Difference between revisions of "Part:BBa M50011:Design"

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===Source===
 
===Source===
  
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This genetic sequence comes from the work of Hydrogen Peroxide Sensor HyPer.  
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This genetic sequence comes from the work of Hydrogen Peroxide Sensor HyPer. The research into the HyPer gene was created by Belousov et al.
  
 
===References===
 
===References===
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Belousov VV, Fradkov AF, Lukyanov KA, et al. 2006. Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nat Methods. 3(4):281-6. http://dx.doi.org/10.1038/nmeth866
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Ravindra Kumar S, Imlay JA. 2013. How Escherichia coli Tolerates Profuse Hydrogen Peroxide Formation by a Catabolic Pathway. Journal of Bacteriology. 195(20):4569-4579. doi:10.1128/JB.00737-13.
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Nakamura J, Purvis ER, Swenberg JA. 2003. Micromolar concentrations of hydrogen peroxide induce oxidative DNA lesions more efficiently than millimolar concentrations in mammalian cells. Nucleic Acids Research. 31(6):1790-1795.
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DNA 2.0 Plasmid Webpage: https://www.dna20.com/eCommerce/catalog/datasheet/248
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BIOE44 Lab Booklet: “Practical 3”
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BIOE 44 Lab Booklet: “Experimental Measurements”

Latest revision as of 05:34, 12 December 2016


This is an E. coli specific sensor for hydrogen peroxide.


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 844
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 25
    Illegal SapI.rc site found at 709


Design Notes

This device is designed as a rhamnose - inducible biological sensor that allows E. coli to detect the presence of hydrogen peroxide. This sensor consists of a strong RBS, ampicillin resistance, a high copy number at the origin of replication, the gene of interest, the HyPer plasmid, and a transcription terminator from DNA 2.0. Specifically, a strong RBS was selected in order to ensure robust expression of the HyPer gene. Additionally, the plasmid is constructed of DNA 2.0’s default terminator, pT-T7, which is derived from the T7 bacteriophage.

This device is based off of the HyPer gene, which allows cells to sense hydrogen peroxide; however HyPer originally formatted the genetic sequence for mammalian cells, so we converted it to E. coli cells. With regards to the design of the HyPer gene, the Belousov et al. researchers used the knowledge that the E. coli regulatory region, OxyR-RD, is especially sensitive to the presence of hydrogen peroxide. The researchers also inserted a cpYFP into the sensor to demonstrate when hydrogen peroxide is detected.

Source

This genetic sequence comes from the work of Hydrogen Peroxide Sensor HyPer. The research into the HyPer gene was created by Belousov et al.

References

Belousov VV, Fradkov AF, Lukyanov KA, et al. 2006. Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nat Methods. 3(4):281-6. http://dx.doi.org/10.1038/nmeth866

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Ravindra Kumar S, Imlay JA. 2013. How Escherichia coli Tolerates Profuse Hydrogen Peroxide Formation by a Catabolic Pathway. Journal of Bacteriology. 195(20):4569-4579. doi:10.1128/JB.00737-13.

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Nakamura J, Purvis ER, Swenberg JA. 2003. Micromolar concentrations of hydrogen peroxide induce oxidative DNA lesions more efficiently than millimolar concentrations in mammalian cells. Nucleic Acids Research. 31(6):1790-1795.

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DNA 2.0 Plasmid Webpage: https://www.dna20.com/eCommerce/catalog/datasheet/248

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BIOE44 Lab Booklet: “Practical 3”

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BIOE 44 Lab Booklet: “Experimental Measurements”