Difference between revisions of "Part:BBa K3409009"

Latest revision as of 22:08, 27 October 2020

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

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 [http://2015.igem.org/Team:NCTU_Formosa 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. 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

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.



Results of the validation assay of phase I
The aim of this assay is to target the his-tag fused to the receptor binding protein gp37, which should be directed to the outer membrane of our transformed bacteria, via a monoclonal anti-his antibody conjugated to fluorescein isothiocyanate (FITC). Thus, after the immunolabelling, the fluorescence is measured at excitation wavelength of 497 nm and emission wavelength of 519 nm.

The controls for this assay are bacteria that have been transformed with the pSB1A3 containing the RFP only and that undergo the same treatment as our samples. The samples are bacteria containing the plasmid construct of phase I within the his-tag fused to the receptor binding protein gp37. Additionally, samples and controls are treated with and without anti-his antibodies diluted at 1/1000 and 1/100. Finally, our samples have been done in triplicate (S1-series, B1-series and B2 series).


We can observe that all the negative control samples, which are the samples that are not treated with the anti-his antibody (S1, B1, B2) and the control (C) emit a similar signal around 27000. Samples treated with the anti-his antibody 1/1000 dilution (S1 Ab 1/1000, B1 Ab 1/1000, B2 Ab 1/1000) and the control that has been treated with the anti-his antibody 1/1000 dilution (C Ab 1/1000), emit a signal around 27000 as well. Finally, samples treated with the anti-his antibody 1/100 dilution (S1 Ab 1/100, B1 Ab 1/100 , B2 Ab 1/100 ) emit a signal around 43,000, 38,000, 40,000 restively, and the control that has been treated with the anti-his antibody 1/100 dilution (C Ab 1/100) emit a signal around 40,000. Thus, S1 Ab 1/100 (43,000) is the only sample of transformed bacteria with the construct of phase I that has a higher signal than its controls (C Ab 1/100 = 40,000 and S1 without antibody = 27,000), which is not concluding enough to suggest that the receptor binding protein is well expressed on the outer membrane of our chassis. However, the fact that we have similar signals for each condition shows that the measured signals are well due to the presence or absence of the anti-his antibody. However, what this data shows is that the antibody might bind unspecifically to other regions of the bacteria or other components in the reaction tube. This specificity issue could not be analysis in more depth because of a lack of time. After having performed this immunolabelling, we thought that this assay was not enough to confirm the presence or not of the receptor binding protein gp37 on the surface of our chassis. We should rather proceed with lysing the cells and differential centrifugation steps to separate the membrane from the bacterial cytoplasm. Then, both samples should by analyzed by performing a western blot by targeting again the his-tag with an anti-his antibody. This would show if the protein is present in the membrane or in the cytoplasm at all.

Experimental perspectives Phase 1 | Targeting and binding to the target bacteria

• Perform a differential centrifuge to extract the fraction with the membrane the fraction with the cytoplasm and detect the presence of the gp37 fused to the his-tag via Western blot in either fraction
• The immunolabelling experiments should be repeated to validate or not the presence of gp37 on the surface of our chassis by optimizing the unspecific binding (Maybe try to use a higher concentration of BSA when performing the blocking step)
• Perform the experiment with the TEV protease and proceed with the protein purification with the Ni2+ chromatography columns specific to the his-tag.
• Validate experiments with confocal microscopy to have the possibility to see any signal at the surface of the chassis
• Validate the binding of the chassis to the target

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