Part:BBa_K3470014

Constitutive Promoter – RBS – HlyB – RBS – HlyD – RBS – TolC – RBS - GFP+HlyA - Double Terminator

HlyB acts as an ATP-binding cassette and recognizes the substrate via its secretion signal peptide (HlyA). It exports any protein (like IL-10) fused to the secretion signal, produced inside the bacteria that must act on targets outside and is responsible for the specificity of the secretion system process. (Bravo et al., 2008, Delepelaire, 2004) HlyD consists of a short cytoplasmic domain at the N-terminus, a membrane anchor and a large periplasmic domain. It acts as a membrane fusion or adaptor protein by linking the HlyB and TolC domains of the Hemolysin transport system and helps in the transportation of proteins. TolC is a specific outer membrane protein which forms a long channel throughout the periplasm and the outer membrane, largely open towards the extracellular matrix for export of required components to targets outside the bacteria. (Gentschev et al., 2002, Schmitt et al., 2003) HlyA is a signal sequence that interacts with the cytoplasmic region of the pre-formed HlyB–D complex. After the binding of the HlyA secretion signal by the HlyB–D complex, HlyD induces the interaction with TolC which then transports it out of the cell. (Hess et al., 1990) The team tested for the efficiency of the control system consisting of HlyB, HlyD, TolC and HlyA against a control with HlyB, HlyD and TolC absent. We analyse the HlyA + GFP transported outside the cells, HlyA + GFP remaining inside the cells and the total HlyA + GFP produced with absorbance vs time graphs to show to efficiency in getting the anti-inflammatory cytokines outside the cell to the target site. The plot determined the amount of HlyA+GFP transported outside. Hence it should be ideally equal to the plot of control showing that the transport system is extremely efficient in getting the anti-inflammatory cytokines outside the cell to the target site.

Referances: Bavro, V. N., Pietras, Z., Furnham, N., Pérez-Cano, L., Fernández-Recio, J., Pei, X. Y., Misra, R., & Luisi, B. (2008). Assembly and Channel Opening in a Bacterial Drug Efflux Machine. Molecular Cell, 30(1), 114–121. https://doi.org/10.1016/j.molcel.2008.02.015 Delepelaire, P. (2004). Type I secretion in gram-negative bacteria. Biochimica et Biophysica Acta - Molecular Cell Research, 1694(1-3 SPEC.ISS.), 149–161. https://doi.org/10.1016/j.bbamcr.2004.05.001 Hess, J., Gentschev, I., Goebel, W., & Jarchau, T. (1990). Analysis of the haemolysin secretion system by PhoA-HlyA fusion proteins. MGG Molecular & General Genetics, 224(2), 201–208. https://doi.org/10.1007/BF00271553 Schmitt, L., Benabdelhak, H., Blight, M. A., Holland, I. B., & Stubbs, M. T. (2003). Crystal structure of the nucleotide-binding domain of the ABCtransporter haemolysin B: Identification of a variable region within ABC helical domains. Journal of Molecular Biology, 330(2), 333–342. https://doi.org/10.1016/S0022-2836(03)00592-8