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Project: Reclaiming Wastewater Reclamation Click here to edit this page!

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Overall Project Description

We are working to optimize the wastewater treatment process. Currently we are addressing some of the difficulties faced by the working microbial community in the bioreactor. These include: the buildup of biofilm, the destruction of the "working class" bacteria by phage, antibiotic discharge into the wastewater, and nitrate production. We will be building our machine in the bioreactor bacterium N. multiformis and optimizing it to address the aforementioned obstacles.

Project: Biofilm Annihilation

Project Background:

Biofilm production by bacteria is a major concern in activated sludge processors (ASPs). The buildup of these bacteria in these processors inhibits the helpful bacteria from being able to effectively break down several components. Of main concern are the biofilms of bacteria such as Nocardia spp., Thiothrix spp., Sphaerotilus natans, and several others. In order to solve this issue we cloned the genes for Alpha Amylase and DispersinB, both which break down the polysaccharide matrix of biofilms, as well as the gene for AiiA, a quorum-sensor blocker, into Nitrosospira multiformis, one of the helpful bacteria in the activated sludge processors. Attached to these genes in their respective chassis is a signaling sequence which dictates the expression of these gene products extracellularly. With the expression of these genes outside the cells there should be a significant decrease in the amount of biofilm buildup by these biofilm-creating bacteria and bacteria such as N. multiformis will be less restricted in breaking down the interested components of ASPs.

AiiA, Alpha Amylase, DispersinB, A visual representation of biofilm inhibition and degradation.

Biofilm Team:

Cam Zenger, Jared McOmber, and Jordan Berg

The focus of the biofilm team was to insert the genes for Alpha Amylase, DispersinB, and AiiA with a signaling sequence for extracellular expression into the pSB1C3 chassis and to assay the efficacy of these genes in biofilm reduction in ASPs. Additionally, site-directed mutagenesis was performed on Alpha Amylase to remove the PstI restriction site found within the gene.

References:

• Donelli G, Francolini I, Romoli D, Guaglianone E, Piozzi A, Ragunath C, Kaplan JB. 2007 Aug. Synergistic Activity of Dispersin B and Cefamandole Nafate in Inhibition of Staphylococcal Biofilm Growth on Polyurethanes. Antimicrobial Agents and Chemotherapy. [accessed 27 Mar 2014]; 51(8):2733–2740. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1932551/pdf/1249-06.pdf.
• Kalpana BJ, Aarthy S, Pandian SK. 2012 Feb. Antibiofilm Activity of α-Amylase from Bacillus subtilis S8-18 Against Biofilm Forming Human Bacterial Pathogens. Appl Biochem Biotechnol. [accessed 27 Mar 2014]; 167:1778–1794. http://download.springer.com/static/pdf/846/art%253A10.1007%252Fs12010-011-9526-2.pdf?auth66=1407375774_bcd3eaa94748c4bb0a1ec0553c81fa5c&ext=.pdf.
• Brown-Elliott BA, Brown JM, Conville PS,Wallace RJ. 2006 Apr. Clinical and Laboratory Features of the Nocardia spp. Based on Current Molecular Taxonomy. Clin Microbial Rev. [accessed 14 Feb 2014]; 19(2):259-282. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1471991/.
• Fazekas E, Kandra L, Gyémánt G. 2012 Dec. Model for β-1,6-N-acetylglucosamine oligomer hydrolysis catalysed by DispersinB, a biofilm degrading enzyme. Carbohydr Res. [accessed 14 Feb 2014]; 363:7-13. http://www.ncbi.nlm.nih.gov/pubmed/23103508.
• Kaplan JB, Ragunath C, Ramasubbu N, Fine DH. 2013. Detachment of Actinobacillus actinomycetemcomitans biofilm cells by an endogenous beta-hexosaminidase activity. J Bacteriol. [accessed 14 Feb 2014]; 185:4693-4698. http://www.uniprot.org/citations/12896987.

Project: Crispy CRISPR's

Project Background:

Nitrosospira multiformis, a key microbe in sewage treatment centers using the activated sludge process, suffers a high rate of bacteriophage induced lysis(7). Between the high titer of bacteriophage found in sewage, prophage incorporated into its genome, and its natural slow rate of growth, N. multiformis does not reach high concentrations in bio-reactors(7). To combat the prevalence of bacteriophage infection we have cloned the CRISPR 3 system found in Streptococcus thermophilus LMD-9 into N. multiformis(6). Using a novel spacer region we will specifically target three prophage that reside in N. multiformis' genome. The CRISPR 3 system has been shown to acquire additional phage spacers and will provide adaptive immunity to N. multiformis(1).

CRISPR TEAM:

Garrett Jensen, Mike Abboud, Michael Linzey.

