Team:Utah State/Project

Introduction

Stains

Chlorophyllase

Chlorophyll is naturally degraded during normal turnover of the pigment, when leaves change colors during fall, when fruit ripens, and during triggered cell death due to extreme temperature or water shortage. The first step in the breakdown of chlorophyll is catalyzed by the enzyme chlorophyllase. Chlorophyll is broken down into chlorophyllide and phytol. Chlorophyll is a dark green and hydrophobic molecule while chlorophyllide is a lighter green and hydrophilic (Arkus et. al, 2007). The chlorophyllase enzyme obtained for our project was cloned from a species of wheat, Triticum aestivum. Chlorophyllide is further broken down by a series of enzyme-catalyzed reactions into colorless final products (Eckhardt et. al, 2004).

Green grass stains are actually chlorophyll stains. When a grass stain occurs the friction caused from sliding the plant over the material breaks cell membranes. This releases chlorophyll and other proteins into the fabric. Because chlorophyll is similar in chemical structure to natural fibers like cotton and wool it binds to the fabric making it difficult to remove with regular detergents (Rodriguez, 2003). Our project aims to synthetically produce the enzyme chlorophyllase that will begin the degradation of chlorophyll. As chlorophyll is degraded into chlorophyllide, it will become more water soluble. The green, water soluble, stain will then be easier to remove with normal washings.

Cellulase

Cellulose is a polymer of β-linked glucose produced by plants, and can be broken down into its subunits by cellulase. Cellulose is not usable by most organisms because of the beta-linked glucose of which it is made up. Mammals use α-linked glucose to store their energy as glycogen while plants create starch the same way; cellulose is produced only for structural support. However, some organisms can enzymatically change beta links to alpha links, after breaking down a chain of cellulose. Our produced enzyme is only the enzyme that helps breakdown cellulose chains into smaller units called cellobiose, two glucose molecules linked by a β 1-->4 bond (Part:BBa_K118023). These smaller units of the large cellulose chain are more soluble in water, helping to remove the components of a plant stain on clothing.

Amylase

Starch is a polymer of α-linked glucose, and is the primary energy storage in plants. This polymer can be broken down by amylase, an enzyme produced in our mouths to help break down food as soon as it enters our body. These long chains of glucose are broken down into single glucose molecules. Any organism that consumes starch produces amylase, including E. coli. The gene we used to produce our amylase is native to E. coli (Part:BBa_K523001). The breakdown of starch helps remove common food stains.

Bioplastic

What is bioplastic?

Bioplastics-as the name entails, means plastic that is biological in origin. One focus of Utah State’s 2014 iGEM team was to study polyhydroxybutyrates (PHBs), which are biodegradable polymers belonging to the group polyhydroxyalkanoates (PHAs). PHAs are naturally produced by some microorganisms in nature as a storage intermediate for energy and carbon. It has been reported that PHAs can accumulate up to 90% of dry cell weight under ideal conditions. PHAs are interesting plastics as they have similar physical and mechanical properties to their petroleum based counterparts such as polypropylene and polystyrene. PHB has a melting temperature of 179℃, a young’s modulus of 3.5 GPa, and a tensile strength of 40 MPa. This compares to polypropylene having a melting point of 170℃ a young’s modulus of 1.7 GPa and a tensile strength of 35 MPa (Khanna and Srivastava 2005). The similarity in properties between PHB and polypropylene is advantageous: 1) PHBs could replace polypropylene, 2) similar properties could mean mixing the two plastics leading to ‘hybrid’ materials, and 3) downstream processing of PHB could be carried out with the same equipment reducing processing costs. The potential applications for PHAs are so vast that many companies are starting to commercialize (Chanprateep 2010).

Figure #. The figure above shows the standard structure of PHA. When R is methyl (CH3) the polymer is known as PHB. ‘n’ represents repeating subunits of the monomer. PHB polymers can up to 900-1000 kDa in size.

The genes from some of these PHA producing species of microbes can be expressed in laboratory based strains (e.g. E. coli) to overexpress the PHA polymer to obtain high yields.

Polyhydroxyalkanoates at iGEM

Polyhydroxyalkanoates (PHAs) have a long established history at the iGEM competition. The first PHA project at iGEM was first carried out by the 2008 Utah State iGEM team (Utah_State 2008). In 2012 the Tokyo Tech team demonstrated a fully functional polyhydroxybutyrate (PHB) production system made out of BioBrick parts using a native promoter system (Tokyo_Tech 2012). Tokyo Tech’s complete operon was released to the iGEM community in the 2013 distribution kit. In 2013 the Imperial College team used a hybrid promoter system to improve upon Tokyo Tech’s 2012 design to get 11x more PHB production (Imperial_College 2013). Imperial’s project also explored degradation of PHB for carbon recycling.

Design

Team Goals

Goal #1 - Produce stain-fighting enzymes in E. coli

To produce stain-fighting enzymes in E. coli, we need to genetically engineer our bacteria using genes that code for the synthesis of our three enzymes: cellulase, amylase, and chlorophyllase. Each construct should include a histidine tag for protein purification. After enzymes are purified, assays with each enzyme’s substrate should be conducted in order to determine if the enzyme is active or not.

