Coding
hcp

Part:BBa_K4739002

Designed by: Huaiyuan Ma   Group: iGEM23_SUSTech-Shenzhen   (2023-10-08)


hcp, the inner tube of T6SS

The T6SS needle's inner tube, vital for the functionality of T6SS, is constructed from hexamers of the Hcp1 protein, which are donut-shaped and stacked in a head-to-tail orientation to form a hollow tube. Its inner diameter is ∼40 Å and has an external diameter of ∼110 Å, limiting the size of the effector <25kD (Cherrak et al., 2019). The Hcp hexameric rings and associated structures form the tube through which toxins or effectors can be transported during the secretion process. The sequence we provide is from the P. aeruginosa genome. In our project, we utilize this structure to carry the drugs we aim to carry. For example, minihepcidin, and we witnessed a successful secretion through western blot. Results can be viewed from the result section of our wiki.

Structure of Hcp

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 142
    Illegal NgoMIV site found at 265
  • 1000
    COMPATIBLE WITH RFC[1000]


Engineering and Results

Fusion protein

We designed a plasmid with Hcp1 and linked it to Minihepcidin using BBa_K4739000, putting a FLAG after Minihepcidin. (The plasmid was built based on pPSV37, an IPTG-induced expression vector.) we predicted the structure of the fusion protein using AlphaFold 2

Then we introduce the plasmid into T6SS positive PAO1 and T6SS negative PAO1 as a negative control, compare the result of western blot result of the cell and supernatant, and retrieve the following results:

Hcp result

The Hcp-fused proteins can only be detected in cells. Even though the Pseudomonas aeruginosa (expressing Hcp1-Minihepcidin) lysed (since we detected the RpoB signal in the supernatant), we still couldn’t detect the signal of FLAG. We have found something we can optimize. We will continue to conduct engineer after the wiki freeze.

Reference

Cherrak, Y., Flaugnatti, N., Durand, E., Journet, L., & Cascales, E. (2019). Structure and Activity of the Type VI Secretion System. Microbiology Spectrum, 7(4). https://doi.org/10.1128/microbiolspec.psib-0031-2019

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