Part:BBa_K1985015

pSB1A3-[AraC-pBAD]-[CsgA-SS-Sup35NM]

This part is an improved version of biobrick (BBa_K1739002), which was designed by the Kent 2015 iGEM team. The promoter BBa_J23104 has been removed and instead an inducible arabinose promoter, part BBa_K1321333, has been used. The second part of this composite biobrick contains 2 segments: the CsgA signal sequence and Sup35NM.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1205
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961
  

Usage and Biology

The part was used in pSB1A3.

For more information on biology and usage of Sup35NM with the CsgA signal sequence, see part BBa_K1739002. For information on the promoter see BBa_K1321333.

Validation

The part was first validated with a diagnostic restriction digest using Spe1 and BamH1 and agarose gel electrophoresis. The expected band sizes from the digest were: 3327kB for the plasmid backbone and 958kB for the insert. A 1kB plus DNA marker was used to verify the sizes of the bands and it was confirmed that the correct plasmid had been produced.

Figure 1. Agarose gel of the restriction digest of BBa_K1985012 in pSB1A3, with EcoRI and PstI.

Congo Red Plate Validation

We also validated our part using a Congo Red agar plate assay with the antibiotics chloramphenicol and ampicillin present in order to select the VS45 strains expression our fusion protein. As a negative control, we used VS45 with pVS105, as this plasmid contains CsgAss and Sup35M, it does not have the ability to self-assemble into amyloid nano-wires. These strains were plated in 3 segments of the plate and left to incubate for 5 days at 22°C. This resulted red colonies of VS45 with CsgAss-Sup35NM due to the binding of Congo Red to the amyloid fibres produced by the colonies. White colonies of VS45 with the negative control plasmid, as expected, showed no export of amyloid-forming protein (shown in fig.1).The Sup35NM cells are a similar colour to the positive control PVS72. These results confirmed that our fusion protein was being produced and targeted to the curli export pathway by CsgAss, as well as self-assembling into amyloid fibres.

Figure 2. shows a Congo red plate split into 3 sections; Sup35NM, PSV105 and PSV72. The PSV105 and PSV72 are the negative and positive controls respectively.

Figure 3. This figure also highlights the similarity in the colour of the positive control PSV72 and the SUP35NM VS45 cells, this is a strong indication that the SUP35NM cells are producing amyloid fibres.

AFM Image Validation

Further validation for protein export and amyloid formation by our fusion protein was achieved using atomic force microscopy (AFM) imaging of our samples. Using the aforementioned E.coli strains a 5 day incubation at 25°C was carried out to make sure that the amyloid fibres were stable for the AFM protocol, as suggested by Sivanathan and Hochschild (2012). The resulting images showed the presence of amyloid aggregates in the VS45 sample with the Sup35NM prion forming domain. The image was compared with samples without amyloid fibers being produced by PVS105 VS45 cells (figure 5) and PSV72 VS45 cells (figure 4) which produced amyloid fibers. From figure 6 it is evident that there is a similar structure beside the cells to the fiber found on the PSV72 image. This strongly indicates that the sup35NM protein does in fact produce amyloid fibers.

Figure 4. Shows PSV72 which is the positive control. The image shown above shows that there is a presence of amyloid fibers.

Figure 5. Shows the negative control PSV105. It is evident that are no fibers present, which was anticipated.

Figure 6. Shows the VS45 cells with the Sup35NM prion forming domain. From the image there are structures beside the cells which resemble the fibers found in the positive control PSV72.