Expression of chloroplast genes is primarily regulated posttranscriptionally, and a number of RNA elements, found in either the 5'- or 3'-untranslated regions (UTRs) of plastid mRNAs, that impact gene expression have been identified. Complex regulatory and feedback mechanisms influence both translation and protein accumulation, making assignment of roles for specific RNA elements difficult. To identify specific contributions made by various UTRs on translation of plastid mRNAs, we used a heterologous gfp reporter gene that is fused combinatorially to chloroplast 5'- and 3'-UTRs. In general, the 5'-UTR, including the promoter, of the plastid atpA and psbD genes produced the highest levels of chimeric mRNA and protein accumulation, while the 5'-UTR of the rbcL and psbA genes produced less mRNA and protein. Varying the 3'-UTR had little impact on mRNA and protein accumulation, as long as a 3'-UTR was present. Overall, accumulation of chimeric mRNAs was proportional to protein accumulation, with a few notable exceptions. Light-regulated translation continues to operate in chimeric mRNAs containing the 5'-UTR of either the psbA or psbD mRNAs, despite translation of these two chimeric mRNAs at very different efficiencies, suggesting that translational efficiency and light-regulated translation are separate events. Translation of some chimeric mRNAs was much more efficient than others, suggesting that interactions between the untranslated and coding sequences can dramatically impact translational efficiency.

In an effort to develop microalgae as a robust system for the production of valuable proteins, we analyzed some of the factors affecting recombinant protein expression in the chloroplast of the green alga Chlamydomonas reinhardtii. We monitored mRNA accumulation, protein synthesis, and protein turnover for three codon-optimized transgenes including GFP, bacterial luciferase, and a large single chain antibody. GFP and luciferase proteins were quite stable, while the antibody was less so. Measurements of protein synthesis, in contrast, clearly showed that translation of the three chimeric mRNAs was greatly reduced when compared to endogenous mRNAs under control of the same atpA promoter/UTR. Only in a few conditions this could be explained by limited mRNA availability since, in most cases, recombinant mRNAs accumulated quite well when compared to the atpA mRNA. In vitro toeprint and in vivo polysome analyses suggest that reduced ribosome association might contribute to limited translational efficiency. However, when recombinant polysome levels and protein synthesis are analyzed as a whole, it becomes clear that other steps, such as inefficient protein elongation, are likely to have a considerable impact. Taken together, our results point to translation as the main step limiting the expression of heterologous proteins in the C. reinhardtii chloroplast.

Description of Proposed PaperAs the drilling industry moves towards extended-reach wells in more challenging formations, the need to identify operational solutions (e.g., drilling fluids/lubricity additives) to reduce friction, torque, and drag has become more important. Additionally, as the industry looks to maximize the use of water-based drilling fluids (to benefit from lower drilling fluid costs and improved environmental profile), the need to find novel additives that can improve lubricity has become more pressing. However, current methodologies for reducing downhole friction in horizontal wells generally involve reactive addition of lubricant products that are broadly acting, that may adversely affect the rheology of the fluid system, or that may dissipate or degrade over time. To address the need for new additives, a novel biotechnology method for encapsulating oil in polysaccharide-based polymers has been developed that selectively delivers high pressure lubricants at areas of high shear, pressure, and friction (e.g., between the drill string and formation or casing and also between drill bit and formation). ApplicationDue to the protection afforded by encapsulation, this targeted friction inhibitor can be proactively added to water-based drilling fluid systems, where it circulates through the system until conditions are met to break the capsule and release lubricant. Observations in laboratory and field testing suggest potential operational improvements in running casing, reduction of torque/drag, reduction of bit balling, and faster Rate of Penetration (ROP) from targeted lubrication. 2 SPE-169547-MS Results, Observations and ConclusionsOur characterization of encapsulated oil has shown the ability to reduce the coefficient of friction by over 80% in water-based mud formulations in the laboratory. In field-scale testing at the Catoosa Testing Facility, we have observed reductions in drag of up to 50%, reductions in torque of up to 45%, and up to 20% improvement in Rate of Penetration (ROP) in horizontal drilling after addition of encapsulated oil to a water-based polymer mud system. These results were substantially improved relative to observed results in an exploratory field well, in which ROP increased by up to 216% after addition of encapsulated oil in a saturated sodium chloride drilling fluid system. Significance of Subject MatterOur field testing and characterization in the laboratory and field have demonstrated the utility of adding encapsulated oil to a water-based mud-system to drill horizontal wells to improve the operational efficiency of drilling.