Phi29 Protein as a Way to Induce Artificial Competency in E.coli

Bacterium Escherichia coli (E. coli) is one of the most popular model organisms used for laboratory work. However, E. coli cells possess a weak tendency to uptake DNA from the surroundings, i.e. it shows a weak competence. Competence can be induced by two common methods: electroporation and chemical transformation using divalent cations. Electroporation requires extra equipment as electroporator, while chemical transformation takes at least one day preparation. It was shown that bacterial virus phi29 DNA packaging motor is capable to pump both double-stranded and single-stranded DNA. However, no data is available for delivering plasmid DNA using phi29 DNA motor. We suggest introducing the new method for the induction of artificial competence in E. coli via DNA pump motors. The series of viral proteins such as a portal protein (gp10) that form the major motor dodecamer channel and hexameric ATPase gp 16 will be expressed in E. coli. Signal leading peptide sequences for outer and inner membrane destination will be added to phi29 DNA motor. The proposed method aims to reduce time and cost in the preparation of competent cells.

E.Coli derived camelid antibodies as a sensor for p53 in saliva

Oral squamous cell carcinoma (OSCC) is a malignant tumor with 640,000 new cases annually in the world. Saliva testing is non-invasive procedure that is capable to detect potential biomarkers for OSCC. The DNA, RNA and proteins derived from the cancerous cells can be obtained from saliva samples. It was shown that elevated level of p53 protein was identified in OSCC patients at different stages of the disease. Camelid antibodies containing only variable regions, nanobodies (VHH) and single-chain variable regions (scFv) with VH and VL, are becoming popular in many biological studies including diagnostic applications. It was identified that VL region alone showed higher affinity to p53 than VHH, and dimerization of VL region with another one increases the affinity up to 10 folds. Camelid antibodies have similiar affinity to its substrate as human antibodies and can be conjugated to other proteins without functional lose. They can be expressed and secreted in many organisms including E.Coli in high amount, which reduces the cost of antibodies production. Thus, the aim of this project is to design a biosensor, based on available sequence of antibodies, to detect p53 in saliva samples for OSCC diagnosis.

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