Developing an Unnatural Amino Acid-Specific Aminoacyl tRNA Synthetase

Unnatural Amino Acids (UAAs), amino acids not present in the human genetic code, have been synthesized to have a broad range of useful properties, in this case, as metal-binders which could have drug delivery applications. In order for the cell to place a UAA into the protein, two components, a unique aminoacyl tRNA synthetase and a corresponding tRNA must be present. If an amino acid is successfully charged to the tRNA, a stop codon is suppressed and a functional protein is built with the UAA at the mutation site. Such a tRNA molecule has previously been developed, as well as many synthetases specific to UAAs. In this work, the range of UAAs which can be incorporated into proteins using the E. coli’s own machinery is expanded by the development of a novel aminoacyl tRNA synthetase. By making a library of synthetase-coding plasmid variants and performing positive and negative screenings, the binding pocket of the synthetase can be modified for specificity to a UAA while not allowing the tRNA to be charged with a natural amino acid. In this work, we are attempting to evolve new tRNA synthetases for the incorporation of metal-binding amino acids by developing the plasmid library and a screening system to find synthetase variants meeting these criteria

Developing an Unnatural Amino Acid-Specific Aminoacyl tRNA Synthetase

Unnatural Amino Acids (UAAs), amino acids not present in the human genetic code, have been synthesized to have a broad range of useful properties, in this case, as metal-binders which could have drug delivery applications. In order for the cell to place a UAA into the protein, two components, a unique aminoacyl tRNA synthetase and a corresponding tRNA must be present. If an amino acid is successfully charged to the tRNA, a stop codon is suppressed and a functional protein is built with the UAA at the mutation site. Such a tRNA molecule has previously been developed, as well as many synthetases specific to UAAs. In this work, the range of UAAs which can be incorporated into proteins using the E. coli’s own machinery is expanded by the development of a novel aminoacyl tRNA synthetase. By making a library of synthetase-coding plasmid variants and performing positive and negative screenings, the binding pocket of the synthetase can be modified for specificity to a UAA while not allowing the tRNA to be charged with a natural amino acid. In this work, we are attempting to evolve new tRNA synthetases for the incorporation of metal-binding amino acids by developing the plasmid library and a screening system to find synthetase variants meeting these criteria

Explorations in Real-Time Polymerase Chain Reactions

Polymerase Chain Reaction (PCR) is a method by which a specific segment of DNA can be replicated for further analysis or to ascertain the presence of that sequence. Real-time (or quantitative) PCR allows researchers to view the amplification of the DNA segments both quantitatively and in real time, providing faster results and an idea of the process and rate of the reaction. This real-time information is gathered using fluorescence detection, as fluorophores attach to the amplifying, double stranded DNA segments. The fluorescence detected is proportional to the amount of DNA replicated. We have used both conventional and real-time PCR to test the identity and genetic composition of various food samples, and to learn more about the process of PCR and its uses in research. First, we used a BioRad Fish DNA Barcode kit to test the identity of five fish samples. From this experiment, only one sample was successfully amplified and sent for sequencing. Next, we used a kit that tests for the presence of genetically modified genes in plants (GMO Test kit), also from BioRad, to test commercially available corn-based snack foods for the presence of GMOs, using SYBRgreen as the fluorophore for the real-time PCR reaction. The results from this genetically modified food experiment positively identified GMO genes in a DoritosTM sample, and gave expected control values which indicated a reliable analysis. In the future, we will use these tests as the basis for developments of new protocols for use in the classroom or in research

Explorations in Real-Time PCR

Real-time PCR (Polymerase Chain Reaction) is a method by which a specific segment of DNA can be replicated and the results of this amplification viewed both in real time and quantitatively using fluorescence. We have used Real-Time PCR to test the identity and genetic composition of various food samples, and to quantify the amout of this DNA present in the food samples. Another goal of this project was to learn more about the process and instrumentation for PCR and its uses in research. Using and Rt-PCR SYBRgreen kit from BioRad, we tested commercially available corn-based snack foods for the presence of GMOs. The results from this genetically modified food experiment positively identified GMO genes in a DoritosTM sample, and gave expected control values, indicating a reliable analysis. In the future, we will use these test as the basis for developments of new protocols for use in the classroom or in research

Screening Unnatural Fluorescent Amino Acids for Incorporation into E. Coli Cellular Machinery

Fluorescence can be used in optical imaging to view cell activity in vivo. Fluorescent proteins and organic dyes are the primary method of visually tracking biomolecules in vivo. However, several research groups have also incorporated unnatural fluorescent amino acids (UFAAs) into proteins for in vivo analysis of said proteins. However, only a handful of UFAAs have been successfully incorporated into proteins using this method (sometimes called the amber suppression method)1. To expand the library of UFAAs viable for cell imaging, several UFAAs were tested in a Green Fluorescent Protein (GFP) screening system to determine if these UFAAs could be incorporated into GFP in response to an amber stop codon using previously developed ‘promiscuous’ tRNA-aaRS (aminoacyl tRNA synthetase) pairs2. This incorporation was quantified by fluorescence readings of mutated green fluorescent protein (GFP). The GFP was mutated to have a TAG (stop) codon deactivating its fluorescence. This deactivation could be overcome by the incorporation of a UFAA in response to the TAG codon, achieved by insertion of the UFAA by the tRNA specific to this codon. Several different UFAAs and several different amber suppression based tRNA/aaRS pairs were analyzed with this screening system, and the viability of these UFAAs to be incorporated into proteins using available amber suppression systems assessed. 1. Lampkowski, Jessica S.; Uthappa, Diya M.; Young, Douglas D.. Site-specific incorporation of a fluorescent terphenyl unnatural amino acid. Bioorg. Med. Chem. Lett. 2015, Available online 25 September 2015, accessed 10 October 2015. 2. Wang, Feng; Niu, Wei; Guo, Jianto; Schultz, Peter G.. Unnatural Amino Acid Mutagenesis of Fluorescent Proteins. Angew. Chem. Int. Ed. 2012, 51, 10132 –10135

