Part:BBa_K3409008
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.
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.
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 4480
Illegal NheI site found at 4503 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1555
Illegal BglII site found at 2749
Illegal BglII site found at 4526 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 438
Illegal NgoMIV site found at 2929
Illegal AgeI site found at 1112
Illegal AgeI site found at 1224
Illegal AgeI site found at 1897
Illegal AgeI site found at 4581 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 3132
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