Team:Utah State/Project

Introduction

Every year, more than one million tons of laundry detergent is used to maintain and clean clothes. Laundry detergent contains many useful components, such as surfactants and enzymes that enhance the cleaning process by emulsifying and degrading components in stains thus removing them from the clothes. However, after each wash, the laundry detergent (including enzymes) is washed out with the dirty water, resulting in a large amount of waste and chemicals being dumped back into the water supply [1]. Our motivation for this project is to design a reusable laundry treatment that will reduce the amount of waste as well as cost that is associated with washing clothes; this reusable treatment will contain a higher concentration of enzymes, which will decrease the amount of detergent needed per laundry cycle thus lessening the impact of waste on the environment.

In today’s society it is very common for laundry detergent to contain certain enyzmes. The most common enzymes included in laundry detergents are proteases, lipases, amylases, and cellulases. Proteases are in charge of degrading proteins like those found in blood stains, lipases degrade fats and oils, amylases take care of starchy and carbohydrate based stains like custard or chocolate stains, and cellulases help maintain the structure of the material by “modifiying the structure of cellulose fiber on cotton”; it also makes the material softer and brighter [2]. Out of these four classes of enzymes, our project includes a cellulose, an amylase, as well as chlorophyllase, which can be used to remove grass stains. We chose these particular enzymes because they were easily attainable, with the amylase and the cellulase genes being available in the distribution kits and because they make up a large portion of enzymes included in regular laundry detergents. We chose chlorophyllase because of its ability to make cleave chlorophyll (the green pigment associated with grass stains) into a water-soluble compound called chlorophyllide. Grass stains are really hard to remove and so we felt like incorporating this enzyme could potentially increase the effectiveness of grass stain removal.

What is a stain?

Stains are those are subtle patches of mustard or dirt that leave a black hole for the attention of anyone who talks to you. So why don’t those stains just come off when we wash them with water in the bathroom after lunch? Well, for the same reason we sometimes get dirt under our fingers. The fabric in our clothes and many other materials can absorb liquid, which is then trapped inside the fabric, unable to leave. These are mechanical stains, where just some water and agitation may remove the stain from inside the fabric. Other stains however, involve some chemical reactions. Severe stains can change the chemistry of the fabric by bonding to it, leaving that lovely grape juice stain, even after washing. In order to reverse the bonding that has taken place, a detergent must be used.

What is a detergent?

Well first of all, is detergent just another name for soap? Although they perform similar jobs, a soap doesn’t always do what detergent can do. To explain more about detergent, let’s first talk about soap.

Before soap, there was water. Water is an amazing cleaning agent. Water is made of Oxygen and Hydrogen, elements that are combined together to form a molecule. What makes elements unique and different from each other are combinations of small charged particles called protons. When one proton is all by itself, we call that Hydrogen. When 8 protons gather together, we call that Oxygen. Protons aren’t the only charged particles; there are oppositely charged electrons that are attracted to protons. Groups of protons have to stay together, their numbers don’t change, but electrons move from group to group fairly easily. This whole dance of protons and electrons is what causes every muscle in your body to move and every plant to grow.

So back to water, how does water dissolve things? A water molecule doesn’t share its electrons very well. One side of a water molecule has a large group of protons, while the other side has 2 pairs of weaker protons. This imbalance causes water to be polar, one side being positively charged the other side more negative. If we want to wash anything with water, it will only be cleaned off if that substance is polar too. Well what if we need to wash something off like oil and dirt? Oil and other stains are made of non-polar molecules where the electrons are spread out evenly among all the different groups of protons. This is where soap comes in. Soap is made of long molecules that have both properties, a polar end and a non-polar end. Finally, water can attach to the polar end while dirt and oil can bind to the non-polar end. This helps us wash our hands clean of grease and grime.

Now back to detergent. What makes detergent different from soap? Soap is made from natural sources, like animal and plant oils (for their non polar end) and salts like sodium (for their polar end). Detergents are made of synthetic molecules not found in plants or animals. Detergents are most often used to clean materials like clothes, parts of your house, or metal. Soap is less harsh and is used to clean our skin. Because detergent is more powerful, less of it can be used in a large volume of water.

Enzymes

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

Figure 2. Schematic of our process from designing DNA constructs to fighting stains with our functionalized bioplastic.

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 - 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

[1] Bajpai, D. and Tyagi V.K. “Laundry Detergents: An Overview.” Journal of Oleo Science 56 (2007): 327-340.

[2] Hasan, F., Shah, A., Javed, S., Hameed, A. “Enzymes Used in Detergents: Lipases.” African Journal of Biotechnology 9 (2010): 4836-4844.

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.