Fabrication, characterization and degradation of PHB and PHBV microspheres for liver cell growth

Master'sMASTER OF ENGINEERIN

Synthesizing non-natural parts from natural genomic template

<p>Abstract</p> <p>Background</p> <p>The current knowledge of genes and proteins comes from 'naturally designed' coding and non-coding regions. It would be interesting to move beyond natural boundaries and make user-defined parts. To explore this possibility we made six non-natural proteins in <it>E. coli</it>. We also studied their potential tertiary structure and phenotypic outcomes.</p> <p>Results</p> <p>The chosen intergenic sequences were amplified and expressed using pBAD 202/D-TOPO vector. All six proteins showed significantly low similarity to the known proteins in the NCBI protein database. The protein expression was confirmed through Western blot. The endogenous expression of one of the proteins resulted in the cell growth inhibition. The growth inhibition was completely rescued by culturing cells in the inducer-free medium. Computational structure prediction suggests globular tertiary structure for two of the six non-natural proteins synthesized.</p> <p>Conclusion</p> <p>To our best knowledge, this is the first study that demonstrates artificial synthesis of non-natural proteins from existing genomic template, their potential tertiary structure and phenotypic outcome. The work presented in this paper opens up a new avenue of investigating fundamental biology. Our approach can also be used to synthesize large numbers of non-natural RNA and protein parts for useful applications.</p

Autofluorescence spectroscopy in redox monitoring across cell confluencies.

Patient-specific therapies require that cells be manufactured in multiple batches of small volumes, making it a challenge for conventional modes of quality control. The added complexity of inherent variability (even within batches) necessitates constant monitoring to ensure comparable end products. Hence, it is critical that new non-destructive modalities of cell monitoring be developed. Here, we study, for the first time, the use of optical spectroscopy in the determination of cellular redox across cell confluencies by exploiting the autofluorescence properties of molecules found natively within cells. This was achieved through a simple retrofitting of a standard inverted fluorescence microscope with a spectrometer output and an appropriate fluorescence filter cube. Through spectral decomposition on the acquired autofluorescence spectra, we are able to further discern the relative contributions of the different molecules, namely flavin adenine dinucleotide (FAD) and reduced nicotinamide adenine dinucleotide (NADH). This is then quantifiable as redox ratios (RR) that represent the extent of oxidation to reduction based upon the optically measured quantities of FAD and NADH. Results show that RR decreases with increasing cell confluency, which we attribute to several inter-related cellular processes. We validated the relationship between RR, metabolism and cell confluency through bio-chemical and viability assays. Live-dead and DNA damage studies were further conducted to substantiate that our measurement process had negligible effects on the cells. In this study, we demonstrate that autofluorescence spectroscopy-derived RR can serve as a rapid, non-destructive and label-free surrogate to cell metabolism measurements. This was further used to establish a relationship between cell metabolism and cellular redox across cell confluencies, and could potentially be employed as an indicator of quality in cell therapy manufacturing