Team:Hong Kong UCCKE/Design
Design
As we have mentioned in the description part, we hope to create a domestic device to digest food waste by anaerobic digestion and convert it into detergent which can be applied in homes. Yet, as the hydrolysis of food waste occurs at a very slow rate, we used an integration of synthetic biology and mechanical approach to speed up the digestion process. For synthetic biology approach, we designed three enzyme parts to facilitate the decomposition of different food waste substrates and to suppress the bad smell generated during the process.You will find the details of the two parts in this page.
For synthetic biology approach, we designed three enzyme parts to facilitate the decomposition of different food waste substrates and to suppress the bad smell generated during the process.You will find the details of the two parts in this page.
Macromolecules in food waste
Macromolecules are very large molecules that typically composed of thousands of atoms or more, and takes a long time to be degraded. In food, the major source of macromolecules are carbohydrates, lipids, proteins. Therefore, we mainly focus on catalyzing the degradation of these food wastes.
Carbohydrates (Polysaccharides)[1]
Polysaccharides are long chains of monosaccharides that may be branched or not branched. There are two main groups of polysaccharides: starches and fibers.
Starch
Starch molecules are found in abundance in grains, legumes, and root vegetables, such as potatoes. There are two types of Plant starch, Amylose and Amylopectin. Amylose, a plant starch, is a linear chain containing hundreds of glucose units. Amylopectin, another plant starch, is a branched chain containing thousands of glucose units. They are contained together in foods, but the smaller one, amylose, is more abundant.
Starch is the most important source of carbohydrates in the human diet and accounts for more than 50% of our carbohydrate intake. It occurs in plants in the form of granules, and these are particularly abundant in seeds (especially the cereal grains) and tubers, where they serve as a storage form of carbohydrates. The percentage of solid mass of starch in food are: potatoes 15%, wheat 55%, corn 65%, and rice 75%.[2]
When Starch is located under high temperature, the crystal structure of starch breaks down, making them much easier to break down in the human body.
Dietary fibers
Dietary Fibres are polysaccharides that are highly branched and cross-linked. Some dietary fibers are pectin, gums, cellulose, and lignin. It can be found mainly in grains, fruits and vegetables.
Vegetables: Split Peas(8.3%), Broccoli (3.3%)
Fruits: Apple(2.4), Asian Pear(3.6%), Avocado (6.7%)
Grains: Oats(10.6%), cooked White Rice(0.2%), cooked brown rice(1.8%), Barley(17.3%), Corn(13.4%)[3]
Periplasmic Alpha-amylase(malS)
Periplasmic alpha-amylase(malS) is a hydrolyse that degrades alpha-linked polysaccharides with at least 3 glucose residues, including starch and glycogen[4], yielding smaller sugars including maltotriose, maltose, and glucose.[4] Therefore, it can catalyse the primary degradation of large carbohydrates in food waste, accelerating the reduction in solid mass, and facilitating the following steps of anaerobic digestion by increasing the surface area to act on. It is especially specified for digesting rice, which contains about 75% of carbohydrates in solid mass.
Mechanisms of malS
malS recognizes substrates from the non-reducing end and hydrolyses the α-1,4 glycosidic bonds between each glucose monomer.
1,4 glycosidic bonds are formed due to condensation reactions between a hydroxyl oxygen atom on carbon-4 on one sugar and the α-anomeric form of C-1 on the other[5], bonding two monosaccharides to form a disaccharide and subsequently a polysaccharide. A water molecule is lost in the reaction.
Fig. 1 Condensation and Hydrolysis reaction of Carbohydrates
There are are two types of glycosidic bonds - α-1,4 and β-1,4 glycosidic bonds. α-1,4 glycosidic bonds are formed when the OH on the carbon-1 is below the glucose ring; while β-1,4 glycosidic bonds are formed when the OH is above the plane[6].
Fig. 2 Structure of Maltose(with α-1,4 glycosidic bonds) and Lactose (with β-1,4 glycosidic bonds)
When two alpha D-glucose molecules join together a more commonly occurring isomer of glucose compared to the L-glucose, form a glycosidic linkage, the term is known as a α-1,4-glycosidic bond[7].
