Transcript
DEPARMENT OF CHEMICAL ENGINEERING CHE 309- Introductory Bioengineering SECTION 01 GROUP 2 Dr. Ginette Turcotte Algae’s Biological Clock Gets Duped Submitted on: February 02, 2014 Due: February 03, 2014 Authors: Table of Contents Introduction………………………………………………………...…........ 3 Importance ……………………………………………………………..…....3 How it Works …………………………………………………………..…....3 Process Diagram ………………………………………………………....….5 Fermentation………………………………………………………………….6 Non-Fermentation………………….…………………………………….….11 Canadian Regulations……………....…………………………………….….16 Midterm Question……………………..…………………………………….17 Assignment Question.…………………………………………………..…...17 References…………………………..……………………………………….18 1.0 Introduction “We have shown that manipulating cyanobacteria’s clock genes can increase their production of commercially valuable biomolecules,” says Carl Johnson Stevenson, Professor of Biological Sciences at Vanderbilt University. Organisms and single cells have endogenous biological “clocks” that allow them to determine the time of day.[16] Many biological processes depend on the time of day. 2.0 Importance Since all the processes of life occur through cells, cells are known as the fundamental process of living organisms.[3] However, one of the significant functions about living organism cells is the time, whether it’s daylight or night time.[3] As a matter of fact, human beings, plants and algae’s body works differently from day and night time due to the biological clock processes.[3] A biological clock is an inner regulator that responsible for the annually, monthly and daily circadian rhythmic which located in the living organism’s hypothalamus of the brain.[3] Circadian rhythm is the daily physical activity changes that occur in 24 hours and can be affected by the external environmental factors such as light, pressure and temperature.[9] However, cyanobacteria cells consist of protoplasm which is composed of several biomolecules including water, amino acids and proteins.[12] 3.0 How it Works 3988435161925To increase the production of these biomolecules, food, drugs and biofuels, scientists and researchers have found a technique to control and manipulate the blue-green algae’s internal biological clock by making it think that it’s always in daylight mode. [1] Many of the pharmaceutical biological drugs that are made of proteins such as human insulin and anticancer medications are produced by algae. [5] Human insulin is a protein that is used to help many people with diabetes by regulating the amount of sugar in human bloodstream due to the disorder of the pancreas organ in production of the insulin.[8] Thus, in order to make human insulin for diabetics, genetic engineering places the human insulin DNA into the DNA of algae. [6] The first step is isolating the gene; part of DNA in the human chromosome that is responsible for the production of insulin protein. [6] Secondly, the cyanobacteria plasmid ring is cut by special enzymes after it is removed from the algae cell. [6] Plasmid is a circular part of cyanobacteria DNA. [10] Then, the human insulin gene is inserted into the plasmid ring to recombine with the algae DNA 6. Using very small needle syringes the scientists insert the combined plasmid into the cyanobacteria cell membrane. [6] In order for the cell to grow, live and thus produce the insulin protein, scientists have tricked the biological clock in algae. [6] They have locked the blue-green algae circadian biological clock by hereditarily downregulating the KaiC protein and upregulating the KaiA protein (photosynthetic genes) into its daytime mode configuration. [2] It was shown that locking the biological clock of cyanobacteria produce five times more human insulin in constant light. [1] In addition, reprogramming the cyanobacteria circadian patterns by improving gene expression can heterologous proteins KaiA and KaiC and thus increases the production of biofuels and medications. [7] Cyanobacteria’s clock consists of three proteins including KaiA, KaiB, and KaiC that help the gene clock to work during the day. [2] However, the KaiA and KaiC proteins acts as switches in which turn the algae cell day and night time gene on and off. [2] Cyanobacteria have six KaiC molecules that combined tougher to form a ring in which makes it functions as the main clock in the algae. [2] Also, the KaiC protein has three phosphorylation sites and since the KaiC ring depends on the biochemical reaction of these sites, any change in these three sites will turns off the timepiece. [2] Lastly, the gene insulin protein is produced in the cell as the algae remains in daytime setting. As the blue-green algae reproduce, the piece of human insulin DNA molecules will replicate in eukaryotic cells. [10] Then, the insulin protein that is generated in the host cell will be purified and will work perfectly as the produced by the human body. [6] Since the process is recent and under study, there are only a few pharmaceutical and energy companies such as BiofulesDigest who have been using cyanobacteria’s biological clock techniques to boost up their product fabrications. 