Part:BBa_K1897007

Complete Has operon (controlling expression of luxR)

Usage and Biology

The Has operon is originally a heme acquisition system from the Serratia Marcescens. In the original system, HasA, a hemophore, is secreted out of the bacteria to capture extracellular heme. This holo-HasA then binds to the cell surface receptor HasR. This causes a conformational change in HasR and activation of anti-sigma factor HasS. HasS then releases the extra cytoplasmic function sigma factor HasI. HasI then allows the expression of genes controlled by the Has promoter (pHas).

NUS_Singapore utilises this system to create the RIOT Sensor, one of its two spatial sensors in the RIOTSystem. THe RIOTSystem is a spatially specific cancer diagnostic that relies upon spatial markers that are unique to the tumour microenvironment to allow for specific detection of the tumour. One of the two sensors employed detects the presence of CD44v6, a commonly upregulated cell surface marker on a variety of cancers (Todaro et al., 2014). This is done by conjugating HasA with a CD44v6 antibody (RIOT Transponder). Therefore, the the conjugate can attach to the surface of cancer cells and the holo-HasA would be able to bind to HasR expressed on the E. coli containing the RIOT Sensor. This would then trigger the expression of luxR which is under the expression of the pHas (Figure 1). The luxR is then used in the RIOT Invader, another component of the RIOTSystem which allows for invasion into the cancer cells.

Figure 1: Schematic of how the RIOT Sensor is used. The HasR, HasS and HasI are expressed constitutively under pconst in the E. coli. When the RIOT Transponder binds to HasR (blue), it causes activation of the HasS (green) which releases hasI (brown). This allows the expression of LuxR which is under the control of pHas.

Apart from containing the Has proteins and luxR, there are also two other genes, the mRFP gene and the Ampicillin resistance gene. The mRFP gene is used as a reporter gene for visualisation of whether the circuit has been successfully induced in the presence of holo-HasA. The Ampicillin resistance gene is used as a selection marker to allow for selection of E. coli that have taken up the plasmid.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1691
  Illegal NheI site found at 1714
  Illegal NotI site found at 3609
  Illegal NotI site found at 4403
  Illegal NotI site found at 4527
  Illegal NotI site found at 5531
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1720
  Illegal BamHI site found at 4476
  Illegal BamHI site found at 4810
  Illegal BamHI site found at 5466
  Illegal XhoI site found at 1
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1767
  Illegal NgoMIV site found at 1771
  Illegal NgoMIV site found at 1827
  Illegal NgoMIV site found at 1945
  Illegal NgoMIV site found at 2099
  Illegal NgoMIV site found at 2164
  Illegal NgoMIV site found at 2500
  Illegal AgeI site found at 1404
  Illegal AgeI site found at 1516
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterisation via fluorescence microscopy

Figure 2: HasA induction of E. coli with the Has System circuit. Top: red fluorescence microscopy pictures of (from left to right) negative control, 10^-4 M HasA, 10^-5 M HasA. Bottom: bright field microscopy pictures of (from left to right) negative control, 10^-4 M HasA, 10^-5 M HasA

To determine if the circuit is indeed functional, it was transformed into E. coli and induced with different concentrations of heme-loaded holo-HasA for 2 hours. The results are seen in Figure 2 where in the negative control where no holo-HasA was added, there is no fluorescence seen. However, in the presence of holo-HasA, the bacteria fluoresce red.

References

Biville, F., Cwerman, H., Létoffé, S., Rossi, M. S., Drouet, V., Ghigo, J. M., & Wandersman, C. (2004). Haemophore‐mediated signalling in Serratia marcescens: a new mode of regulation for an extra cytoplasmic function (ECF) sigma factor involved in haem acquisition. Molecular microbiology, 53(4), 1267-1277.

Cescau, S., Cwerman, H., Letoffe, S., Delepelaire, P., Wandersman, C., & Biville, F. (2007). Heme acquisition by hemophores. Biometals, 20(3-4), 603-613.

Rossi, M. S., Paquelin, A., Ghigo, J. M., & Wandersman, C. (2003). Haemophore‐mediated signal transduction across the bacterial cell envelope in Serratia marcescens: the inducer and the transported substrate are different molecules. Molecular microbiology, 48(6), 1467-1480.

Todaro, M., Gaggianesi, M., Catalano, V., et al., (2014). CD44v6 is a marker of constitutive and reprogrammed cancer stem cells driving colon cancer metastasis. Cell stem cell, 14(3), 342-356.

Wandersman, C., & Delepelaire, P. (2004). Bacterial iron sources: from siderophores to hemophores. Annu. Rev. Microbiol., 58, 611-647.