Part:BBa_K4387987

Hemolysin A secretion system for E. coli

The hemolysin A secretion machinery is a one-step secretion system (T1SS), originally isolated from uropathogenic E. coli strains. [1] It comprises three main peptides, the inner membrane proteins HlyB and HlyD, and the outer membrane protein TolC. Together, these three proteins build a continuous channel through which originally the HlyA toxin is secreted in a one-step manner. Interestingly, the secretion signal is not found on the N-terminal site, instead it is found at the C-terminal end and the signal sequence is not removed during secretion. Scientists have identified the secretion signal and were able to secrete various proteins of different sizes with this secretion machinery. [1]

This composite part contains the 2 membrane proteins HlyB (BBa_K4387999) and HlyD (BBa_K4387998) necessary for the channel formation. TolC is not included because it is already genomically expressed in many E. coli strains. [1] The formation of the secretion machinery is regulated by the constitutive promoter BBa_J23100 together with the RBS BBa_B0030. For a successful secretion, the c-terminal HlyA-tag (BBa_K4387997) has to be fused to the end of the protein intended to be secreted.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1160
  Illegal AgeI site found at 1330
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

Western blot

Figure 1: Double transformed E. coli MC1061 (left) and E. coli Nissle 1917 (right) were induced by arabinose and incubated at 37°C overnight. Anti-myc antibodies were used in the Western blots to stain secreted nanobodies in the bacterial supernatant. N1 = VHH#2B, N2 = VHH#3E, N3 = VHH#12B, N4 = biv. VHH#2B, N5 = biv. VHH#3E, N6 = biv. VHH#12B, N7 = linked VHH#3E + VHH#2B, N8 = linked VHH#2B + VHH#12B, N9 = linked VHH#3E + VHH#12B

We double transformed the common lab strain E. coli MC1061 and the probiotic E. coli Nissle 1917 with the high copy plasmid containing the composite parts BBa_K4387979 until BBa_K4387986 required for induced nanobody expression, and the medium copy number plasmid containing this composite part required for the secretion system. Liquid overnight cultures of the transformed strains were grown and induced by adding the appropriate amount of arabinose. On the next day, the cells were centrifuged, and the supernatant was run on a gel. To see if nanobodies of the correct size have been secreted, we conducted a Western blot detecting the myc-tag fused to the nanobodies with anti-myc antibodies (Figure 1).

As seen in figure 1, we received bands with the size of approximately 45 kDa for monovalent nanobodies and of approximately 65 kDa for bivalent constructs, which fits the expected size of the nanobodies together with the myc-tag and HlyA-tag. We can therefore assume that both bacterial strains were able to secrete proper nanobodies of different sizes.

In the corresponding part sections BBa_K4387979 until BBa_K4387986 further experiments proving the functionality of the secreted nanobodies are displayed. The secretion hence does not negatively affect the properties of the secreted nanobodies.

References

• [1] Ruano-Gallego, D., Fraile, S., Gutierrez, C. et al. Screening and purification of nanobodies from E. coli culture supernatants using the hemolysin secretion system. Microb Cell Fact 18, 47 (2019). https://doi.org/10.1186/s12934-019-1094-0