HTGAA 2015 Adolfo Benítez Final Project presentations Bacteodorant Title: Bacteodorant The idea: Find the metabolic path and change the DNA of a Bacteria to create banana odor from human sweat STEPS Step 1: Find the right bacteria Step 2: Find the Genome Sequence of the bacteria Step 3: Find the enviroment to grow up Step 4: Pathway design Step 5: Gen Drive What we need to create the bacteriodor? • A bacteria that is very common in our enviroment, NOT DANGEROUS • Can survive in dry enviroment in order to keep them in a bootle. • We can change the genoma for our porpouse. According to: Microbiome Topographical and Temporal Diversity of he Human Skin Elizabeth A. Grice, et al. Science 324, 1190 (2009); DOI: 10.1126/science.1171700 There are 19 bacterial phyla on human skin. The most sequences were assigned to four phyla: Actinobacteria (51.8%), Firmicutes (24.4%), Proteobacteria (16.5%), and Bacteroidetes (6.3%). 205 bacterial species identified represented by at least five sequences, three were associated with more than 62% of the sequences: Corynebacteria (22.8%; Actinobacteria), Propionibacteria (23.0%; Actinobacteria), and Staphylococci (16.8%; Firmicutes) Bacterias to test: 1. Staphylococcus aureus 2. Staphylococcus epidermis 3. Nitrosomonas eutropha Staphylococcus aureus. Medium needed 1 • http://aac.asm.org/content/10/6/915.full.pdf • Bacteria: Staphylococcus aureus • Strain PS84 • Enviroment: • Shake wáter • 37ºC • SM4 . Ratio of the inoculum to the médium was: 1:26 • Formula to SM4: • • • • • • • • • • • • • • • • • • • DL-Aspartic acid 0.180 (g/l) L-Valine 0.170 L-sodium glutamate 0.090 DL-f3-Phenylalanine 0.010 L-Proline 0.070 L-Arginine monohydrochloride 0.025 L-Histidine monohydrochloride 0.050 Glycine 0.050 DL-Leucine 0.050 L-Cystine 0.005 L-a-Alanine 0.080 Nicotinic acid 0.0002 Thiamine hydrochloride 0.00001 Sodium citrate 0.5 Ammonium sulfate 1.0 Magnesium sulfate 0.1 D-Glucose 2.0 Potassium dihydrogen orthophosphate 3.0 Dipotassium hydrogen orthophosphate 7.0 Staphylococcus aureus in dry medium • According to: • http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2134531/pdf/jhyg00121-0003.pdf • Rountree PM (1963) The effect of dessication on the viability of Staphylococcus aureus. J Hyg 61:265–272 • Strain number: PP7/1 Page Pattern: 42E Source: Air Sample With 36 to 84 days of drying death-rate K/day: 0,029 • K is: 𝐵 +𝐵 • K = 2 · 3 0𝑡 𝑡 • Where t = time of drying in days, Bo = log10 count at time 0, and B1 = log10 count at time t. Is easy to keep alive this bacteria in a dry medium. • Staphylococcus aureus is a gram-positive coccal bacterium that is a member of the Firmicutes, and is frequently found in thenose, respiratory tract, and on the skin. It is often positive for catalase and nitrate reduction. Although S. aureus is not alwayspathogenic, it is a common cause of skin infections such as abscesses, respiratory infections such as sinusitis, and food poisoning. Pathogenic strains often promote infections by producing potent protein toxins, and expressing cell-surface proteins that bind and inactivate antibodies. The emergence of antibiotic-resistant forms of S. aureus such as MRSA is a worldwide problem in clinical medicine. • Staph infections may cause disease due to direct infection or due to the production of toxins by the bacteria. Boils, impetigo, food poisoning, cellulitis, and toxic shock syndromeare all examples of diseases that can be caused byStaphylococcus. • S. aureus is responsible for many Staphylococcal infections, but it may also occur as a commensal. The presence of S. aureus does not always indicate infection. It can survive from hours to weeks, or even