Team:AUC TURKEY/Project/Introduction

The core principle of our project is the development of a novel reporter system. We developed a biological system which is a reporter system more agile and recognizable in comparison to the present reporter systems using E.coli. Although the currently present reporter systems bear many incapabilities and disadvantages, the lack of alternative reporter systems forces the science community to use these systems. Our project can perhaps obscure this situation.

 

 White rot fungus degrade several large molecules such as cellulose, hemicellulose and lignin to use as a source of nutrition and continue their existence. The breakdown of lignin, an amorphous substance and a polimer, through lignin peroxidase, an enzyme of the oxireductase enzyme group, is crucial.

 

 

 

 

 

 

 

 

 

 

 

 

In the examination of the simbiotic relationship of white rot fungus and ligneous trees, scientist uncovered the mycoremediational breakdown that takes place in fungi through laccase enzymes. After recognizing the possibilities that arose due to this discovery, scientists utilized the mentioned function of fungi on the remediation of dyes. If enzymes could be used to breakdown dyes, than it would be possible to form a biological reporter system that would be built around the breakdown of dyes instead of synthesizing it, which would in turn speed up the process, make it easier to observe changes and give the ability to mediate the integration of the method to bioremediation systems. With this design, we will have formed a reporter system from scratch based on the breakdown of dyes instead of their synthesis.

 

PRESENT REPORTER SYSTEMS

Commonly used reporter genes that induce visually identifiable characteristics usually involve fluorescent and luminescent proteins. Examples include the gene that encodes jellyfish green fluorescent protein (GFP), which causes cells that express it to glow green under blue light, the enzyme luciferase, which catalyzes a reaction with luciferin to produce light, and the red fluorescent protein from the gene dsRed. The GUS gene has been commonly used in plants but luciferase and GFP are becoming more common. [i]

A common reporter in bacteria is the E. coli lacZ gene, which encodes the protein beta-galactosidase. This enzyme causes bacteria expressing the gene to appear blue when grown on a medium that contains the substrate analog X-gal. An example of a selectable-marker which is also a reporter in bacteria is the chloramphenicol acetyltransferase (CAT) gene, which confers resistance to the antibiotic chloramphenicol.

Most of the reporter systems are based on the use of visual data. To understand if whether the necessary conditions were met, change in visual context must occur. In the appropriate conditions, these report systems give response through the synthesis of specific color material that was not present in the environment.

 

 

A prominent reporter system that is the green fluorescent protein, a protein that does not carry a complex and long sequence structure that is responsible for its prominence, is a protein composed of 238 amino acid residues (26.9 kDa) that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range.[ii] [iii] In cell and molecular biology, the GFP gene is frequently used as a reporter of expression. [iv] In modified forms it has been used to make biosensors, and many animals have been created that express GFP as a proof-of-concept that a gene can be expressed throughout a given organism. The GFP gene can be introduced into organisms and maintained in their genome through breeding, injection with a viral vector, or cell transformation. To date, the GFP gene has been introduced and expressed in many Bacteria,Yeast and other Fungi, fish (such as zebrafish), plant, fly, and mammalian cells, including human. Martin Chalfie, Osamu Shimomura, and Roger Y. Tsien were awarded the 2008 Nobel Prize in Chemistry on 10 October 2008 for their discovery and development of the green fluorescent protein.

The GFP reporter systems which started a new era in biological reporter systems with the works of Martin Chalfie, Osamu Shimomura, and Roger Y. Tsien are still widely used today. GFP reporter systems which can be used in many processes ranging from the confirmation of the synthesis of a sequence to the validation of the binding of a protein to the cell membrane of a cell, still command great importance as a reporter system, however these reporter systems also carry several disadvantages. The most crucial of these disadvantages is the problem of time restriction. To overcome this hurdle in front of scientific research exponentially increases the development in scince. Reducing the time allocated for experiments and increasing their efficiency are the most important matters in science research. Currently, the issue of time has become more prominent. The incubation process following cloning protocols can take up to 16 hours, significantly increasing the time required to carry out projects hence restricting the conduction of many of these projects. If an example was to be given to the current problem, the time restriction prevents the use of synthetic biology for the development of toxic gas sensors as the sensors would need to give immidiate response. It is not possible to state that this problem has been solved as alternative methods either require highly expensive chemicals to operate or have too complex systems preventing implementation

 

OUR MODULE

Reporter systems carry great significance in the world of biology. As previously mentioned, the problems that currently exist in the development of reporter systems slow down the development of Biology, specifically synthetic biology and genetics.

We aimed to overcome the issue of time with our system. We had to abandon certain project ideas due to the insufficiency of the available time that inspired us to find a possible way of overcoming this issue which would open doors to many scientists interested in this area.

Our focus was on the GFP reporter system as it had been used numerous times by scientists worldwide and therefore carried great attention in reporter systems. If the issue of time was to be tackled, it had to address the issue in the GFP reporter system. The report system had to respond in a shorter period of time if progress was to be accelerated. It was not crucial to see a 100% complete result, even the glimpse of a change would have been enough for our system.. After observing the currently present GFP reporter systems, it came to our notice that the bacteria try to produce an inexisting substance from scratch, step by step and the presence of the substance can only be recognized after the substance has reached a certain concentration. If the process was to be reversed, however, it would take a significantly reduced amount of time. After reaching this state of recognition, we decided that the breakdown of the color instead of its production would be a lot more agile. Allthe enzyme produced would be capable of breaking down color in a short time interval. The exponentially increasing levels of enzyme presence would increase the enzyme activity, hence reducing the time of the process even more.

There is another advantage to this new system, while the necessity of the colored substance reaching a certain density was a disadvantage in the previous system, in the new system even the change in the hue of the color would prove that our system was functioning and the density issue would then become an advantage. The only external addition to the system required is the addition of a specific dye. The system therefore bears the potential to eliminate 16-hour long waiting times and makes life easier for scientists.

If our goal within this project was accomplished it would be a revolution for biological reactions. Who wouldn't want to decrease the required time by 75%?

Our project has the potential to be adapted for other areas of science because of its relation with color. When we think of the importance of bioremediation processes, to decrease the harm of chemical waste to the ecology of the Earth, these alternative systems are important. It won't be hard to integrate this system.

If we are able to optimize and develop the steps of our project professionally, we will be able to open a new door within the science of biology.