4.0 Process Diagram Algae Biomass Extraction [13] [17] [22] ALGAE BIOREACTOR CULTIVATION PROCESS ALGAE HARVESTING & OIL EXTRACTION ALGAE OIL PROCESSING Upregulate KaiA protien and downregulate KaiC protein to turn on 95% of the cell’s genes active during daylight. Tested in hydrogenase, insulin, and luciferase and produced 200%, 500% and 700% more product respectively. 5.0 Fermentation Fermentation is the process when organic molecules serve as both electron donors and electron accepters and where the molecule which is being metabolized is not fully oxidized have all its potential energy extracted from it.[18] Cyanobacteria also known as blue green algae have a variety of fermentation pathway. Fermentation pathway is a pathway where cellular respiration occurs. [24] The fermentation pathway includes: Homolactic acid fermentation, Heterolactic acid fermentation, Mixed acid fermentation and Homoacetate fermentation. The homolactic acid fermentation is when a true homolactic bacterium (or bacteria) metabolizes glucose to make ATP, NADH and several biosynthetic precursors. The heterolactic acid fermentation when a true heterolactic bacterium (or bacteria) metabolizes glucose for the formation of NADPH for synthesis of fatty acid and steroids.[11] The mixed acid fermentation is when the product of the organism is a mixture of different acids usually acetate, lactate and formate. The homoacetate fermentation has two genera that are aerobes that oxidize sugar, sugar alcohols and ethanol but their major end product is acetic acid. In all species investigated, fermentation is constitutive. [11] Below is a list of cyanobacteria capable of fermentation [24] Organism Strain, Origin Fermentation pathway Products Anabaena azollae AaL symbiont from Azolla caroliniana homoacetate acetate (lactate, CO2, H2) Anabaena azollae AaN Anabaena azollae AaS symbiont from Azolla filiculoides Anabaena siamensis Asl paddy field acetate (CO2, H2) Cyanothece PCC 7822 (Inst. Pasteur) mixed acid H2, ethanol, lactate, formate, acetate Microcoleus chthonoplastes microbial mat Microcystis aeruginosa PCC 7806 (Inst. Pasteur) H2, ethanol, acetate Nostoc sp. Cc symbiont from Cycas circinalis homoacetate acetate (lactate, CO2, H2) Nostoc sp. Al2 symbiont from Anthoceros laevis Nostoc sp. Efl symbiont from Encephalaratos ferox Nostoc sp. MAC symbiont from Macrozamia lucida Nostoc sp. Mml symbiont from Macrozamia moorei Nostoc sp. Ml symbiont from Macrozamia sp. acetate (CO2, H2) Nostoc sp. Gm symbiont from Gunnera manicata acetate (lactate) Nostoc sp. Tl paddy field acetate (formate, CO2, H2) Nostoc sp. Bali acetate (CO2, H2) Oscillatoria limnetica hypolimnion Solar Lake homolactate Lactate Oscillatoria limosa microbial mat heterolactate homoacetate lactate, ethanol, acetate, Oscillatoria sp. not known lactate, ethanol, acetate, formate Oscillatoria terebriformis hot spring microbial mat Spirulina platensis not known mixed acid H2, ethanol, lactate, formate, acetate Spirulina mimosa not known Lactate, acetate Metabolism of Cyanobacteria during the fermentation The metabolism of cyanobacteria occurs when the cyanobacteria organisms possess many types of enzymes that catalyze the hydrogen production by biological process and the nitrogen fixation process. Hydrogen is very useful in this metabolism because it does not pollute the environment, recyclable processes and the biological process of hydrogen production is environmentally friendly and requires less energy compared to thermo chemical and electrochemical.[14] Nitrogen is needed in this metabolism because it is required to biosynthesize the structure of living organisms, plants and animals. Due to the useful of nitrogen, nitrogen fixation is essential for the manufacturing of explosives and agriculture organic or inorganic materials.