months, on dry environmental surfaces, depending on strain. Wikipedia May cause disease die to direcinfection or due to the production of toxins by the bacteria. For example: Boils, impetigo, food poisoning, cellulitis, and toxic shock syndromeare. Have many Virulance factors: Enzymes (coagulase, deoxyribonuclease, lipase, beta-lactamase, etc.), Toxins, immunoevasive strategies, and so on. MRSA (strain of Staphylococcus aureus) has become resistant to most β-lactam antibiotics. Staphylococcus aureus Staphylococcus epidermis 2 • It is part of the normal human flora, typically the skin flora, and less commonly the mucosal flora.[2]Although S. epidermidis is not usually pathogenic, patients with compromised immune systems are at risk of developing infection. • The most common treatment for these infections is to remove or replace the infected implant, though in all cases, prevention is ideal. The drug of choice is often vancomycin, to which rifampin or aminoglycoside can be added.[citation needed] Hand washing has been shown to reduce the spread of infection. • Preliminary research also indicates S. epidermidis is universally found inside affected acne vulgaris pores, where Propionibacterium acnes is normally the sole resident Staphylococcus epidermis Nitrosomonas eutropha 3 • Nitrosomonas eutropha is not known to have any pathogenic qualities. • Living bacterial skin tonic, developed by AOBiome, a biotech start-up in Cambridge, Mass. The tonic looks, feels and tastes like water, but each spray bottle of AO+ Refreshing Cosmetic Mist contains billions of cultivated Nitrosomonas eutropha, an ammonia-oxidizing bacteria (AOB) that is most commonly found in dirt and untreated water. AOBiome scientists hypothesize that it once lived happily on us too — before we started washing it away with soap and shampoo — acting as a built-in cleanser, deodorant, anti-inflammatory and immune booster by feeding on the ammonia in our sweat and converting it into nitrite and nitric oxide. The new York Times (May 22, 2014) According to Joint Genome Institut (US department of energy): http://genome.jgi.doe.gov/nitec/nitec.home.html • Nitrosomonas eutropha C91 (Kingdom: Bacteria) is an autotrophic nitrifying bacterium. As a member of the ammonia-oxidizing bacteria, it catalyzes the oxidation of ammonia to meet its energy requirements for growth. According to Joint Genome Institut (US department of energy): http://genome.jgi.doe.gov/nitec/nitec.home.html Chain, P., J. Lamerdin, F. Larimer, W. Regala, V. Lao, M. Land, L. Hauser, A. Hooper, M. Klotz, J. Norton, L. Sayavedra-Soto, D. Arciero, N. Hommes, M. Whittaker, D. Arp. 2003. Complete genome sequence of the ammonia oxidizing bacterium and obligate chemolithoautotroph Nitrosomonas europaea . J. Bacteriol. 185: 2759-2773 • Nitrosomonas eutropha C91 (Kingdom: Bacteria) is an autotrophic nitrifying bacterium. As a member of the ammonia-oxidizing bacteria, it catalyzes the oxidation of ammonia to meet its energy requirements for growth. Nitrosomonas eutropha. 3 In order to use genome technics, we need to know the complete genome sequence of the bacteria!! Here it is the paper that had found it: Chain, P., J. Lamerdin, F. Larimer, W. Regala, V. Lao, M. Land, L. Hauser, A. Hooper, M. Klotz, J. Norton, L. Sayavedra-Soto, D. Arciero, N. Hommes, M. Whittaker, D. Arp. 2003. Complete genome sequence of the ammonia oxidizing bacterium and obligate chemolithoautotroph Nitrosomonas europaea . J. Bacteriol. 