Engineering

Christopher Werner, Brigham Young University Engineering Preparation of DNA linear expression templates (LET) via Polymerase Chain Reaction (PCR) is significantly faster than procurement of DNA via cell growth and plasmid purification. Unfortunately, linear templates have not consistently achieved protein yields comparable to plasmids in cell-free protein synthesis (CFPS). Possible reasons for lower LET yields were investigated by producing a number of different extracts. Extracts were differentiated by varying harvest time after induction and modifying the extract preparation procedure. These extracts were tested with py71 sfGFP plasmid (producing a reporter protein) and LET’s created through PCR from the same plasmid. Protein yields obtained by fluorescence measurement were plotted against combined tRNA and rRNA amounts obtained through DNA electrophoreses and densitometry. A correlation was seen between tRNA and rRNA amounts and a normalized LET yield (LET yield divided by the plasmid yield under identical conditions). We considered two reasons for this correlation. First, the increased tRNA and rRNA indicated and increase in the concentration of cell translation machinery present, which increased the kinetics of the reaction, allowing LET’s to produce protein quicker before degradation by Deoxyribonucleases (DNAse). Second, the increased tRNA and rRNA amounts acted as a shield for mRNA from Ribonucleases, allowing more of the mRNA to be translated before LET’s were degraded by DNAse’s. Further work must be done to verify the accuracy of this correlation, as well as to determine the cause for increased LET yields in extracts with higher tRNA and rRNA amounts.

Ryan Putman, Utah State University Engineering Spider silk possesses superior mechanical properties to most other biological or man-made materials. In particular, research has demonstrated that spider silk is as strong as Kevlar, yet much more elastic. The unique feature of both strength and elasticity naturally piques interest in numerous scientific fields (e.g. medical sutures, automobile seat belts, or athletic performance wear). However, the limiting factors in using spider silk on a commercial scale are the production of sufficient protein product and the ability to do so in a cost-effective manner. An additional challenge is due to the territorial and cannibalistic nature of spiders, which makes harvesting their silk from large “spider farms” an infeasible task. To overcome these limitations, an approach using synthetic biological engineering principles has been employed. This emerging field of study provides a powerful tool, the use of standard biological parts called BioBricks. Using these standard DNA parts, a genetic circuit with the necessary regulatory components was engineered to recombinantly produce a synthetic spider silk protein in the microorganism Escherichia coli, which will provide a more consistent and sustainable source of spider silk than by harvesting directly from spiders. Expression of this recombinant protein has been verified through SDS-PAGE protein gels and subsequent protein immunoblot. The next step is to create a genetic circuit that will be used to secrete the spider silk protein outside of the E. coli bacterium. This could greatly reduce the downstream processing costs associated with protein purification as well as potentially increase overall yield. Therefore, a genetic tag that targets products for secretion has been fused to the spider silk coding regions. Further testing is required to determine the difference in overall protein yield from secreting as opposed to non-secreting strains of the engineered bacteria. Once these values are determined, the production can be optimized and scaled-up.

Connor George, Utah State University Electrical and Computer Engineering The SABER instrument, aboard the TIMED satellite, measures optical data regarding parameters of the Earth’s atmosphere with respect to altitude. Approximately once per minute, SABER performs a limb-scan measurement on the Earth’s atmosphere from which altitude emission profiles of key atmospheric gasses, including hydroxyl at wavelengths of 1.6 μm and 2.0 μm, are derived. Most hydroxyl profiles within the SABER dataset contain a single peak in the airglow altitude profile centered near an altitude of 87 km, but a significant portion of the profiles display two or more local maxima. MATLAB code was written to analyze the geophysical and temporal global distribution of the multiple-peak profiles. Graphs have been created which display relationships between the percentage of multiple-peak profiles and the local time, the cardinal orientation of the SABER device, and the latitude and longitude at which the atmospheric profile was measured. Patterns have been observed in multiple-peak profile distribution with respect to these variables. Possible causes of the multiple-peak occurrences in the hydroxyl altitude profiles include waves, geometrical effects of the SABER instrument, and/or chemistry of the atmosphere. In addition to graphing software, analysis software was written which counts the number of peaks present in any given altitude profile, and which ascertained the percentage of profiles displaying multiple-peak characteristics. A small (<1%) portion of hydroxyl altitude profiles were found to have abnormal distributions due to erroneous or noisy data collected by SABER. Software has also been written to remove such exceptions from the dataset. Additional investigation into the relationship between multiple-peak occurrences and cardinal direction orientation of the SABER device is required in order to further identify the causes for multiple peak profiles. An investigation into seasonal patterns for multiple-peak profiles is to be conducted. As the dataset grows, exception software will be updated to identify invalid altitude profiles. Also, ozone has been found to have multiple-peak altitude profiles similar to those of hydroxyl, and studies complementary to those performed on hydroxyl altitude profiles will be performed on ozone.