[11] Anabaena azollae is a cyanobacteria organism capable of fermentation. Anabaena azollae is a genus of filamentous phototrophic cyanobacteria that has two main cell types: photoautotrophic cell and a heterocyst cell. The photoautotrophic cell has a circular structured filament which the organism uses to perform photosynthesis in the cytoplasm with the help of solar energy, bacteriochlorophyll and CO2. [3] The solar energy is used to generate Hydrogen (H2) a source of combustible energy. Cyanobacteria have been highly tested for their ability to capture and convert solar energy to hydrogen. During the conversion, the cyanobacteria undergo biophotolysis process. The biophotolysis process is the process whereby light energy is captured using the two photosystem and split into water which produces oxygen and hydrogen (via a hydrogenase enzyme).[15] “A likely better strategy is to exploit the use of radiant solar energy by the photosynthetic electron transport system to enhance the rates of H2 formation and so improve the chances of utilizing cyanobacteria as a source for the generation of clean energy.” [20] The heterocysts cell is produced by Anabaena in the fixation of atmospheric nitrogen. The nitrogen fixation process is catalyzed by nitrogenase, an enzyme highly sensible to oxygen. [3] Some microorganism fixes atmospheric nitrogen and converts it to ammonium (NH4). Heterocrysts cell is also able to fixed nitrogen into other compounds such as ammonia, nitrate which gets absorbed and converted to protein and nucleic acids by plants. [5] 2679700211455 The cyanobacteria obtain its bluish colour from a pigment called phycocyanin. This pigment is used to capture sunlight for photosynthesis process. During photosyntheisis, water is its electron donor and it’s by product is oxygen (in some other organism hydrogen sulfide). The carbon dioxide gotten is reduced to carbohydrates. During a dark anaerobic energy generation in cyanobacteria, carbohydrate is used as the substrate to help increase the life span of the dark anaerobic. [24] Conditions of the fermentation of Cyanobacteria Inorder for bloom formation to occur, some conditions needs to take place. Bloom is when cyanobacteria grow profusely and congregate thereby making a lake look green during the summer season. These conditions include: Light Intensity Cyanobacteria contains pigments such as phycobili proteins which include allophycocyanin (blue), phycocyanin (blue) and sometimes phycoerythrine (red) but their main pigment is cholorophyll which is used for harvesting light and photosynthesis process. Many cyanobacteria are sensitive to prolong period of high light intensities but if they are exposed to high light intensity periodically; they can grow at their maximal rate. [18] Gas vesicles Many planktonic cyanobacteria contain gas vessel. This gas vessel helps to lower the cyanobacteria density lesser than water because A gas vehicle has a density of one tenth that of water. [18] Growth rate The cyanobacteria bacteria unlike single-celled green algae cannot bloom in water with short retention time because they have a slow growth rate and require water with a long retention times. [18] Phosphorus and Nitrogen Cyanobacteria need a sufficient among of phosphorus and nitrogen for its bloom formation to occur. From past experiments conducted, many cyanobacteria have higher nitrogen or phosphorus than many other photosynthetic organisms. But they can out-compete their phytoplankton organisms under conditions of phosphorus or nitrogen limitation because they have a substantial storage of nitrogen which can be use in produce two to four cell division. [18] Population stability Cyanobacteria are attacked by viruses, bacteria and actino-mycetes. This attack slows the growth rate of the cyanobacteria and in return helps balance the high vogue of their already established population. [18] Temperature In order for most cyanobacteria to achieve maximum growth, the environment temperature must be more than 25oC. Other cyanobacteria like green algae and diatoms require higher temperature. [18] 5.3 The Biological Product from the Fermentation Broth The biological product from the fermentation broth The recovery and purification of a product from the fermentation broth is a complex, aqueous mixtures of cell which comprises of different compounds. It also requires many processing steps and major expenses in the production of most fermentation product. The fermentation broth is a multiple mixture of nutrients cells and their debris in which microorganisms grow and reproduce.[24] There are different methods of recovering the biological product from the fermentation broth. These methods depend on the product, its solubility, size of the process and value of the product. Some product requires high purity because of where they are used/what they are used for (e.g. pharmaceutical) while others doesn’t.