Food Substrates of malS
Here are the polysaccharides that contains α-1,4-glycosidic bond and can be hydrolysed by malS:
Starch: Amylose has a linear structure with α-1,4 linkages, while Amylopectin contains a branched structure with α-1,4 linkages and α-1,6 linkages. It takes up 15% in potatos, 55% in wheats, 65% in corn, and 75% in rice. [2]
Fig. 3 Structure of Amylose[10]
Fig. 4 Structure of Amylose[10]
Dietary Fibre: Most Dietary Fibre are polysaccharides linked by beta bonds. Yet, Pectin is a linear chain of alpha(1-4)linked D-galacturonic acid that forms the pectin-backbone, a homogalacturonan.[8]
It is the component of primary cell walls, thus can be found in fruits, vegetables, legumes, sugar beets, potatos. Studies found that oranges and carrots has the highest amount of pectin among fruits and vegetables respectively. [9]
Glycogen: Glycogen is the energy reserve carbohydrate of animals.It can be found as granules in the animal liver (4%–8% by weight of tissue) and in skeletal muscle cells (0.5%–1.0%).Other than that, we don’t obtain much glycogen from food.
Exceptions (can’t be hydrolysed):
Lactose in milk and dairy products:
Fig. 5 Structure of Lactose[10]
Cellulose and other dietary fibres in vegetables and fruits:
Fig. 6 Structure of Cellulose
Lipid
Lipids, including Animal and vegetable fats and oils, are big and complicated esters, so we can study them by studying esters.
Esters
Esters are compounds formed from a reaction between carboxylic acids and (usually) alcohol. The hydrogen in the compound's carboxyl (-COOH) group is replaced with a hydrocarbon group, forming an ester bonds that links the hydrocarbon group to the center carbon atom. A water molecule is lost in the process. [11]
Fig. 7 Formation of Esters[12]
An example is ethyl ethanoate, where the hydrogen in the -COOH group has been replaced by an ethyl group. [11]
Fig. 8 Structure of ethyl ethanoate[11]
There are mainly three types of Lipids:
Triacylglycerols
Triacylglycerols (also known as triglycerides) make up more than 95 percent of lipids in the diet and are commonly found in fried foods, vegetable oil, butter, whole milk, cheese, cream cheese, and some meats. [13]
Phospholipids
Phospholipids make up only about 2 percent of dietary lipids. They are water-soluble and are found in both plants and animals. Phospholipids are crucial for building the protective barrier, or membrane, around your body’s cells. [13]
Sterols
Sterols are the least common type of lipid. Cholesterol is perhaps the best well-known sterol. Cholesterol is an important component of the cell membrane.[13]
Fig. 9 Types of Lipid[13]
Lipase and Esterase both hydrolyse ester bonds, but they can be differentiated on the basis of their substrate specificity.[14] Lipase display high activity towards complex water-insoluble long chain triacylglycerols, and works best at lipid-water interface. While Esterase display highest activity towards water-soluble short acyl chain esters, and are inactive against water-insoluble esters as vinyl laurate and trioctanoylglycerol. Therefore, we designed one lipase and one esterase part to perform a continuous hydrolysis of ester bonds in the anaerobic digestion of food waste, with Lipase primarily degrading large triglycerides into glycerol and fatty acids,to degrade lipids more comprehensively by digesting ester bonds of esters of different chain lengths and in different media.
Lip8 Esterase
Lip8 is a cytoplasmic esterase that catalyzes the hydrolysis of ester bonds, especially that of water-soluble short acyl chain esters. Yet, it still works against various triacylglycerols and long-chain fatty acid methyl esters. Among triacylglycerols with different fatty acids, Lip8 was found to have the highest activity against triacetin (C2). While methyl acetate (C2), methyl propionate (C3), and methyl butyrate (C4) are the substrates with higher hydrolytic activity among different fatty acid methyl esters. Among four Natural oils, Lip8 showed highest activity against tung oil, whose principal fatty acid component is eleostearic acid (C18: 3)[15].
Fig.10 Hydrolytic activity of Lip8 against various triglycerides, fatty acid methyl esters, and natural oils. The activities are shown as values relative to that against tri-n-caproin. The thin bars at the ends of the bold bars indicate variation.
