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Project: Reclaiming Wastewater Reclamation Click here to edit this page!
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Project Description		Content
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. References iGEM teams are encouraged to record references you use during the course of your research. They should be posted somewhere on your wiki so that judges and other visitors can see how you though about your project and what works inspired you.		You can use these subtopics to further explain your project Overall project summary Project Details Materials and Methods The Experiments Results Data analysis Conclusions It's important for teams to describe all the creativity that goes into an iGEM project, along with all the great ideas your team will come up with over the course of your work. It's also important to clearly describe your achievements so that judges will know what you tried to do and where you succeeded. Please write your project page such that what you achieved is easy to distinguish from what you attempted.

Project: Biofilm Annihilation

Project Background:

Biofilm production by bacteria is a major concern in ASPs (activated sludge processors). 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.

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:

1. 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
2. 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
3. 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
4. "Addgene: Addgene's CRISPR Guide." Addgene: Addgene's CRISPR Guide. Web. 8 Apr. 2014.
5. 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
6. "Streptococcus Thermophilus LMD-9, Complete Genome." National Center for Biotechnology Information. U.S. National Library of Medicine, 24 Oct. 2006. Web. 8 Apr. 2014. .
7. 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. .