Goal #2 - Demonstrate that enzymes can help remove stains

In order to show that enzymes can remove stains, multiple assays will be conducted to demonstrate each enzyme’s ability to degrade its specific substrate (i.e. cellulase degrading cellulose in CMC agar plates, amylase degrading starch in starch agar plates, and chlorophyllase cleaving the phytol tail of chlorophyll to make it water soluble. These assays will provide a proof of concept; pairing this proof of concept with background literature reviews will help demonstrate that enzymes can in fact remove stains.

Goal #3 - Immobilize enzymes to bioplastic

To immobilize enzymes to bioplastic, we can build upon previous iGEM team's work regarding the production of bioplastic in E. coli. After using green fluorescent protein (GFP) as a reporter to illustrate attachment to the bioplastic granules inside the cell, we can go forward with the same strategy for each of our enzymes. To further solidify our end goal, we can chemically immobilize our BioBrick-produced enzymes to PVC plastic and show that the enzymes retained their efficacy after attachment (chemical immobilization would result in a covalent bond between enzyme and activated PVC plastic, the same type of bond that would occur in our biological immobilization strategy).

Goal #4 - Manufacture a reusable bioplastic laundry treatment

The objective of creating a reusable bioplastic laundry treatment is to reduce the quantity of harsh detergents, minimize wastes of non-reusable "free" enzymes, and improve upon the removal of tough clothing stains. To create this treatment, we first must prove that we can immobilize our stain-fighting enzymes to bioplastic while maintaining their efficacy. Second, we must be able to produce a sufficient amount of enzyme-immbolized bioplastic to allow for rigorous testing. We can then take this enzyme-immobilized bioplastic and construct an additive device to traditional laundry cycles that can be reused in multiple washes. Furthermore, we can use the stain-fighting bioplastic to surface coat scrubbing brushes and buckets for use in under-developed countries and for those who cannot afford traditional laundry machines.

Goal #5 - Win 6th straight Gold Medal

To win our 6th straight Gold Medal, the Utah State iGEM team will need to continue to go above and beyond the requirements necessary for a Gold Medal. We will need to ensure that our project is novel, clearly developed and completed by the students, and helps to build upon previous iGEM team projects by adding to the growing wealth of information and BioBrick parts in the Registry.

Goal #6 - Win Best Manufacturing Project

In order to win the Best Manufacturing Project at the Giant Jamboree, we have to make sure that our project is well-rounded and provides a novel solution to an ongoing problem.

Goal #7 - Have fun and LEARN

The whole iGEM experience should be about learning, enjoying yourself, and giving back. There have been countless opportunities for our student team members to work as a group, learn about Synthetic Biology, teach Synthetic Biology to others, and participate in cutting-edge laboratory research that will benefit us now and into our professional careers. Even before attending the International Jamboree in Boston this year, all of us have taken away lessons and experiences that will greatly improve upon how we conduct research and work with others in the future.

References

Agnew, D. E. and B. F. Pfleger (2013). "Synthetic biology strategies for synthesizing polyhydroxyalkanoates from unrelated carbon sources." Chemical Engineering Science 103: 58-67. Chanprateep, S. (2010). "Current trends in biodegradable polyhydroxyalkanoates." Journal of Bioscience and Bioengineering.

Jacquel, N., C.-W. Lo, et al. (2008). "Isolation and purification of bacterial poly(3-hydroxyalkanoates)." Biochemical Engineering Journal 39(1): 15-27. Khanna, S. and A. K. Srivastava (2005). "Recent advances in microbial polyhydroxyalkanoates." Process Biochemistry 40(2): 607-619.

Linton, E., A. Rahman, et al. (2012). "Polyhydroxyalkanoate quantification in organic wastes and pure cultures using a single-step extraction and 1H NMR analysis." Water Science and Technology 66(5): 1000-1006.

Peters, V. and B. H. A. Rehm (2005). "In vivo monitoring of PHA granule formation using GFP-labeled PHA synthases." FEMS Microbiology Letters 248(1): 93-100.

Rehm, B. H. A. (2009). Microbial Production of Biopolymers and Polymer Precursors: Applications and Perspectives. Caister Academic Press, Norfolk, UK.

Tomizawa, S., M. Hyakutake, et al. (2011). "Molecular weight change of polyhydroxyalkanoate (PHA) caused by the PhaC subunit of PHA synthase from bacillus cereus YB-4 in recombinant Escherichia coli." Biomacromolecules 12(7): 2660-2666.

York, G. M., B. H. Junker, et al. (2001). "Accumulation of the PhaP phasin of Ralstonia eutropha is dependent on production of polyhydroxybutyrate in cells." Journal of Bacteriology 183(14): 4217-4226. York, G. M., J. Stubbe, et al. (2001). "New insight into the role of the PhaP phasin of Ralstonia eutropha in promoting synthesis of polyhydroxybutyrate." Journal of Bacteriology 183(7): 2394-2397.