Nanobodies
Plasmid design
RFP – engineered mutant of red fluorescent protein from Discosoma striata (coral)
HlyA- C-terminal signal sequence of alpha-hemolysin
The construct was synthesized by GenScript company in pUC57 vector
The next step is to induce protein expression with IPTG and to purify the proteins using Ni/NTA columns
After that, we are planning to test Vhp53 affinity to p53 that is present in the saliva of cancer patients.
We want to use the saliva because more than 400 types of proteins, including: Cancerous markers (CEA, SCC, IAP, Cyfra, p16, p53 and others - 81% sensitive), Diabetic markers (Procalcitonin, Carbonic anhydrase 6 and othres (more than 60)), Periodontal, disease markers (He lactoferrin, TIMP and others), Sjogren’s syndrome (decreased lipocalin 1, calgranulin and etc.), Markers for other systemic diseases, Different bacteria (Streptococcus mutans and lactobacilli) and viruses can be tested.
We expect the secretion of proteins with dimerization capacity by the haemolysin type I transport system of Escherichia coli
Here is how do we plan to prepare saliva for testing:
Centrifuge saliva samples (800 g, 10 min, 4°C), and suspend the pellets in 150 μl of lysis buffer (45 mM HEPES, 0.4 M KCl, 1 mM EDTA, 10% glycerol, pH 7.8). May add protease inhibitors if available (Halt Protease Inhibitor Cocktail, EDTA-Free (100X). Incubate for 30 min at room Temperature
Centrifuge the samples at (11 000 g, 10 min, 4°C)
Measure [protein] concentrations in the supernatants
Transfer a volume containing 50 ng of protein to a 1.5 ml vial and bring all samples to the same volume of 500 μl with the addition of PBS
Mix the solutions well and add 100 μl of each sample to ELISA-plate wells (nunc-immunoplate; Thermo Fisher Scientific). Cover the plate and store overnight at 4°C
On the next day, wash each well three times with 100 μl PBS–Tween solution (PBS-T, PBS containing 0.05% Tween 20) and add a volume of 100 μl of 1% BSA PBS-T blocking solution (PBS containing 0.05% Tween 20 and 1% BSA) to each well. Incubate 1 h at room Temperature
Add 100 μl of ab to each well. Incubate 2 h at room Temperature
Wash the plate as described above
Add 100 μl of 3,3′,5,5′-tetramethylbenzidine solution (Southern Biotech) to each well. They used a peroxidase-conjugated goat anti-rabbit secondary antibody (1 : 5000; Jackson Immunoresearch). Incubate 1-2 min.
Add 100 μl of stopping reagent to each well (10% sulphuric acid).
Measure the absorbencies of the samples at the wavelength 450 nm directly after the addition of the stopping reagent, using a Zenith 200 ELISA reader
Adapted from: Shpitzer, T., Hamzany, Y., Bahar, G., Feinmesser, R., Savulescu, D., Borovoi, I., ... & Nagler, R. M. (2009). Salivary analysis of oral cancer biomarkers. British journal of cancer, 101(7), 1194-1198.
Introducing permanent competence into E. coli
Making construct
The gene for Gp16 ATP-ase protein was ordered from GenScript company.Then, it was combined with the constitutive Anderson promoter + INP, and the constructed part was cloned into standard pSB1C3 plasmid with Circular polymerase extension cloning (CPEC).
References
Fraile, S., Muñoz, A., De Lorenzo, V., & Fernández, L. A. (2004). Secretion of proteins with dimerization capacity by the haemolysin type I transport system of Escherichia coli. Molecular microbiology, 53(4), 1109-1121
Schwartz C, De Donatis GM, Fang H, Guo P. (2013). The ATPase of the phi29 DNA packaging motor is a member of the hexameric AAA+ superfamily. Virology. 443: 20–27.
Quan J, Tian J. (2009) Circular Polymerase Extension Cloning of Complex Gene Libraries and Pathways. PLoS ONE 4(7): e6441. doi:10.1371/journal.pone.0006441
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