Part:BBa_K5252024

pipaH*

Part of a 3-input AND gate [1]. The gate serves as an indicator of landmine or UXO degradation, with it only activating when TNT and RDX are absent and when the degradation products of these compounds are present, ensuring the marker is a reliable indicator of task completion.

The logic gate is based on the layering of two 2-input AND gates, in which promoters serve as inputs and outputs. In these systems, one input controls the expression of a transcriptional activator and the other induces the expression of a chaperone protein. The chaperone protein is necessary to activate the output promoter.

By controlling the activity of the input promoters with transcriptional repressors downstream of the TNT sensitive-promoter (yqjF3rd) BBa_K3350862 and RDX-activated riboswitch BBa_K5252007, we can maintain the signal in an off-state until these compounds are fully degraded. We selected the formaldehyde promoter (frmPromoter) as our third input promotor. It is repressed by frmR, which we chose to express constitutively (downstream of J23100 promoter). frmR itself is inactivated by formaldehyde, a degradation product of RDX [2], which serves as an inducer for the frm promoter.

And gate to identify landmine degradation

Thus, all three inputs are active only in the absence of TNT and RDX and in the presence of formaldehyde, which comes from degraded RDX. We used mScarlet as a fluorescent marker.

References

[1] Moon, T., Lou, C., Tamsir, A. et al. Genetic programs constructed from layered logic gates in single cells. Nature 491, 249–253 (2012). https://doi.org/10.1038/nature11516

https://www.nature.com/articles/nature11516#citeas

[2] Seth-Smith HM, Rosser SJ, Basran A, Travis ER, Dabbs ER, Nicklin S, Bruce NC. Cloning, sequencing, and characterization of the hexahydro-1,3,5-Trinitro-1,3,5-triazine degradation gene cluster from Rhodococcus rhodochrous. Appl Environ Microbiol. 2002 Oct;68(10):4764-71. doi: 10.1128/AEM.68.10.4764-4771.2002. PMID: 12324318; PMCID: PMC126434.

https://pubmed.ncbi.nlm.nih.gov/12324318/

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 361
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 361
  Illegal NheI site found at 355
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 361
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 361
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 361
  Illegal NgoMIV site found at 124
  
• 1000
  COMPATIBLE WITH RFC[1000]