[23] The most useful method for the recovery of cyanobacteria from the fermentation broth will be separation and purification method. A more economical method for the recovery process will be the crystallisation of the product from the concentrated broth.[24] During the separation and purification method, the products have to be processes and passed through several steps. They are as follows: Separation of the insoluble product is usually the first step. Sometimes, the broth maybe pre-treated to aid solid separation. Since the product of cyanobacteria is biomass, separation of solids is the major step in the product recovery. The separation techniques mostly used are filtration, centrifugation and coagulation.[23] After the cells have been separated, they product has to be isolated for the desired product concentration to increase. Then the substances with different polarities are separated from the product.[23] The next step is to further purify the product concentration using the fractional precipitation, chromatography and absorption methods. The last step is to isolate the final product and drying the crystallized product by a vacuum-tray drier, freeze drying, rotary drum driers, spray dryers or pneumatic. The vacuum-tray drier is most used in pharmaceutical product were the product loss and heat damage must be minimized. The freeze drying is when the water is removed from the frozen solution through sublimation.[23] Either the vacuum-tray drier or the freeze drying should be used in this case for cyanobacteria product. 6.0 Non-Fermentation 6.1 Biological Clocks Circadian rhythms are defined as physical, mental and behavioral changes that follow a 24-hour cycle. These changes are determined by the light and dark conditions in an organism’s environment. [4] The biological clock in algae controls the circadian rhythms. Biological clocks or “Circadian clocks” are autonomous internal daily timekeeping mechanisms that allow organisms to adapt to external daily rhythms of light, temperature, and other environmental factors.[28] A mechanism is defined as a system that consists of interacting biological processes, which as a result produce one or more effects on the organism. A mechanism consists of three major components: A central oscillator. An oscillator is referred to the repetitive variation, with respect to time, of a biological process between two or more different states. Circadian oscillators are abundant in tissues of the body where they are synchronized by both endogenous signals to regulate specific transcriptional activity. Transcription is the first step of gene expression, in which a particular segment of DNA is copied to RNA by the enzyme RNA polymerase. A series of input pathways to this central oscillator to allow entrainment of the clock. Entrainment is referred to the rhythmic physiological or behavioural events that match their period and phase to that of an environment oscillation. [28] In terms of biochemistry, there are a series of output pathways that are synchronized by different phases of the central oscillator throughout an organism. In terms of the molecular level, the function of circadian clocks is controlled by feedback loops involving both positive and negative elements. [28] In many organisms including algae, most parameters must stay under control within a narrow range around a certain optimal level under certain environmental conditions. [28] The deviation of the optimal value of the controlled parameter can result from the changes in both internal and external environments. Biological systems contain many types of regulatory circuits, both positive and negative. A negative feedback look refers to the reaction that slows down a process; whereas the positive feedback look tends to accelerate it. [28] Circadian rhythms have a genetic component. Researchers identified genes that direct circadian rhythms in cyanobacteria. A biological clock serves the function of regulating light-dark activities. These light-dark activities include: gene expression metabolism cell division development and behavior The circadian clock is responsible for all gene expression in a photoautotrophic cyanobacterium Synechococcus elongates.