Composite
NO Sensor

Part:BBa_M36210:Design

Designed by: Michele Dragoescu, Shaheen Jeeawoody, Lizzie Peiros   Group: Stanford BIOE44 - S11   (2011-12-07)

Nitric oxide sensing construct for use in Bacillus subtilis


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Input: Nitric oxide Output: PoPS signal (polymerases per second)

The construct can be implemented in a plasmid containing Gemini (Martin et al), a hybrid reporter that gives off a fluorescent signal from GFP and produces Lac-Z, a protein that interacts with the enzyme B-galactosidase to produce a color change. In the absence of NO, tyhe repressible pLac promoter BBa_K0911108 is constitutively on and Gemini is transcribed and translated, giving off both a colorimetric and fluorescent signal. Our following description of this NO sensor assumes the use of Gemini as a reporter, though any other reporter could be used due to the sensor output of PoPS.

When nitric oxide is present in cells, it binds to the NO promoter BBa_K2560009 and initiates transcription by recruiting a ribosome to the 5' bicistronic UTR ribosome binding site (RBS) BBa_M36282. The bicistronic RBS uses RNA polymerase as a physical barrier to unwanted secondary structure and was chosen because of its high efficiency. The gene BBa_K08200410, which codes for LacI repressor protein, is transcribed. Transcription will stop when RNA polymerase reaches the strong terminator BBa_B100211.

When LacI is produced it binds to the pLac promoter BBa_K091110 and represses it, stopping the transcription of the Gemini gene. Hence, the presence of NO can be detected by a lack of visual fluorescent or colorimetric signal due to the lack of PoPS signal.

The form of LacI produced in this construct is found endogenously in wild type E. coli, so this construct was designed for experiments in Bacillus subtilis that contains a homologous LacI protein that cannot bind to that pLac promoter. Therefore, the absence of NO can be detected by a visual colorimetric or fluorescent signal due to the presence of a PoPS signal.

Source

These are all of the individual parts used in this composite NO sensor:

Bodapati S, Joung J, Esquivel-Reynoso D (2011) 5' bicistronic UTR including ATG start codon. Available: parts.igem.org/wiki/index.php?title=Part:BBa_M36282.

Cool R (2008) LacI Promoter. Available: parts.igem.org/wiki/index.php?title=Part:BBa_K091110.

Ee S, Kodaganti NS, Li M, Goh FM, et al. (2009) Sensing device by NTU iGEM Singapore 2009. Available: 2009.igem.org/Team:NTU-Singapore/Project.

Ee S, Kodaganti NS, Li M, Goh FM, et al. (2009) NO sensing promoter. Available: parts.igem.org/wiki/index.php?title=Part:BBa_K256000.

Huang H (2006) Terminator. Available: parts.igem.org/wiki/index.php/Part:BBa_B1002.

Xue Y, Shen H (2008) LacI wild type. Available: parts.igem.org/wiki/index.php?title=Part:BBa_K082004.

References

Martin L, Che A, Endy D, 2009 Gemini, a Bifunctional Enzymatic and Fluorescent Reporter of Gene Expression. PLoS ONE 4(11): e7569.doi:10.1371/journal.pone.0007569 Gemini, a Bifunctional Enzymatic and Fluorescent Reporter of Gene Expression