Part:BBa_K1897007

Complete Has operon (controlling expression of luxR)

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]

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 to capture extracellular heme. This holo-HasA then binds to the cell surface transceptor HasR. This causes a conformational change in HasR and deactivation of anti-sigma factor HasS. HasS then releases the sigma-like factor HasI. HasI then allows transcription initiation at the Has promoter (pHas).

NUS_Singapore utilises this system to create the RIOT Responder circuit, 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). The conjugate can attach to the surface of cancer cells while the holo-HasA would be able to bind to HasR expressed on the E. coli containing the RIOT Responder circuit. This would then trigger the expression of luxR which is under the control 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 in the E. coli. When the RIOT Transponder binds to HasR, it causes activation of the HasS which releases HasI. This allows the expression of LuxR which is under the control of pHas (Has operon promoter).

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 seq3 was digested with XbaI and PstI. In the middle lane it shows the two bands obtained when the plasmid containing seq2 + seq3 was digested with the same enzymes. Finally, the full construct was successfully ligated into pSB1C3 as an insert band of the correct size was dropped out upon RE digestion. BB: BioBrick

The construct was synthesised in 3 different sections named seq1, seq2 and seq3. These were added sequentially by restriction enzyme (RE) digestion and ligation into pSB1C3. The first stage involved the RE digestion of pSB1C3 and seq3 and subsequent ligation into the pSB1C3. Seq2 was then added into the plasmid already containing seq3 by RE digestion and ligation. Finally, the full construct was obtained upon the addition of seq1 into the Biobrick plasmid containing seq2 + seq3. 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 seq3 in pSB1C3, RE digestion was done with XbaI and PstI to drop out the insert as seen through the insert band of approximately 3 kbp (band a), close to the theoretical size of 2888 bp. Similarly, RE digest using the same enzymes was carried out to confirm the presence of seq2 + seq3. The insert band (band b) was approximately 5.5 kbp which is similar to the expected size of seq2 + seq3 which is 5281 bp. Finally, seq1 was added to the seq2 + seq3 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 SDS-PAGE and Western Blot

Figure 3: Western blot of his-tagged HasR, HasS, and HasI expression and purification. The wells were loaded with (from left to right) total lysate, flow through, wash 1, wash 2, and elute.

The plasmid was transformed into NEB® Stable Competent E. coli (High Efficiency) and allowed to grow in 400mL of LB liquid media at 37°C. The bacteria were then pelleted and lysed using B-PER Bacterial Protein Extraction Reagent. Centrifugation was then done and the supernatent ran through the Nickel Chelated Column (flowthrough). The column was then washed twice with wash buffer (wash 1 and wash 2) and eluted.

SDS-PAGE of the samples was run on a polyacrylamide gel (4% stacking, 10% resolving) at 70V for 30 minutes and followed by 120V for 1 hour. Subsequently, semi-dry transfer was conducted at 15V for 1 hour to transfer the protein bands to a PVDF membrane. After blocking and incubation with the relevant primary and secondary antibodies, the blot was visualised with chemiluminescence.

As seen in figure 3, bands corresponding to the approximate sizes of each protein component (HasR: 98kDa, HasS: 34kDa, HasI: 20kDa) were observed. The result implies that the HAS operon pathway was present and expressed in the RIOT Responder bacteria.

Characterisation via fluorescence microscopy

The DNA gel above has shown that the designed construct is indeed in the pSB1C3 backbone. Fluorescence microscopy was done to determine if the circuit is working as expected. In this case, we are testing firstly whether HasR, HasS and HasI have been correctly expressed and secondly if the circuit is only induced in the presence of HasA. Theoretically, exposure to holo-HasA should lead to the activation of HasR which induces the HasI to allow expression of genes under pHas. This would therefore allow mRFP to be produced, leading to a detectable fluorescence in bacteria that was not detected previously before induction. Fluorescence also indicates that HasR, HasS and HasI are functional as the signal can also be transduced if the proteins have been produced correctly.

Figure 4: 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 4 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.

Figure 5: Mean fluorescent intensities of RIOT Responder bacteria cells at 2 hours after induction by HasA at three concentrations (0M negative control, 10-4M, and 10-5M ). The measured fluorescence intensities were normalised to a background value in the images. The black square points indicate average mean fluorescence in each sample and standard deviation. Asterisks indicate ρ<0.05 significance between the samples according to one-tail Mann-Whitney U test.

Quantification of the fluorescence obtained was also done and the fluorescence of the RIOT Responder cells induced at the different HasA concentrations were compared as shown in Figure 5. The results suggest that higher concentration of HasA corresponded to higher fluorescent intensities and implies that the RIOT Responder circuit can be activated by addition of HasA.

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.