[2] The rhythms of phosphorylation in cyanobacteria relies on the time keeping system of circadian clocks. Phosphorylation is the addition of a phosphate (PO43-) group to a protein or any other organic molecule. The activation of enzymes relies on the addition of the phosphate group. Translation activity is referred to the event of when the messenger RNA (mRNA) is produced as a result of transcription. The mRNA is decoded by a ribosome complex to produce a specific amino acid chain, or polypeptide that will fold into an active protein. Therefore, protein phosphorylation is one type of post-translational activity. In this case, phosphorylation is a positive feedback loop and dephosporylation is a negative feedback loop. "Circadian clocks contain an autonomous oscillator consisting of negative feedback circuits that can be entrained to environmental synchronizers (zeitgebers) via input pathways and that produce overt rhythms via output pathways. Circadian regulation of the expression of ccgs (clock-controlled genes) is thought to play an important role in generating output”. [28] 6.2 Conditions Required for Activation of Daylight Genes Researchers today have been focusing on the objective of tuning gene expression. In order for the bacterium to operate optimally, they must be put in one of the following conditions: constant-light conditions being exposed to periodic light-dark cycling Work by Johnson and his team in 2004 involved developing a bioluminescent reporter strain that expresses a daily rhythm of light emission. [16] (The bioluminescent reporter strain was used as a marker, in which the clock mutants were identified. [16] They mapped the bioclock genes of S.elongatus. One conclusion that they came to was that the clock is formed by the Kai protein complex (periodosome), which consists of 3 proteins: KaiA KaiB KaiC It was discovered that 2 of the proteins, KaiA and KaiC can be used to activate the cell’s daytime and nighttime genes. [2] KaiC is capable of autophosporylation, which also involves KaiA and KaiB. The rhythmic expression of KaiB and KaiC proteins and the circadian control of the subcellular localization of KaiB may contribute to the periodic autophosporylation of KaiC. The Kai protein complex regulates gene expression by periodically altering supercoiling and condensation of the entire chromosome. [28] KaiC acts as a duplicate DNA recombinase/DNA helicase protein that can assemble into homo-hexamers and bind DNA. [28] In Professor Johnson’s experiment, KaiC was crystallized to determine its three-dimensional structure and phosphorylation sites.[16] All three proteins showed circadian oscillations in a test tube. The over expression of the “dusk protein” KaiA and the under-expression of KaiC led to the activation of 95% of the blue-green algae’s daylight genes. [2] 6.3 Cell Division Controlled by Circadian Clocks In cyanobacteria, the cell division cycle is also controlled by circadian clocks. The process of cell division slows down during dusk. The presence of the clock protein KaiC terminates cell division under constant light conditions. Cell division is also controlled by the circadian input kinase CikA, which mediates entrainment of the circadian clock to light. [28] The CikA protein does not function as a photoreceptor, but is able to detect light-controlled changes in photosynthetic activity which involves redox reactions. “In cyanobacteria the minimal autonomus oscillator that persists in constant darkness functions by maintaining a phosphorylation rhythm in the KaiA-KaiB-KaiC periodosome. In the presence of environmental light, an extended loop comes into play that involves genome-wide regulation of gene expression via the histidine kinase SasA and chromosome compaction. Photic input is thought to be mediated via redox signals produced as a result of photosynthesis that can be sensed by LdpA and relayed to the histidine kinase CikA “.[28] The interaction of histidine kinase SasA with KaiC plays a significant role in linking the Kai periodosome to circadian clock-controlled gene expression. SasA, are referred to as null mutants that experience loss of circadian gene expression. However, SasA maintains low-amplitude rhythmicity of a few promoters including KaiBC and it maintains obvious rhythms of chromosomal compaction. S. elongatus is an obligate photoautotroph, and its metabolic activity, transcription, and translation are dramatically suppressed in constant darkness. [28] In addition to recombinant KaiA, KaiB, KaiC proteins, adenosine triphosphate (ATP) is also required to assemble a working clock. The cyanobacterium consists of a timekeeping loop that maintains rhythms in the periodosome and produces clock output in the form of gene expression rhythms. The loop is self-sustained under constant light conditions, which enables it to run constantly and not be affected by changes in temperature in the surrounding environment. 