Recent changes in climate patterns pose an enormous threat to the agricultural industry. Increasing temperatures and lower soil water content systematically decrease crop yields. This project aimed to tackle this problem from the bottom up by engineering a common soil bacterium, Pseudomonas putida, to overexpress plant growth-promoting enzymes under a temperature-dependent system. P. putida, with its known root interactions with Arabidopsis thaliana, was utilized to promote plant growth through the production of indole-3-acetic acid (IAA), 1-aminocyclopropane-1-carboxylate (ACC) deaminase, and trehalose synthase. A program that couples genetic algorithms and NUPACK was created to design and optimize low-temperature RNA thermometers. Using molecular cloning techniques, necessary part plasmids were designed, implemented and tested in E. coli. Three of our RNA thermometers with melting temperatures of 30°C and one with a melting temperature of 37°C show promise for having better fold efficiencies than thermometers currently found in the iGEM registry. Maximizing crop yields now will ensure better food availability and distribution in the future.
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The unique properties of non-model bacteria can expand the applications of synthetic biology. However, currently there are few reliable tools for engineering non-model bacteria. A central obstacle to the development of such tools is the dependence of circuit expression on host machinery. To address this problem, we developed PORTAL, a system which uses T7 transcription and orthogonal ribosomes to insulate the circuit from host processes. We characterized PORTAL in four E. coli strains, Shewanella oneidensis, and Pseudomonas putida, comparing PORTAL-driven and host-driven expression of a reporter. To design orthogonal ribosomes, we created software that analyzes binding energies of 16S rRNA and determines the optimal orthogonality-promoting anti-Shine-Dalgarno mutations. We created a model that simulates the performance of PORTAL and shows that the system is minimally sensitive to metabolic differences. PORTAL presents a tunable “virtual machine” to facilitate insulated synthetic gene circuit expression in non-model bacteria.
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A recent analysis of America’s drinking water for carcinogenic hexavalent chromium (CrVI) revealed over 218 million Americans consume CrVI at levels exceeding a de minimis lifetime cancer risk. Our team attempted to solve this problem by engineering Shewanella oneidensis to reduce CrVI to its less toxic form (CrIII) in contaminated wastewater. We enhanced reduction via expression of a mutated chromate reductase enzyme (chrR6) and increased chromate permeability via over-expression of sulfate transporter and binding proteins (cysP,U,W,A and sbp). Addressing biocontainment, we included a “kill switch” where low CrVI levels signaled expression of a “toxin” (BamHI). Laboratory work has been completed in model organism Escherichia coli; future work includes characterizing the circuit in S. oneidensis. To assess real-world feasibility, we developed bioreactor- and cell-scale simulations of our engineered bacteria in an activated sludge secondary treatment system aimed at determining the kinetics of Cr(VI) remediation and cell death.
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The HKUST-Rice iGEM team is the first transcontinental team in iGEM, comprising of 31 student members– 18 from the Hong Kong University of Science and Technology and 13 from Rice University. The benefit of forming a large joint team is that we have members coming from different disciplines who contribute different perspectives to the project. This has enabled us to tackle a broad subject matter effectively.
Nitrogen (N), phosphorus (P) and potassium (K) are three essential macronutrients needed for healthy plant growth. By creating a biological sensor that can quickly provide the status of these macronutrients in the soil, we can provide farmers with the tools to monitor and respond to potential nutrition deficiencies. In previous iGEM projects, nitrate- and phosphate-responsive promoters have been utilized extensively. However, a potassium-responsive promoter was still lacking. Our team submitted the first ever potassium-sensing promoter to the Part Registry, in addition to providing more comprehensive characterization data for the existing phosphate- and nitrate-responsive promoters. Our biological sensors are constructed in E. coli, to detect NPK levels in the surrounding environment and produce a measurable level of reporter protein. To simulate the expression of multiple sensors in a single system, we characterized the effects of a parallel sensors system, in contrast to a single output system.
Check out our 2015 wiki here