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Complete Has operon (controlling expression of luxR)

Sequence and Features

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 Responder, one of its two spatial sensors in the RIOT System. The RIOT System 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 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 Responder. 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 RIOT System which allows for invasion into the cancer cells.

Figure 1: Schematic of how the RIOT Responder is used. The HasR, HasS and HasI are expressed under a constitutive promoter 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.

Creating the construct

Figure 2: DNA gel showing the stages in construction of the full construct. In the leftmost lane it shows the two bands obtained when the plasmid containing AS3 was digested with XbaI and PstI. In the middle lane it shows the two bands obtained when the plasmid containing 2AS3 was digested with the same enzymes. Finally, the full construct was succesfully ligated into pSB1C3 as an insert band of the correct size was dropped out upon RE digestion.

The construct was synthesised in 4 different sections named 1, 2, AS and 3. These were added sequentially by restriction enzyme (RE) digestion and ligation into pSB1C3. The first stage involved the 3 way ligation of AS and 3 into the pSB1C3. 2 was then added into the plasmid already containing AS and 3 by RE digestion and ligation. Finally, the full construct was obtained upon the addition of 1 into the Biobrick plasmid containing 2AS3. After every digestion and ligation, the ligation mix was transformed into E. coli and colony PCR done to determine which colonies contained the plasmid of interest. The final construct obtained was sent for sequencing to determine the exact sequence of the construct and mutations if any.

To determine the presence of AS3 in pSB1C3, RE digestion was done with XbaI and PstI to drop out the insert as seen through the insert band of 2.888 kbp (band a). Similarly, RE digest using the same enzymes was carried out to confirm the presence of 2AS3. The insert band (band b) was approximately 5.5 kbp which is similar to the expected size of 2AS3 which is 5281 bp. Finally, 1 was added to the 2AS3 in biobrick via RE digestion and ligation and this was ascertained by RE digestion. The Has operon full length insert (expected size 7147 bp) was successfully excised out as band c which was slightly higher than 7 kbp.

In all cases, a common plasmid backbone was also seen around 2 kbp (boxed in black).

Characterisation via fluorescence microscopy

The DNA gel above has shown that the designed construct is indeed in the pSB1C3 backbone. However, to test if the proteins are functional and the circuit working as expected, fluorescence microscopy was done to view the bacteria before and after induction with holo-HasA.

Figure 3: 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 3 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.