Screening and selection of artificial riboswitches.

Synthetic riboswitches are engineered to regulate gene expression in response to a variety of non-endogenous small molecules, and a challenge to select this engineered response requires robust screening tools. A new synthetic riboswitch can be created by linking an in vitro-selected aptamer library with a randomized expression platform followed by in vivo selection and screening. In order to determine response to analyte, we developed a dual-color reporter comprising elements of the E. coli fimbriae phase variation system: recombinase FimE controlled by a synthetic riboswitch and an invertible DNA segment (fimS) containing a constitutively active promoter placed between two fluorescent protein genes. Without an analyte, the fluorescent reporter constitutively expressed green fluorescent protein (GFPa1). Addition of the analyte initiated translation of fimE causing unidirectional inversion of the fimS segment and constitutive expression of red fluorescent protein (mKate2). The dual color reporter system can be used to select and to optimize artificial riboswitches in E. coli cells. In this work, the enriched library of aptamers incorporated into the riboswitch architecture reduces the sequence search space by offering a higher percentage of potential ligand binders. The study was designed to produce structure switching aptamers, a necessary feature for riboswitch function and efficiently quantify this function using the dual color reporter system.

The Influence of Inlet Asymmetry on Steam Turbine Exhaust Hood Flows.

It has been widely recognized for some decades that it is essential to accurately represent the strong coupling between the last stage blades (LSB) and the diffuser inlet, in order to correctly capture the flow through the exhaust hoods of steam turbine low pressure cylinders. This applies to any form of simulation of the flow, i.e., numerical or experimental. The exhaust hood flow structure is highly three-dimensional and appropriate coupling will enable the important influence of this asymmetry to be transferred to the rotor. This, however, presents challenges as the calculation size grows rapidly when the full annulus is calculated. The size of the simulation means researchers are constantly searching for methods to reduce the computational effort without compromising solution accuracy. However, this can result in excessive computational demands in numerical simulations. Unsteady full-annulus CFD calculation will remain infeasible for routine design calculations for the foreseeable future. More computationally efficient methods for coupling the unsteady rotor flow to the hood flow are required that bring computational expense within realizable limits while still maintaining sufficient accuracy for meaningful design calculations. Research activity in this area is focused on developing new methods and techniques to improve accuracy and reduce computational expense. A novel approach for coupling the turbine last stage to the exhaust hood employing the nonlinear harmonic (NLH) method is presented in this paper. The generic, IP free, exhaust hood and last stage blade geometries from Burton et al. (2012. "A Generic Low Pressure Exhaust Diffuser for Steam Turbine Research,"Proceedings of the ASME Turbo Expo, Copenhagen, Denmark, Paper No. GT2012-68485) that are representative of modern designs, are used to demonstrate the effectiveness of the method. This is achieved by comparing results obtained with the NLH to those obtained with a more conventional mixing-plane approach. The results show that the circumferential asymmetry can be successfully transferred in both directions between the exhaust hood flow and that through the LSB, by using the NLH. This paper also suggests that for exhaust hoods of generous axial length, little change in Cp is observed when the circumferential asymmetry is captured. However, the predicted flow structure is significantly different, which will influence the design and placement of the exhaust hood internal "furniture."