Team:University College London/LabBook

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Expt 4.1 - Transformation of Key Aggregation Module BioBricks

4.1 will be transforming the Curli Cluster, Green Fluorescence Protein and Ribosome Binding Site BioBricks into our W3100 competent cell line. Transformed cells will be cultured under the selective pressure of antibiotic, after which the plasmids are purified and the presence of the correct BioBricks will be diagnosed with an Analytical Restriction Enzyme Digest and Gel Electrophoresis.

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-= Research =
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-== Project Overview ==
+<div class="experiment">Expt 4.1 - Transformation of Key Aggregation Module BioBricks</div>
+<div class="experimentContent>4.1 will be transforming the Curli Cluster, Green Fluorescence Protein and Ribosome Binding Site BioBricks into our W3100 competent cell line. Transformed cells will be cultured under the selective pressure of antibiotic, after which the plasmids are purified and the presence of the correct BioBricks will be diagnosed with an Analytical Restriction Enzyme Digest and Gel Electrophoresis.  </div>
-In many of the worlds oceans, currents carry debris and pollution originating from coastlines. This waste accumulates in  regional gyres, where the worlds ocean currents meet, and can reach extremely high concentrations. Plastic is estimated to account for 60-80% of this debris, and is known to be gradually broken down by solar energy and the mechanic action of the sea. This means the majority of the plastic waste are several millimetres in size or less, which has made efforts to clean them from the ocean largely unsuccessful. These tiny plastic fragments - microplastics - enter the digestive systems of resident organism, which are affected either by the physical size of the plastic or its toxicity from adsorbing organic pollutants.
-UCL iGEM proposes a synthetic biology approach for the bioremediation of micro-plastic pollutants within the marine environment, with emphasis on regions of excessive debris accumulation, such as the North Pacific ‘garbage patch’.
-We intend to engineer enhanced adhesive properties in ''Escherichia coli'' and marine bacteria ''Roseobacter denitrifican''s & ''Oceanibulbus indolifex'', of the ''Roseobacter'' clade. To alter the composition and dynamics of resultant biofilms for the adhesion of micro-plastic pollutants, with an extended vision of creating mass aggregates, or ‘Plastic Islands’.
-We will attempt to demonstrate micro-plastic particle aggregation and several additional genetic components, including plastic degradation, salinity/osmotic tolerance in ''E. coli'', bacterial buoyancy and novel active biological containment strategies, for an integrative approach to marine bioremediation.
-== Biobrick Overview ==
-== Module 1: Detection ==
-Our Detection Module will allow our bacteria to detect the presence of plastic. The purpose of this is to control the production of our adhesive – Curli (Module 2) – which binds non-specifically to an extraordinary array of surfaces.  By producing adhesive only when plastic is present, we prevent our bacteria binding to non-plastic materials.
-== Aggregation Module ==
-'''Curli expression for the aggregation of plastic and production of biofilm
-BBa_K729003'''
-The Aggregation Module will enable our bacteria to construct islands from smaller plastic fragments. To do so we have decided to transform our bacterium with a circuit to produce an adhesive protein called Curli. As Curlis are non-specific in the surfaces they bind, we also have a module (Module 1) to ensure they are produced only in the presence of plastic
-== Plastic Degradation ==
-'''Multi-copper oxidase/Laccase for the degradation of polyethylene and other plastics BBa_K729002'''
-As an alternative to Island Formation Modules (Modules 1 and 2), we are also developing an alternative solution – Degradation of plastic. This will investigate enzymes expressed by numerous organisms that have been demonstrated to degrade plastics.
-==Buoyancy Module==
-'''Temperature dependent buoyancy for localisation of engineered bacteria within the water column BBa_K729000'''
-The Buoyancy Module is key to both the Degradation (Module 4) and the Island Formation systems (Module 1 and 2). Buoyancy is required to position our bacteria in the water column, alongside the plastic fragments, and also to enable them to buoy the plastic aggregates (Module 2).
-==Salt Tolerance==
-'''irrE a global regulator from ''Deinococcus radiodurans'' for increased salinity tolerance in E. coli BBa_K729001'''
-A core module for our project is enabling E.coli to tolerate the salt content of the ocean; without this ability E.coli could not survive in a marine environment. Due to the widespread use of E.coli as a chassis for Synthetic Biology this Module is being developed to demonstrate that E.coli, as well as marine bacteria, could be used as the chassis for this project.
-==Containment Components==
-UCL iGEM 2012 addresses fundamental barriers to the implementation of traditional biological containment systems.
-'''A novel threefold active biological containment system
-BBa_K729010, BBa_K729009'''
-As our project suggests the release of genetically modified bacterium into the environment, we feel it is necessary to contain the risk of spreading genetic information by horizontal gene transfer. To do so we are suggesting a trio of protective systems – consisting of two toxin/anti-toxin pairs  - Colicin-E3/Colicin Immunity E3 and Holin/Anti-Holin endolysin– and an excreted nuclease.
-'''Nuclease from Staphylococcus aureus for genomic deletion
-BBa_K7290011'''
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