LipIAF5-2 Lipase
LipIAF5-2 is a new heterologous lipase with no more than 52% identity with other lipases[15]. It showed higher hydrolytic activity with long-length acyl chains, showing maximal activity with p-NPM (C14) and about 70% activity with p-NPM (C16) and p-NPM (C18)[15]. It is extracellular and is secreted out of the cell[15].
Fig.11 Effect of chain length on LipIAF5-2
It is also outstanding in the efficient synthesis of short chain esters which produces sweet-smelling aromas by transesterification and esterification reactions in organic media. It showed good affinity toward glyceryl trioctanoate and the highest conversion yields were obtained for the transesterification of glyceryl triacetate with methanol. [16]
Fig.12 Reaction scheme for the transesterification of glyceryl triacetate into isoamyl acetate in presence of isoamyl alcohol.
It is solvent-tolerant and thermostable, and has maximum activity at 40℃ on triglycerides, with a drastic decrease observed above this temperature when acting on triglycerides. [16]
Fig.13 Effect of temperature on the hydrolytic activity of the LipIAF5-2 on triglycerides.
Composite Part Design
All of the above 3 enzyme expressing genes are added a RBS, B0034[17], and a double terminator, B0015[18].
B0034 is one of the strong RBS with the highest relative strength in the iGEM ribosome binding site catalog. It is chosen to achieve a higher expression level, thus enhances the synthesis of the enzyme gene to further speed up the anaerobic digestion process. B0015 is a double terminator combining B0010 and B0012. It is one of the most commonly used and reliable terminator in the iGEM catalog.
BBa_K3077000[19], BBa_K3077100[20]
Over-expressing the gene will exert metabolic stress on the cell and the cell may eventually die. Therefore, in order to keep the cell healthy for continual synthesis of protein, we added a conditional promoter R0010 to prevent over-expression.
Lac operon R0010
Lac Operon, registered as BBa_R0010, is a conditional promoter that promotes transcription in the absence of LacI protein and the presence of CAP protein, which can be maintained by the presence of IPTG with a low glucose level. [22]
Fig.14 Transcription conditions of Lac Operon
CAP binding site
When glucose levels are low, cAMP are produced as a “hunger signal”. The cAMP attaches to CAP, allowing it to bind to DNA. CAP helps RNA polymerase bind to the promoter, resulting in high levels of transcription. With high glucose level, no cAMP is produced, CAP cannot bind DNA and is inactive, so transcription occurs only at low level.[22]
Lacl binding site
When IPTG/Lactose is absent, the lacl binds tightly to the operator. It gets in RNA polymerase’s way, preventing transcription. IPTG/Lactose binds to lacl and makes it let go of the operator. RNA can now transcribe the operon.[22]
Still, this is just a temporary design to this project. When we investigated the the way of enzyme transport, we plan to design a new part with a conditional promoter to enhance the anaerobic digestion of food waste. We also added a 10X His-tag K844000[23] at the N terminus to allow protein purification in the experiments.
The lac operon R0010, RBS B0034, gene of interest, 10X His-tag K844000 and double terminator B0015 forms the composite part.
Fig.15 Pathway of BBa_K3077000 and BBa_K3077100
BBa_K3077200 [24]
We added J23111, a constitutive promoter, to lipIAF5-2.
JJ23111 is a consensus promoter sequence and one of the strongest member of the family. It promotes a continual and high rate of gene expression, thus enhances the synthesis of the enzyme gene to further speed up the anaerobic digestion process.
The constitutive promoter J23111, RBS B0034, gene of interest, and double terminator B0015 forms the composite part.
Fig.16 Pathway of BBa_K3077200
Process
We ordered two DNA from IDT to perform our assays. Below shows the process of how we used the ordered DNA and what problems we faced during handling the gene.
For malS coding gene, we first get the sequence from IGEM library, which this gene was designed by Team Utah State and registered as BBa_K1418020[26] in 2014. Then, we designed a composite part, which was registered as BBa_K3077000, for the expression of periplasmic alpha-amylase malS. However, it can’t be ordered from IDT, so related assays can’t be performed to test the effectiveness of the design.
