Team:Wisconsin-Madison/biobricks

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Revision as of 19:55, 9 August 2011

Project >> Overview, Ethanol Sensor, Alkane Sensor, Microcompartment

Biobricks

BioBricks are DNA sequences with a standardized interface that allows for simple cut and paste construction of biological systems. The interface consists of six specific restriction enzyme sites that flank the DNA coding region upstream and downstream; Along with restriction sites, BioBrick parts are also cloned into a specific plasmid backbone. There are two classifications of BioBricks: parts and devices. Parts are the basic constituents of larger systems that code for basic biological functions, and devices consist of multiple parts that work synergistically to serve a specific application. In order to functionalize these BioBricks, they are implemented into living cells, most commonly E. coli bacterial cells. Every year, each iGEM team submits their standardized BioBricks to the iGEM parts registry; this registry constitutes an open source library of synthetic biological parts that any future team can order and use for their project.

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-Ethanol Sensor
+Biobricks
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-The primary source of results thus far has been a plate reader. This is a device which can record the optical density (OD – a measure of cell concentration) as well as the fluorescence of up to 96 cultures at once. Ideally, we hope to produce such a robust fluorescent response as to not need the plate reader for qualitative assessments.
+BioBricks are DNA sequences with a <a href="https://2011.igem.org/Team:Wisconsin-Madison/cutsites">standardized interface</a> that allows for simple cut and paste construction of biological systems. The interface consists of six specific restriction enzyme sites that flank the DNA coding region upstream and downstream; Along with restriction sites, BioBrick parts are also cloned into a specific plasmid backbone. There are two classifications of BioBricks:  parts and devices. Parts are the basic constituents of larger systems that code for basic biological functions, and devices consist of multiple parts that work synergistically to serve a specific application.  In order to functionalize these BioBricks, they are implemented into living cells, most commonly E. coli bacterial cells. Every year, each iGEM team submits their standardized BioBricks to the iGEM parts <a href="https://2011.igem.org/Team:Wisconsin-Madison/registry">registry</a>; this registry constitutes an open source library of synthetic biological parts that any future team can order and use for their project.
-<p><br>
-This graph shows the fluorescent response of cell cultures containing two plasmids: pBAD33bb: exaDE and PexaA: TagRFP in pSB1A2 (pEtRv2.1). The fluorescence of the system at varying ethanol concentrations was normalized to the optical density of their respective cell culture. The cells’ fluorescence was measured over a range of ethanol concentrations from 0%-5% EtOH. This ethanol sensing system is “turned on” or induced in the presence of arabinose by the PBAD promoter.  The blue data points on the graph represent the fluorescence of cell cultures without any arabinose and therefore the ethanol sensor is uninduced; the red data points represent the fluorescent response of the induced system in the presence of .2% arabinose.
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-<img src="https://static.igem.org/mediawiki/2011/0/00/Pets_2.1_graph.jpg" align="left" width="500">
-From the graph, it can be seen that there is an increase in fluorescence between the induced and uninduced system. This is the result that was expected, however, there isn’t a large difference in fluorescence between the two systems. To further increase this difference in fluorescence, we intend to decrease the “leakiness” of our promoter through directed evolution.  Additionally, the graph shows a positive correlation between fluorescence and ethanol concentration; therefore, our ethanol sensor exhibits a linear response in fluorescent intensity with varying ethanol concentrations. The magnitude of the slope of the data points represents the degree of our systems response. Currently, the slope of the data is smaller than desired. This will  also be the target of a directed evolution upon the ethanol sensor.