References:

• Rimantas Sapranauskas, et. Al. The Streptococcus thermophilus CRISPR/Cas system provides immunity inEscherichia coliNucl. Acids Res. (2011) 39 (21): 9275-9282 first published online August 3, 2011doi:10.1093/nar/gkr606
• Hongfan Chen, Jihoon Choi, and Scott Bailey. Cut Site Selection by the Two Nuclease Domains of the Cas9 RNA-guided EndonucleaseJ. Biol. Chem. jbc.M113.539726. First Published on March 14, 2014,doi:10.1074/jbc.M113.539726
• Shah SA, Erdmann S, Mojica FJ, Garrett RA. Protospacer recognition motifs: Mixed identities and functional diversity. RNA Biology 2013; 10:891 - 899; PMID: 23403393; http://dx.doi.org/10.4161/rna.23764
• "Addgene: Addgene's CRISPR Guide." Addgene: Addgene's CRISPR Guide. Web. 8 Apr. 2014.
• Krzysztof Chylinski, Kira S. Makarova, Emmanuelle Charpentier,and Eugene V. Koonin. Classification and evolution of type II CRISPR-Cas systemsNucl. Acids Res. first published online April 11, 2014 doi:10.1093/nar/gku241
• "Streptococcus Thermophilus LMD-9, Complete Genome." National Center for Biotechnology Information. U.S. National Library of Medicine, 24 Oct. 2006. Web. 8 Apr. 2014. .
• Choi, Jeongdong, Shireen M. Kotay, and Ramesh Goel. "Various Physico-chemical Stress Factors Cause Prophage Induction in Nitrosospira Multiformis 25196- an Ammonia Oxidizing Bacteria." Science Direct. Water Research, 4 Aug. 2010. Web. 1 Feb. 2014. .

Project: Denitrification

Project Background:

Nitrification, the process of oxidizing ammonia to produce nitrates, occurs naturally in many of the bioreactor bacteria, including N. multiformis. These soluble nitrates leave the sewage treatment facility in the effluent, becoming nutrients to aquatic plants and algae downstream. When accumulated in high concentrations, these nitrates lead to eutrophication, a dangerous shift in the balance of the local ecosystem that can be seen in the photograph below. To prevent eutrophication downstream from water treatment facilities, we have designed a plan to engineer N. multiformis to also perform denitrification, the process of converting nitrate into nitrogen gas, as a means of removing nitrogen without negative effects on the environment. According to scientific literature (see below), denitrification can be accomplished by the proteins coded by the nirS, norB, norC, and nosZ genes from Pseudomonas aeruginosa. These genes convert nitrite (NO₂^-) to nitric oxide (NO), then to nitrous oxide (N₂O), and finally to nitrogen gas (N₂). The nitrification pathway in N. multiformis converts ammonia (NH₃) to nitrite (NO₂^-) and then to nitrate (NO₃^-), meaning that the denitrification pathway begins with an intermediate of the nitrification process; a diagram of both pathways can be seen below. The enzyme in N. multiformis that converts nitrite to nitrate in nitrification performs a reversible reaction, so Le Chatelier's principle suggests that the denitrification pathway will work because equilibrium will drive the reaction to produce nitrogen gas. Knocking out the nitrate reductase in N. multiformis will also ensure that our chassis is forced to proceed through the denitrification pathway instead of producing nitrate.

Denitrification Team:

Cameron Sargent and Julie Roberts

References:

• Z. Chen et al., Differentiated response of denitrifying communities to fertilization regime in paddy soil. Microbial ecology 63, 446 (Feb, 2012).
• H. Arai, Regulation and Function of Versatile Aerobic and Anaerobic Respiratory Metabolism in Pseudomonas aeruginosa. Frontiers in microbiology 2, 103 (2011).
• V. Kathiravan, Pseudomonas aeruginosa and Achromobacter sp.: nitrifying aerobic denitrifiers have a plasmid encoding for denitrifying functional genes. World journal of microbiology & biotechnology 30, 1187 (2014).

Project: Serine Auxotrophy

Project Background:

The auxotrophy project aims to create a safety mechanism that will provide a barrier between the proposed modified organism and the environment at large. As a precaution, we have engineered our chassis to be dependent on the environment found in the bioreactor, thus preventing it from escaping and spreading uncontrollably. We found that the amino acid serine is abundant in the water of the bioreactor and is not present in the effluent. We also found that the serA gene is essential to Nitrosospira multiformis production of serine. By removing the serA gene Nitrosospira multiformis will be unable to produce its own serine, which is essential for growth outside of the bioreactor.

Biofilm Team:

Christian Boekweg, Tanner Robinson, and Mark Murdock

The focus of the auxotrophy team was to knock out the gene responsible for the biosynthesis of serine. We found the serA gene to be crucial to Nitrosospira multiformis production of serine.

References:

• Andersson D.I., Hughes D. (2012) Evolution of antibiotic resistance at non-lethal drug concentrations. Drug Resistance Updates Volume 15, 162-172.
• World Health Organization (2005) Containing antimicrobial resistance (10), WHO Policy Perspectives on Medicine 1–5.
• Kümmerer, K. (2004) Resistance in the environment. Journal of Antimicrobial Chemotherapy (2004) 54 (2): 311-320.
• Fishman, N. (2006) Antimicrobial stewardship. Am. J. Infect. Control 34, S55–63
• Antibiotic Prescribing and Use, Price of Care Issue Brief, February 2011, The Center for Healthcare Research & Transformation
• Goel, R., Kotay, S.M, Choi, J. (2010) Various physic-chemical stress factors cause prophage induction in Nitrosospira multiformis 25196- an ammonia oxidizing bacteria. Water Researchhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC1932551/pdf/1249-06.pdf.