Difference between revisions of "Part:BBa K3409008"

Latest revision as of 08:28, 26 October 2020

Expression of receptor binding protein (gp37) on the outer membrane of Escherichia coli with RFP

Contains the elements necessary to recognize and bind an Escherichia coli bacteria.The major component is the gene product (gp) 37 which codes for the T4 bacteriophage recpetor binding protein (RBP) conferring the ability to recongize another target bacteria (E.coli in this case). Gp37 will be directed to the outer membrane of our model organism, E. coli K12. This will be achieved thanks to the co-expressed Lpp-OmpA gene. The Lpp-OmpA is an existing part designed by the NCTU-Formosa team in 2015 (Part: BBa_K1694002). It consists of a N-terminal acids of the lipoprotein (Lpp) sequence followed by amino acids of outer membrane protein A (OmpA). By fusing gp37 to the C-ter end of Lpp-OmpA, it can be displayed on the outer membrane of E. coli. Between the Lpp-OmpA and the gp37, is a red fluorescent marker (RFP) allowing to localize the LTF in the chassis. This BioBrick also contains a cleavage site, the TEV cleavage site, which is a unique cleavage site of the cysteine protease from the Tobacco Etch Virus (TEV). This cleavage site is followed by a His-tag in case of a purification step which can be performed with Ni2+ chromatography columns.

Two chaperones proteins (gp38 and chaperone protein 57A) are present as well for the physical appearance of the gp37.

Usage

This part was designed to engineer a chassis being able to specifically recognize target bacteria, the same way bacteriophages do. In this case, the receptor binding protein, gp37, allows to specifically recognize E. coli non pathogenic strains such as E. coli B or K12.

Characterization

THEORETICAL VERIFICATION OF THE GP37 LTF DISPLAY ON THE SURFACE OF E COLI

To know if displaying the T4 bacteriophage’s LTF tip on the surface of E. coli was sufficient to reach the outer membrane of the target bacteria (i.e. long enough according to the elements found on E. coli outer membranes). We found that the longest known component of a bacterial cell wall is the lipopolysaccharide conjugated with an O antigen which has a length of 49nm. T4 phage’s LTFs have a length of 145nm. Additional characterization on the exact length of the gp37 was not available so we had to proceed by assumptions. Based on the molecular mass of the whole LTF (307 kDa) and the molecular mass of the gp37 product (109 kDa), we can hypothesize that the gp37 protein product has an average length of 51 nm which would mean that our modified E. coli must be able to sense other bacteria thanks to the gp37.

REVELATION OF THE PRESENCE OF GP37 VIA IMMUNOFLUORESCENCE LABELLING

The aim was to detect the receptor binding protein gp37 expressed and directed to the membrane thanks to the co-expressed Lpp-OmpA genes. This is achieved using fused His-tag placed after the OmpA and before the gp37. Thus, the His-tag was targeted with a primary monoclonal anti-his antibody conjugated to a fluorescence marker, fluorescein isothiocyanate (FITC), which has excitation and emission spectrum peak wavelengths of approximately 495 nm/519 nm, detectable with a fluorescence spectrophotometer.

Bibliography

The, T. H., & Feltkamp, T. E. (1970). Conjugation of fluorescein isothiocyanate to antibodies. I. Experiments on the conditions of conjugation. Immunology, 18(6), 865–873.

Islam, M. Z., Fokine, A., Mahalingam, M., Zhang, Z., Garcia-Doval, C., Van Raaij, M. J., Rossmann, M. G., & Rao, V. B. (2019). Molecular Anatomy of the Receptor Binding Module of a Bacteriophage Long Tail Fiber. PLoS Pathogens, 15(12), 1–21. https://doi.org/10.1371/journal.ppat.1008193

Strauss, J., Burnham, N. A., & Camesano, T. A. (2009). Atomic force microscopy study of the role of LPS O-antigen on adhesion of E. coli. Journal of Molecular Recognition, 22(5), 347–355. https://doi.org/10.1002/jmr.955

Sequence and Features