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Team:Cornell/testing/project/wetlab/3/1
From 2012.igem.org
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Wet Lab
- Overview
- Chassis
- DNA Assembly
- Testing & Results
- Future Work
- Animation
Arsenic Reporter
Background and Previous Arsenic Sensors/h3> Several arsenic biosensors have been developed that rely on the Escherichia coli R773 arsenic-resistance plasmid. The ars operon encodes an arsenic and antimony-specific membrane pump that confers resistance by expelling these toxic metals from the cell. Expression of the ars operon is regulated by the ArsR protein which inhibits transcription of the arsR operon in the absence of arsenic. However, when arsenic is present, it sequesters ArsR, allowing transcription of the arsR operon [1,2]. Previously, the University of Edinburgh's iGEM team developed a pH-based arsenic biosensor relying on the fusion of the ars promoter with a lactose degrading fermentation pathway. pH changes can be measured either with indicator solutions or with an electrode. While their biosensor was both sensitive and easy to use, its reliance on pH made it vulnerable to changes in phosphate and bicarbonate levels and required on-site testing [4]. Siegfried et al., 2012, have recently developed a whole-cell arsenic sensing test kit that relies on bioluminescence. Both these test-kits however, lack in their ability to deliver real-time data and require periodic testing.
Design and Construction of our Arsenic Sensor
To expedite the construction of our arsenic reporter system, we employed two existing BioBricks: BBa_J33201 (Edinburgh, 2006) to function as the arsenic sensor and to drive the production of BBa_K098994 (Harvard, 2008), which encodes MtrB. ArsR acts as a negative auto-regulator, repressing the expression of downstream mtrB. Because ArsR activity is repressed in the presence of arsenic, MtrB will be upregulated when arsenic is present, increasing the rate of electrode-reduction.We inserted our arsenic-sensing BioBrick into the pBBRBB cloning vector (Vick et al., 2011) due to its past success in complementation studies [7]. Upon ligation, we transformed competent DH5a E. coli and attempted to transform JG700, our mtrB knockout strain. Because we had no successes electroporating JG700 with our construct, we decided to append a mobility gene and use conjugation to transfer our construct. We first transformed WM3064 with our mobility-enabled arsenic sensing construct. Finally, we conjugated WM3064 with JG700 to create our arsenic-sensing Shewanella strain.
