclical fashion. For its implementations, it has been bracketed into smaller goals:

1. Through the use of a computer program, generate an experimental design. This statistical approach allows for the optimization of variations of different factors simultaneously in order to obtain optimal values. In other words, it allows for a clean design which minimizes outside errors to factor into the results.

2. Experiment editing different types of genes and quantities so as to analyze which  have an actual potential to boost the bacteria’s energy efficiency without harming critical metabolic pathways.

3. Generate prototype which includes the most efficient combination of removed genes, along with the genetic circuit in place. Try functionality on water or soil samples, through the use of a color-coding system which allows an uncomplicated understanding for the analysis of the results. Implement said protoype in bioremediation processes in authorized habitats.

The main, and seemingly only barriers in place preventing the applications of this new product are mostly financial. Although arrangements have been made to make laboratories and equipment available for using, high costs for regents and mediums for modifying, cultivating and analyzing bacteria have prevented the project from advancing further. 

This segment is divided into two parts: social and academic/work experiences.

First off, through multiple social encounters and volunteer work among those affected by this type of pollution has helped shape the project as a whole, as well as intensified the importance of finding a solution. Some of those experiences include volunteer work cleaning up polluted sites and working on projects concentrating on the remediation of contaminated bodies of water. Having a personal involvement with the issue and those affected allows for generating empathy towards the environment, animals and communities very much harmed by toxic pesticides, specifically oranphospate compounds, by which they are contaminated. 

As for the academic/work experiences the team has been able to accumulate, they have shaped the way the idea was processed and designed. Being biotechnological engineering students, new knowledge, concepts and applications of biology-related processes are never far, and help is always ready. Because of this, along with excellent tuition and many accessible laboratory experiences, this project was able to take shape. Said work experiences include working inside professional investigations. Understanding not only how to manipulate laboratory equipment, but also where and how to obtain regents or other necesites has helped develop social and financial skills. Furthermore, these experiences, which are extracurricular activities, have allowed for an expansion on how to work inside a laboratory, how to work independently from professors and how to achieve results.

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