Team:REC-CHENNAI/Integrated-HP
HUMAN PRACTICES
We are proposing an alternate delivery method for the transfection of cargo using Cell-Penetrating Peptide (CPP) as the delivery agent. We consulted several research scholars and scientists working with genetics as well as synthetic biology to gauge what they feel that could help improve our project. The final project that is put up in our wiki is the collation of diverse expert opinions on the grounds of conceptualization of the idea, designing of parts and experiments and safety of our project. We also gained expert inputs with regard to the characterization of biobricks.
We planned to conjugate the cargo and CPP for ensuring certainty of delivery. Our conversation with Dr.Srujan Marepally, Scientist, Centre For Stem Cell Research, Christian Medical College, Vellore who specializes in the mechanisms involved in lipid mediated transfection. We also met Dr.P.Rajasekar, Assistant Professor, Department of Biotechnology, Rajalakshmi Engineering Collegewho works on Nanoparticle based delivery systems of drugs for type II diabetes. They advised against conjugation of cargo with CPP but instead bind them through non-covalent interactions between the same. They also stated the advantages of using a CPP-cargo complex as a mixture to increase the efficiency of delivery and prevent the loss of biological activity.
Dr.D.Karunakaran is an eminent scientist and professor who works on the role of miRNAs in cancer apoptosis. When we presented the system we chose to validate our idea to him, which involved the design of interfering RNA sequence targeting the c-myc mRNA in HeLa cell line, he preferred that we choose some other gene for validating our idea as c-myc mRNA are short-lived. Thus we switched back to brainstorming for other options and choose to target survivin mRNA owing to its high expression and prolonged sustenance in cancer cell lines.
Dr.Rohith, a notable expert in Anna University, Chennai gave valuable inputs on designing our construct as shRNA like siRNA. His valuable tips were to improve the stability of the silencing RNA and also prevent any loss of function of the same. He gave comprehensive inputs on the design of the fluorophore conjugated shRNA.
Our team members had a discussion with Dr.M.Balasubramanyam, Senior Scientist, Indian Council of Medical Research Advanced Centre of Genomics of Type II Diabetes who is working on the molecular pathogenesis of diabetes and RNAi and miRNA mechanisms in which he suggested the optimisation of N/P ratio (Nitrogen/Phosphate ratio) to enable an efficient and effective usage of our CPP.
Dr. Madhavan Jagadeesan was of help in our experimental design as he suggested the use of plate reader and fluorescence spectroscopy instead of a confocal fluorescence microscope to enable quantification of the fluorescence and better understanding of the results as our construct with the fluorophore is the silencing RNA which is a very small molecule to ensure clarity of the image produced by confocal microscopy. This was higly useful and made us restructure the experimental protocols for validation.
The inputs of Ms. Priya, a molecular biologist at Eppendorf, were also incorporated in our experimental design for validation of our proposed system. Her opinion was that the Western Blot test is not essential in our project as the protein is not expressed once the mRNA is silenced. Instead, Reverse Transcriptase PCR or Quantitative Real-Time PCR results will be a direct indicator of the silencing of gene expression at the RNA level.
Dr.V.Gopikrishnan works in bioprospecting and has helped us with the experimental design for the characterization experiment of the Anti-Microbial Peptide [AMP] Latarcin. He extended our experiments studying the antibacterial activity to incorporate yeast. Thus we estimated the antimicrobial activity.
The experiment we designed for assessing Antimicrobial activity of peptides covered a narrow range of dilution of the peptide. The meet up with Dr. Naveen Kumar, Post Immugenix laboratories made us switch to the universal system of double diluting test samples for MIC assay.
Dr. Johanna Rajkumar our Dean and also the Chair of Bio-safety Committee of our college helped us solve certain ethical issues regarding performing Haemolytic Assays and advised us to outsource the haemolytic assay experiment.
Our diversified Integrated Human Practices with relevant experts made our project an ideal delivery system for transient transfection and increased the robustness of our experimental design both in terms of validating our biobrick and characterization of the existing biobrick. All these inputs were inculcated in our project presented in the wiki page.
