Team:REC-CHENNAI/Parts-Characterization
PART CHARACTERIZATION
To characterize the part BBa_K3224003 (Cell Penetrating Peptide - BP100-(KH)9 in HeLa (immortalized human cervical cancer) and C2C12 (immortalized mouse myoblast) cell lines.
When HeLa cells were treated with lower concentrations of CPP (4-16 µM) for 24 hours, ~15% cytotoxicity was observed. Higher concentrations of CPP (32-120 µM) showed ~25% cytotoxicity on the cells.
When HeLa cells were treated with CPP for a time period of 48 hours, the cytotoxicity levels lie below 50% for lower concentrations of the peptide (4 and 8µM). For the concentrations from 16 to 120µM the cytotoxicity shoots up to ~100%.
The action of lower concentrations of CPP (4 and 8µM) on C2C12 cells showed ~35% cytotoxicity. At higher concentrations of CPP (16 to120µM) ~135% cytotoxicity was observed.
When C2C12 cell lines were treated with lower concentrations of CPP (4-32µM) for 48 hours, CPP showed ~100% cytotoxicity. At higher concentrations (64 and 120µM) the cytotoxicity goes beyond 150%.
The Cell Penetrating Peptide BP100-(KH)9 (BBa_K3224003) was synthesized in-vitro and purified by High-Performance Liquid Chromatography (HPLC). The in-vitro synthesized shRNA-like siRNA with a fluorophore and a quencher (BBa-K3224002) was mixed at 100 µM concentration, with varying concentrations of the chimeric CPP BP100- (KH)9 to make up different N/P ratios: 0.5, 1 and 2. Also, only siRNA without the CPP was added to the cells as delivery control. The fluorescence emitted was measured in Arbitrary Fluorescence Units (AFU). The other nucleotide construct, survivin shRNA (BBa_K3224001), was not used for this experiment as it is devoid of a flurophore.
The intensity of the fluorescence emitted is proportional to the amount of cargo successfully delivered into the cells, and hence an indicator of the optimum N/P ratio in order to achieve improved transfection efficiency. From the graph, we observe that the N/P ratio of 0.5 gives the higher intensity of fluorescence which can be attributed to the higher extent of complexation resulting in efficient transfection. Thus the efficient delivery of the nucleic acid has taken place by the CPP.
In addition to optimizing the N/P ratio, the current experiment also confirms that the two submitted parts work: CPP in terms of delivering the cargo, and shRNA-like siRNA in terms of emitting fluorescence. The binding and silencing efficiencies of this nucleotide construct were validated in the subsequent experiments, the results of which have been discussed in the Project Demonstration section.
