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Overall Project
Our aim is to construct a targeted gene therapy vector with high cellular specificity, considerable capacity and the potential for mass production and universal modification. Analogizing the characteristics of bacteriophage lambda and adenovirus, we genomically engineered the fiber protein of adenovirus with the pC of bacteriophage lambda, with the knob region modified by cell-specific peptides generated by phage display (called targeted bioBrick). After inducing the vector genome (generated by bottom-up or top-down approach) into BL21 DE3 E.coli strain, we applied a co-transformed therapeutic DNA (namely a cosmid with a capacity of 40-50 kb) for mass production of our targeted gene therapy vectors containing the desired genes to be delivered. With the targeted bioBrick mediating the attachment and RGD domain mediating the internalization of the targeted vector, we are able to accomplish the targeted gene therapy.
ModuleI: Synthesis of GenSniper Viroin
Using two synthetic biological strageties (bottom-up and top-down), we will construct a genome of our desired gene therapy vector, which consists of the structural proteins for bacteriophage lambda head assembly and lysis genes (and targeted bioBrick later). Our aim in this module is to generate the viroin of our gene therapy vector (namely the head structure of bacteriophage lambda).
ModuleII: Therapeutic DNA
In this module, we plan to construct a cosmid (a molecular cloning with a cos site) encoding the conceptual therapeutic genes, because the structural proteins of bacteriophage lambda will only package the circular DNA with cos site and 40-50 kb in size. Here we will also identify the function of this module through the expression of RFP.
ModuleIII: Targeted BioBrick
This module is the modification of bacteriophage lambda protein C by fiber shaft at its N termini. When constructing this targeted bioBrick, we will apply PCR to add the targeted peptide (in wetlab we just use TAT peptide) and RGD sequence into the fusion protein, forming a peptide-fiber-RGD-C reading frame.
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