For lip8 coding gene, we first get the sequence from [27]. After obtaining the sequence, we designed a composite part, which was registered as BBa_K3077100, for the expression of the Triacylglycerol lipase lip8. After that, we ordered the plasmid containing BBa_K3077100 which is ligated to a non-iGEM backbone from IDT. At the time the DNA was shipped, we verified the gene by colony PCR with prefix forward and suffix reverse, which showed it to be a positive insert. We then transformed the plasmid to the E.coli, then cultivate the bacteria to have enough amount for performing assays of the catalytic activity of transformed E.coli on Tributyrin agar, which will be detailedly explain on the assay page.
For LipIAF5-2 coding gene, we first get the sequence from ENA (Gene ID EU660533)[27]. Then, we designed a composite part, which was registered as BBa_K3077200, for the expression of the heterologous lipase LipIAF5-2. After that, we ordered the plasmid containing BBa_K3077200 which is ligated to a non-iGEM backbone from IDT. At the time the DNA was shipped, we transformed the plasmid to the E.coli, then cultivate the bacteria to have enough amount for performing assays.
References
[1]: https://2012books.lardbucket.org/books/an-introduction-to-nutrition/s08-carbohydrates.html
[3]: https://www.studenthealth.gov.hk/english/health/health_dn/health_dn_dfayb.html
[4]: Freundlieb S, Boos W.(1986) a-Amylase of Escherichia coli, Mapping and Cloning of the Structural Gene, mal& and Identification of Its Product as a Periplasmic Protein*. J Biol Chem.261(6):2946-53.
[5]: Biochemistry. 5th edition.erg JM, Tymoczko JL, Stryer L. New York: W H Freeman; 2002 - Section 11.2Complex Carbohydrates Are Formed by Linkage of Monosaccharides https://www.ncbi.nlm.nih.gov/books/NBK22396/
[6]: Jeremy M. Berg, John L. Tymoczko, Lubert Stryer. Biochemistry Seventh Edition Freeman
[7]: Berg JM, Tymoczko JL, Stryer L. Biochemistry. 5th edition. New York: W H Freeman; 2002. Section 11.2, Complex Carbohydrates Are Formed by Linkage of Monosaccharides. Available from: http://www.ncbi.nlm.nih.gov/books/NBK22396/
[8]: https://pubchem.ncbi.nlm.nih.gov/compound/Pectin
[9]: https://www.ncbi.nlm.nih.gov/pubmed/2993399
[10]: https://www.sciencedirect.com/topics/nursing-and-health-professions/glycosidic-bond
[11]: https://www.chemguide.co.uk/organicprops/esters/background.html
[12]: https://socratic.org/questions/what-is-ester-linkage-in-lipids
[13]: https://2012books.lardbucket.org/books/an-introduction-to-nutrition/s09-01-what-are-lipids.html
[14]: Chahinian H1, Sarda L.Distinction between esterases and lipases: comparative biochemical properties of sequence-related carboxylesterasesProtein Pept Lett. 2009;16(10):1149-61.
[15]: Meilleur C., Hupe J.F., Juteau P., Shareck F(2009). "Isolation and characterization of a new alkali-thermostable lipase cloned from a metagenomic library."J. Ind. Microbiol. Biotechnol. 36:853-861(2009)
[16]: Brault G, Shareck F, Hurtubise Y, Lépine F, Doucet N (2014) Short-Chain Flavor Ester Synthesis in Organic Media by an E. coli Whole-Cell Biocatalyst Expressing a Newly Characterized Heterologous Lipase. PLoS ONE 9(3): e91872. https://doi.org/10.1371/journal.pone.0091872
[17]: http://parts.igem.org/Part:BBa_B0034
[18]: http://parts.igem.org/Part:BBa_B0015
[19]: http://parts.igem.org/Part:BBa_K3077000
[20]: http://parts.igem.org/Part:BBa_K3077100
[21]: https://parts.igem.org/Part:BBa_R0010
[23]: http://parts.igem.org/Part:BBa_K844000
[24]: http://parts.igem.org/Part:BBa_K3077200
[25]: http://parts.igem.org/Part:BBa_J23111
[26]: http://parts.igem.org/Part:BBa_K1418020
[27]: Ogino H1, Mimitsuka T, Muto T, Matsumura M, Yasuda M, Ishimi K, Ishikawa H.Cloning, expression, and characterization of a lipolytic enzyme gene (lip8) from Pseudomonas aeruginosa LST-03. J Mol Microbiol Biotechnol. 2004;7(4):212-23.