Team:Mississippi State/Proposal 2

An Engineered Bacterial System for Lignin Biodegradation

MSU iGEM Team

The emerging field of synthetic biology seeks to apply a logical, engineering based approach to the design and construction of novel biological systems. This is accomplished through the use of standardized biological parts called BioBricks. These parts, or genes, make up systems that perform some biological function, and they can be logically manipulated to produce a product under stringent environmental controls. Already, synthetic biologists have engineered a biological system to produce the anti-malarial drug, artemisinin, at a tenth the cost of conventional production. This field shows incredible promise in lifting the haze from biology, and allowing people to actually engineer systems that benefit all.

Lignin is a nearly ubiquitous biopolymer found throughout the biosphere. It is a major constituent of plant tissue, and proves to be the major limitation of wood biodegradation. Also, its resistance to degradation decreases the availability of other plant tissue components, most notably cellulose and hemicellulose. Lignin is found in many wood products, including newspaper print, and it lends the ‘hardness’ to wood materials. Its limitation on wood product bioavailability is due to the complex polymeric structure which proves difficult to break. Therefore, it presents a formidable physical boundary to enzymes directed at the other constituents of plant matter.

In this research, we seek to gather the enzymes and cofactors necessary for the complete degradation of Lignin. These will be integrated into a bacterial genetic system using the BioBrick standard of assembly. The system will consist of necessary genetic sensors and controls to ensure optimal enzymatic activity in the presence of lignin. It will also improve upon existing methods which use only the White Rot Fungi or enzymes alone. This method is specific for lignin, and will not degrade other plant matter. Also, it will be more economic than purely enzymatic means due to the self-replicating and control factors built into the microorganism.

Significance

The significance of this project is twofold. First of all, we seek to benefit the uses of non-food material biomass as a source of energy. Though ethanol and other biofuels offer an alternative to fossil fuels, their extraction from food crops is unrealistic and puts enormous economic strain on both food products and the further development of natural fuels. Biomass waste contains a huge amount of unused cellulose and hemicellulose, the raw materials for biofuel production. In addition, Lignin has been shown to be a source of biogasoline, which conforms better than any current biofuel to the existing energy infrastructure. As a result, this project will develop a better method for natural degradation of biomass to reduce the costs and complications involved with current methods.

Secondly, this project advances the emerging field of Synthetic Biology. In roughly ten years, the field has seen enormous growth as technologies have emerged which decrease the costs of working with and understanding genetic material. This work is unique in that is seeks to design and build biological systems using the principles of engineering in addition to those of conventional biology. Furthermore, Mississippi State University has the only undergraduate research opportunity for synthetic biology in the South. Therefore, we are leaders in the development of the discipline and its future minds in the state and region. This project offers the unique opportunity for this University to participate in and shape the development of an important new science by supporting the work of the first generation of synthetic biologists.

Prior Experience

Mississippi State University has conducted research in Synthetic Biology for two years, and is currently the only non-medical university in the Southeast to do so. I have participated in Synthetic Biology research for one and one-half years. In the winter of 2006-2007, I participated in follow up research on a Bacterial Hydrogen Detector. This design incorporated Synthetic Biology techniques to engineer bacteria which fluoresced green in the presence of hydrogen. In this work, I gained valuable experience into the fundamental principles and techniques involved in engineering biological systems. In the summer and fall of 2007, I worked on developing a genetic reporting system for ubiquination. This system used E-coli to produce the protein ubiquitin along with a fluorescence tag protein. Proteins destined for degradation were tagged with another fluorescence protein so that the ubiquination process could be seen visually, eliminating the need for further costly and time consuming tests. This work further developed my understanding of Synthetic Biology techniques, and prepared me to take a leading role in further research at Mississippi State

Methods

The Lignin Peroxidase Enzyme (LiP) will be isolated and modified to standard BioBrick format by the addition of standardized pieces of DNA to each end of the gene. White Rot Fungi possess the genes necessary for the production of LiP. This gene will either be isolated from strains of the organism available at Mississippi State, or the genetic material will be bought from a DNA production company. The BioBrick format requires genes be capped with prefix and suffix genetic sequences to allow for ease of use and simplified assembly. The caps allow several parts to be easily assembles like lego bricks, hence the name BioBricks. When digested with restriction enzymes, parts automatically connect in the desired format according to their prefixes and suffixes.

Also, secretion systems will be investigated to optimize the production of LiP. The type III secretion system used by pathogenic bacteria is a possible use for this project because it is specialized for secretion into eukaryotic cells. However, research into an optimal secretion system is one goal of this research, and possible mechanisms and applications will be investigated. These constituents will then be implanted to a host microorganism. The host organism will be investigated depending on the secretion system chosen. Ideally, E-coli bacterial offers the best means of research due to its rapid growth and high level of characterization. However, yeast may also be of use because it possesses a secretion system.

Testing will consist of protein assays to ensure functional LiP is produced. Furthermore, testing will discover the success of an implanted secretion system, as well as the ability of the organism to survive under these modified conditions. From that point, the part will be characterized for admission to the Registry of Standard Biological Parts. Also, the system will be modeled on computer to aid the optimization process. Finally, this work will be entered in the International Genetically Engineered Machines Competition at the Massachusetts Institute of Technology.

Scholarships Received:

Structural Steel Services Scholarship

Mississippi Eminent Scholars Grant

Mississippi Tuition Assistance Grant

Awards:

MAFES Undergraduate Research Award (2008)

Bronze Medal, International Genetically Engineered Machines Competition (2007)