Difference between revisions of "Part:BBa K174015"

Revision as of 19:38, 17 October 2019

Cadmium Sensor

This part is designed to allow B. subtilis to sense cadmium.

To our knowledge, there is no specific cadmium sensing protein. We therefore combined two repressors that each sense a range of metals, but with only cadmuim in common. This cadmium sensor is therefore based on an AND gate configuration.

The part was built by combining operator binding sites for two metal sensor repressor proteins, ArsR and CzrA [1,2].

They both work as repressors and they are relieved from binding to the DNA when they are bound to various metals. ArsR can bind to cadmium, silver, copper and arsenic whereas CzrA can bind to zinc, cobalt, nickel and cadmium. Cadmium can bind to both proteins and using a combinatorial approach enables us to filter out sensing any metals other than cadmium.

When cadmium is present both proteins are unbound from this AND gate and the promoter region controlled by the repressors becomes free to drive the expression of downstream genes such as Gfp as an indicator, or other transcription factors.

To create our device we combined ArsR binding site (BBa_K174016) with cadA promoter (BBa_K174017) from B. subtilis. CadA works as an efflux system to take the metals out in the cell. The cadA promoter was used since it has binding site for CzrA just after the promoter region. The ArsR binding site was positioned just upstream of the cadA promoter.

For more information about this part, go to Newcastle iGEM 2009 [http://2009.igem.org/Team:Newcastle/Project/Overview Overview] and [http://2009.igem.org/Team:Newcastle/Metalsensing Metal Sensing] pages.

Usage and Biology When combined with an RBS (B0036), RFP (E1010), and a double terminator (B0014), the Gaston Day School 2019 team has shown that this promoter is sensitive to cadmium down to 0.25M (p<0.01). Work by earlier Gaston Day School teams had shown that the promoter did work, but the data was not clean and the minimum level of detection was significantly higher (approximately 10mM). Cultures were grown for 24 hours in the presence of 0, 0.25, or 0.75 mM cadmium. Absorbance was measured using a Vernier SpectroVis Plus and data was collected and analyzed using Microsoft Excel’s t-test function. Results are reported as absorbance at 517 nm normalized using absorbance at 700 nm. Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 200
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

Safety

Cadmium is a toxic metal. There are regulations and restrictions to work with heavy metals. To test this part these regulations should be followed. For more information please read [http://www.hse.gov.uk/pubns/indg391.pdf HSE's guidance] to work with cadmium.

References

Moore, C. M. and J. D. Helmann (2005). "Metal ion homeostasis in Bacillus subtilis." Current Opinion in Microbiology 8(2): 188-195.
Harvie, D.R., et al., Predicting metals sensed by ArsR-SmtB repressors: Allosteric interference by a non-effector metal. Molecular Microbiology, 2006. 59(4): p. 1341-1356.

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 Usage and Biology
+When combined with an RBS (B0036), RFP (E1010), and a double terminator (B0014), the Gaston Day School 2019 team has shown that this promoter is sensitive to cadmium down to 0.25M (p<0.01). Work by earlier Gaston Day School teams had shown that the promoter did work, but the data was not clean and the minimum level of detection was significantly higher (approximately 10mM). Cultures were grown for 24 hours in the presence of 0, 0.25, or 0.75 mM cadmium. Absorbance was measured using a Vernier SpectroVis Plus and data was collected and analyzed using Microsoft Excel’s t-test function. Results are reported as absorbance at 517 nm normalized using absorbance at 700 nm.
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 <span class='h3bb'>Sequence and Features</span>