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Team:Cornell/project/wetlab/assembly/arsenic
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| - | <a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/ | + | <a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/transcription">Transcriptional Characterization</a> |
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| - | <a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/ | + | <a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/currentresponse">Current Response</a> |
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<a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/protein">MtrB Protein Expression</a> | <a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/protein">MtrB Protein Expression</a> | ||
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<h3>Background and Previous Arsenic Sensors</h3> | <h3>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. | 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. | ||
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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. | 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. | ||
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| + | <p><b>Click the image below to see how our arsenic sensitive plasmid works!</b></p> | ||
| + | <a href="https://static.igem.org/mediawiki/igem.org/1/18/Together.png" rel="lightbox"><img class="inline" src="https://static.igem.org/mediawiki/igem.org/2/21/BLANK_backbone_500x500.png"></a> <br> | ||
| + | In the figure above is a map of our arsenic detecting plasmid.<p><b>Click the image to see how it works!</b></p> | ||
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Latest revision as of 03:09, 27 October 2012
Arsenic Reporter
Background and Previous Arsenic Sensors
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.
Click the image below to see how our arsenic sensitive plasmid works!
In the figure above is a map of our arsenic detecting plasmid.
Click the image to see how it works!
References
1. Xu, C., Shi, W., & Rosen, B. P. (1996). The chromosomal arsR gene of Escherichia coli encodes a trans-acting metalloregulatory protein. The Journal of Biological Chemistry, 271(5), 2427-32. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/8576202 2. Cai, J., & DuBow, M. S. (1996). Expression of the Escherichia coli chromosomal ars operon. Canadian Journal of Microbiology, 42(7), 662–671.
3. Coursolle, D., and Gralnick, J.A. (2012). Reconstruction of extracellular respiratory pathways for iron(III) reduction in Shewanella oneidensis strain MR-1. Frontiers in Microbiology 3(56)
4. Joshi, N., Wang, X., Montgomery, L., Elfick, A., & French, C. E. Ã. (2010). Novel approaches to biosensors for detection of arsenic in drinking water, 252(May 2008), 100-106.
5. Siegfried, K., Endes, C., Bhuiyan, A. F. M. K., Kuppardt, A., Mattusch, J., van der Meer, J. R., Chatzinotas, A., et al. (2012). Field testing of arsenic in groundwater samples of Bangladesh using a test kit based on lyophilized bioreporter bacteria. Environmental Science & Technology 46(6), 3281-7.
6. Vick, J., Johnson, E., Choudhary, S., Bloch, S., Lopez-Gallego, F., Srivastava, P., Tikh, I., et al. (2011). Optimized compatible set of BioBrickTM vectors for metabolic pathway engineering. Applied Microbiology and Biotechnology, 92(6), 1275-1286. Springer Berlin / Heidelberg. doi:10.1007/s00253-011-3633-4
