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 ten folds. Camelid antibodies have similiar affinity to its substrate as human antibodies and can be conjugated to other proteins without loss of function. 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.

The biosensor will be similar to ELISA, but instead of antibodies, dimerized VL regions will be used. Vhp53 with zipper-containing polypeptide, His-tag, red fluorescent protein (RFP) and HlyA-C-terminal signal sequence of alpha-hemolysin will be expressed in E. coli. We expect that zipper will allow dimerization of two Vhp53 parts and hemolysin secretion system will aid in secretion of nanobodies. Purified nanobodies will be used for detection of p53 protein is saliva samples.

GP16 Protein as a Way to Induce Artificial Competency in E.coli

E. coli is one of the most widely used model organism in Synthetic Biology. E. coli can be engineered in various ways. Apparently, to make recombinant E. coli, one needs to transform recombinant DNA into a competent bacterium. However, E. coli itself has a low tendency to uptake DNA from the environment. The competence needs to be induced by either electroporation or treatment with divalent cations. Electroporation can make the bacteria competent only for a very brief period, while chemical treatment takes at least two days and then requires storage of bacteria at -800C, otherwise, they will lose the competence. Therefore, our team decided to find a way of introducing inducible competence into E. coli.

Gp16 protein is phi29 ATPase, which is a part of viral DNA packaging motor. Hydrophobic protein gp16 was previously introduced into a lipid bilayer in vitro. We are planning to introduce signal leading peptides from the iGEM distribution kit upstream of p16 gene in order to incorporate gp16 protein into bacterial inner and outer membranes. In addition, p16 will be expressed IPTG-inducible promoter, so that the bacteria will become competent only when needed. Furthermore, chaperonin GroEL/ES will be co-expressed in order to ensure correct folding of gp16 protein.

Overall, the proposed method aims to reduce time and cost in the preparation of competent cells.

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