6.4 The Production of Industrial Chemicals The daytime genes are responsible for producing industrially useful chemicals. Some industrially useful chemicals are human insulin, the fluorescent protein luciferase, and hydrogenase. The biological clock must be in daytime mode in order to produce these important compounds. Therefore, researchers inserted genes into cyanobacteria whose biological clocks were altered to remain in daytime mode. As a result, “altered clocks produced 200% more hydrogenase, 500% more insulin, and 700% more luciferase when grown in constant light than they did when the genes were inserted in cells with unaltered clocks”.[2] 7.0 Canadian Regulation Although algae’s growth has a great environmental and industrial benefits, its excessive bloom could lead to serious pollution to the environment. Since controlling and screening this process is essential to maintain the environment healthy and pollution-free, Environment Canada has regulated a series of progressive regulations that will help maintain the growth of algae under control. Throughout this action, algae growth could give the useful side without affecting the health and environment standards. Different processes and factors could lead to algae’s excessive growth in marine and freshwater environment such as increasing specific nutrients in the growth medium (mostly Phosphorus) or changing the algae’s biological clock. Back in the 1960’s an extensive algae growth in the Great Lakes was a big concern that the government of Canada had to address and solve. This excessive growth led to water pollution, and as a result Environment Canada regulated solutions and actions to control and address any issue at the level of existence of the algae in the marine and freshwater environments. [10] These regulations included three parts: governmental, scientifical and human actions. For the governmental part, Ministry of Environment Canada has been working with the domestic, provincial, national partners to develop analytical measurements of the chemicals, toxic and non-toxic elements in the water. These data could be used as standards for monitoring the water environment from pollution. [10] Scientifically, the regulation included monitoring and reporting the environment standards regularly which will help an immediate identification of any changes in the measurements that could lead to a serious issue. [10] We can help our environment as well by projects and research, raise local awareness of environment quality standards especially near shore areas and checking our septic system regularly. 8.0 Midterm-Exam Question What is the function of the Cyanobacteria protein KaiA and KaCi in controlling the biological clock?!! Answer: KaiA protein (photosynthetic genes) hyperphosphorylates KaiC and activates “dusk” gene expression KaiC- regulates “dusk” genes down and “dawn” genes up 9.0 Assignment Question What is the difference between a positive feedback loop and a negative feedback loop? Please use a biological process within an organism to explain the difference. 10.0 References Adult Stem Cells. (n.d.). Adult Stem Cells. Retrieved January 25, 2014, from http://miraclecell.net/tricking-algaes-biological-clock-boosts-production-of-drugs-biofuels/ "Algae's Biological Clock Gets Duped." CEP Magazine- An AIChE Publication Dec. 2013: 8. Print. Anabaena azollae. (n.d.). MicrobeWiki. Retrieved January 25, 2014, from http://microbewiki.kenyon.edu/index.php/Anabaena_azollae Article (2012). Circadian rhythms fact sheet. National Institute of General Medical Sciences, Retrieved from http://www.nigms.nih.gov/Education/Pages/Factsheet_CircadianRhythms.aspx Azolla-Anabaena as a Biofertilizer for Rice Paddy Fields in the Po Valley, a Temperate Rice Area in Northern Italy. (n.d.). International Journal of Agronomy. Retrieved January 25, 2014, from http://www.hindawi.com/journals/ija/2010/152158/ Biological clock. (n.d.). Biological clock. Retrieved January 25, 2014, from http://www.glimmerveen.nl/le/biological_clock.html Cell (biology). (2014, January 27).Wikipedia. Retrieved January 28, 2014, from http://en.wikipedia.org/wiki/Cell_(biology) Connect. (n.d.). Algal Bioenergy. Retrieved January 24, 2014, from https://connect.innovateuk.org/web/algal-biotechnology-special-interest-group/article-view/-/blogs/tricking-blue-green-algae%E2%80%99s-biological-clocks-into-increase-biomolecule-production;jsessionid=A514CF7F477331D21B39751844508F0C.2?