185: 2759-2773 Circular representation of the 2,812,094-bp genome of N. europaea ATCC 19718 Nitrosomonas eutropha. Enviroment to Grow According to: Key Physiology of Anaerobic Ammonium Oxidation MARC STROUS, J. GIJS KUENEN, AND MIKE S. M. JETTEN APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1999, p. 3248–3250 6,7 < pH < 8,3 20ºC < temperature < 43ºC Sustrate: ammonium<0.1gr/liter, nitrite<0.1gr/liter (higher values inhibited ANaerobic AMMonium Oxidation process) After that inhibition, addition of traces restore activity: hydrazine <1.4mg of nitrogen/liter and hidoxylamine < 0.7mg of nitrogen/liter Nitrosomonas eutropha. Enviroment to Grow: CHEMOSTAT I need A CHEMOSTAT • One of the most important features of chemostats is that microorganisms can be grown in a physiological steady state under constant environmental conditions. • In this steady state, growth occurs at a constant specific growth rate and all culture parameters remain constant Sensors: pH, temperature, optical density for bacteria grow control and nitrogen in sustrate. Stream IN Media Feed Sensor optical density Sensor pH Stream IN AIR compressed Sensor Nitrogen Sensor temperatura Stream OUT Product Heat • Info from: https://en.wikipedia.org/wiki/Chemostat https://controls.engin.umich.edu/wiki/index.php/Bacterial_Chemostat_Model Nitrosomonas eutropha. CHEMOSTAT Chemostat, list of components: Nitrosomonas eutropha. Pathway design Find the pathway on: http://www.kegg.jp/ http://www.kegg.jp/dbget-bin/www_bfind_sub?mode=bfind&max_hit=1000&dbkey=kegg&keywords=Nitrosomonas%20eutropha Nitrosomonas eutropha strain C91 http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=335283 Whole-genome analysis of the ammonia-oxidizing bacterium,Nitrosomonas eutropha C91: implications for niche adaptation Lisa Y. Stein1,*, Daniel J. Arp2, Paul M. Berube3, Patrick S. G. Chain4,5,6, Loren Hauser7, Mike S. M. Jetten8, Martin G. Klotz9, Frank W. Larimer7, Jeanette M. Norton10, Huub J. M. Op den Camp8,Maria Shin4,5 and Xueming Wei2 Environmental Microbiology, Volume 9, Issue 12, pages 2993–3007, December 2007 Genome is complete, according to PATRIC: https://www.patricbrc.org/portal/portal/patric/Genome;jsessionid=jqs2yuhrMJUGMK6jgQyotg**?cId=335283.9&cType=genome Genome Sequence: http://prodigal.ornl.gov/genomes/CP000450.gbk Perfums are aldehyds: vanillin, cinnamaldehyde (cinnamon), geranium (lemon) bourgeonal (lily of the valley) and nonanal (Rose) And Nitrosomonas eutropha produces aldehyds ( according to: Nitrification and degradation of halogenated hydrocarbons—a tenuous balance for ammonia-oxidizing bacteria Luis A. Sayavedra-Soto & Barbara Gvakharia & Peter J. Bottomley & Daniel J. Arp & Mark E. Dolan Appl Microbiol Biotechnol (2010) 86:435–444 It seems possible to find a pathway to produce perfum with Nitrosomonas eutropha Nitrosomonas eutropha. CRISPR gen drive CRISPR/Cas9 (*) is a gene-editing technique that has revolutionized the field of genetic engineering since 2013. The approach involves expressing the RNA-guided Cas9 endonuclease along with guide RNAs directing it to a particular sequence to be edited. When Cas9 cuts the target sequence, the cell repairs the damage by replacing the original sequence with an altered version. Making a guide RNA to direct Cas9 to cut any specific gene is straightforward, the unmodified target sequence is introduced into a CRISPR-positive cell and then the CRISPR-cas complex can be isolated via several protein isolation techniques. CRISPR tremendously simplifies the process of deleting, adding, or modifying genes. As of 2014 it had successfully been tested in cells of 20 species, including humans. In many of these species, the edits modified their germline, allowing them to be inherited. Wikipedia (*) Elizabeth Pennisi (2013-08-23). "The CRISPR Craze". Sciencemag.org. Retrieved 2014-07-18.