_33_redirect=https% Current Biology - Circadian Yin-Yang Regulation and Its Manipulation to Globally Reprogram Gene Expression. (n.d.). Current Biology - Circadian Yin-Yang Regulation and Its Manipulation to Globally Reprogram Gene Expression. Retrieved January 26, 2014, from https://www.cell.com/current-biology/abstract/S0960-9822(13)01251-7?switch=standard Excess Algal Growth. (n.d.). Government of Canada, Environment Canada. Retrieved January 26, 2014, from http://www.ec.gc.ca/grandslacs-greatlakes/default.asp?lang=En&n=6201FD24-1 . Fermentation Pathways. (n.d.). Microbial Physiology. Retrieved January 24, 2014, from http://docencia.izt.uam.mx/hgm/bq_fisiol_microbiana/documentos/pdf/Moat_microbial_physiol/cap_11.pdf Genetic Engineering to Produce Insulin. (n.d.). ResourceImpor. Retrieved January 26, 2014, from http://www.abpischools.org.uk/res/coResourceImport/modules/hormones/en-flash/geneticeng.cfm Hanson, Chris. "Algae Tricked into Staying up Late to Produce Biomaterials." Biomass Magazine. BBI International, 19 Nov. 2013. Web. 25 Jan. 2014. <http://biomassmagazine.com/articles/9708/algae-tricked-into-staying-up-late-to-produce-biomaterials>. Hydrogen production by biological processes: a survey of literature. (n.d.).International Journal of Hydrogen Energy. Retrieved January 25, 2013, from http://journals2.scholarsportal.info.ezproxy.lib.ryerson.ca/tmp/14870214388699590324.pdf Hydrogen Production by Cyanobacteria". (n.d.). Springer. Retrieved January 25, 2013, from http://www.springer.com%2Fcda%2Fcontent%2Fdocument%2Fcda_downloaddocument%2F9781461412076-c1.pdf%3FSGWID%3D0-0-45-1265656-p174138566&ei=lnjqUuyKC6bS2QXgkoEI&usg=AFQjCNGWvZVwiQkpLaQzvWQULaPHwiXNXg&bvm=bv.60444564,d.b2I Johnson, C. (2010). Cellular, molecular and evolutionary analyses of biological clocks.Vanderbilt University, Retrieved from http://as.vanderbilt.edu/johnsonlab/ "Mission 2013 - Algae." Mission 2013 - Algae. Massachusetts Institute of Technology (MIT), n.d. Web. 25 Jan. 2014. <http://igutek.scripts.mit.edu/terrascope/?page=Algae>. Mur, L. R., Skulberg, O. M., & Utkilen, H. (n.d.). Chapter 2. CYANOBACTERIA IN THE ENVIRONMENT. Toxic Cyanobacteria in Water:. Retrieved January 25, 2014, from http://www.who.int/water_sanitation_health/resourcesquality/toxcyanchap2.pdf "News Medical." Insulin - What is Insulin?. N.p., n.d. Web. 25 Jan. 2014. <http://www.news-medical.net/health/What-is-Insulin.aspx>. Nitrogen Fixation and Hydrogen Metabolism in Cyanobacteria. (n.d.).Microbiology and Molecular Biology Review. Retrieved January 25, 2014, from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3008169/?tool=pmcentrez&report=abstract Recombinant DNA: Example Using Insulin » In-Depth » Explore More: Genetic Engineering. (n.d.). Recombinant DNA: Example Using Insulin » In-Depth » Explore More: Genetic Engineering. Retrieved January 25, 2014, from http://www.iptv.org/exploremore/ge/what/insulin.cfm Scott, Willie. "Algae Biofuels vs Fossil Fuels." Bright Hub. Bright Hub Inc., 4 Dec. 2010. Web. 25 Jan. 2014. <http://www.brighthub.com/environment/renewable-energy/articles/68532.aspx>. Shuler, M. L., & Kargi, F. (2002).Bioprocess engineering: basic concepts(2nd ed.). Upper Saddle River, NJ: Prentice Hall. Stal, L. J., & Moezelaa, R. (n.d.). Fermentation of Cyanobacteria. FEMS Microbiology Review. Retrieved January 24, 2014, from http://journals2.scholarsportal.info.ezproxy.lib.ryerson.ca/tmp/3102156255750558481.pdf The circadian clock of cyanobacteria. (n.d.). Review articles. Retrieved January 26, 2014, from http://www.math.utah.edu/~keener/lectures/ima/Problems/papers/Kondo_Ishiura.pdf Thylakoid/Citable Version. (n.d.).Thylakoid. Retrieved January 31, 2014, from http://en.citizendium.org/wiki/Thylakoid/Citable_Version Tricking algae’s biological clock boosts production of drugs, biofuels | Research News @ Vanderbilt | Vanderbilt University. (n.d.). Vanderbilt Research. Retrieved January 24, 2014, from http://news.vanderbilt.edu/2013/11/algaes-clock-drugs-biofuels/ Using Green Algae as Drug Factory Could Cut Pharma Costs by 1,000 Times. (n.d.).Popular Science. Retrieved January 25, 2014, from http://www.popsci.com/science/article/2010-03/producing-drugs-green-algae-could-cut-costs-1000-times-over Wijnen, H., & Young, M. W. (2006). Interplay of circadian clocks and metabolic rhythms. In The annual review of genetics (Vol. 40, pp. 412-431). New York: Annual Reviews. Retrieved from http://groups.molbiosci.northwestern.edu/bass/labMem/PDFs/AnnRevGenet11-10-06.pdf Wijnen, H., & Young, M. W. (2006). Interplay of circadian clocks and metabolic rhythms. In The annual review of genetics (Vol. 40, pp. 412-431). New York: Annual Reviews. Retrieved from http://groups.molbiosci.northwestern.edu/bass/labMem/PDFs/AnnRevGenet11-10-06.pdf