Analysis of the Stability of Natural and Unnatural Amino Acids Under Extraterrestrial Conditions

Extraterrestrial life is an area of interest in many scientific disciplines, and learning more about how that life may be built is a valuable pursuit. With this in mind, we look to see which amino acids or unnatural amino acids are more stable in extraterrestrial conditions. For this, we use Ultra Performance Liquid Chromatography (UPLC) with UV-VIS and MS detectors. The UPLC data shows which amino acids have degraded and which have maintained their shape. In our initial tests of five representative natural amino acids, a 10% acetone bath caused the most degradation in the structures, while extreme cold and pH change caused the least

Screening for Unnatural Amino Acid Incorporation in Cellular Machinery

Optical imaging using fluorescence is limited by fluorophore size and unsuitability for in vivo analysis. Unnatural fluorescent amino acids (UFAAs) provide an alternative to bulkier fluorophores for cellular and protein imaging. Additional small, red-shifted, nontoxic, and cell-permeable fluorophores that can be incorporated into proteins in vivo would be a significant advancement in the field (1-4). In order to find these fluorophores, the viability of pairings of UFAAs and the aminoacyl tRNA synthetases (tRNA-aaRS) which attach them to tRNA molecules were tested. Our work has screened seven UFAAs with four tRNA-aaRS developed for specific UFAAs, looking for incorporation into green fluorescent protein (GFP) plasmids using the amber suppression method. A stop (TAG) codon in a GFP plasmid inserted into the host E. Coli causes incomplete formation of the barrel-shaped protein, which then does not fluoresce. If the stop codon, however, is suppressed by the incorporation of a UFAA, GFP fluorescence is expressed from the complete protein, and this fluorescence can be measured and is proportional to the amount of incorporation. Overall, positive and negative controls behave as expected, and other promising hits will be further examined. Miniaturization of the screening has allowed the process to be performed in a microtiter plate, reducing screening time from 3 days to 1.5 days and enabled analyzation by a single instrument. 1.Lang, Chin. Chemical Reviews 2014. 2.Young. Biochemistry 2011. 3.Miyake-Stoner. Biochemistry 2010. 4.Speight. JACS 2013

Concentration of Circulating Anodic Antigen for Low-Resource Diagnosis of Schistosomiasis

Schistosomiasis is a water-borne parasite disease found in tropical regions, especially those with diminished access to clean water. Symptoms, including abdominal pain and chronic diarrhea, affect the ability of infected people to work or learn. Traditionally, schistosomiasis is diagnosed by a fecal smear, but this method requires trained personnel and an advanced infection. To overcome these challenges, a lateral flow assay has been developed which tests for Circulating Anodic Antigen (CAA), a negatively-charged glycoprotein produced by the parasites. This assay is easy to use and has high sensitivity, but concentrating CAA in the urine samples used would further improve it. Our objective was to use positively-charged dendrimers to concentrate CAA onto magnetic beads, which could then be deposited onto the assay. Using an ELISA, we determined the most effective conditions for capture and elution of CAA from the beads. Based on our optimizations, we chose a method which gave 75% recovery of CAA, approximately a 30-fold concentration increase. We also calculated single pg/mL limits of detection, comparable to the levels observed from a single worm pair. The method is currently being incorporated into a paper lateral flow assay which will be deliverable to endemic areas

Concentration of Circulating Anodic Antigen for Low-Resource Diagnosis of Schistosomiasis

Schistosomiasis is a water-borne parasite disease found in tropical regions, especially those with diminished access to clean water. Symptoms, including abdominal pain and chronic diarrhea, affect the ability of infected people to work or learn. Traditionally, schistosomiasis is diagnosed by a fecal smear, but this method requires trained personnel and an advanced infection. To overcome these challenges, a lateral flow assay has been developed which tests for Circulating Anodic Antigen (CAA), a negatively-charged glycoprotein produced by the parasites. This assay is easy to use and has high sensitivity, but concentrating CAA in the urine samples used would further improve it. Our objective was to use positively-charged dendrimers to concentrate CAA onto magnetic beads, which could then be deposited onto the assay. Using an ELISA, we determined the most effective conditions for capture and elution of CAA from the beads. Based on our optimizations, we chose a method which gave 75% recovery of CAA, approximately a 30-fold concentration increase. We also calculated single pg/mL limits of detection, comparable to the levels observed from a single worm pair. The method is currently being incorporated into a paper lateral flow assay which will be deliverable to endemic areas

Architectures for Dynamic Data Scaling in 2/4/8K Pipeline FFT Cores

This paper presents architectures for supporting dynamic data scaling in pipeline fast Fourier transforms (FFTs), suitable when implementing large size FFTs in applications such as digital video broadcasting and digital holographic imaging. In a pipeline FFT, data is continuously streaming and must, hence, be scaled without stalling the dataflow. We propose a hybrid floating-point scheme with tailored exponent datapath, and a co-optimized architecture between hybrid floating point and block floating point (BFP) to reduce memory requirements for 2-D signal processing. The presented co-optimization generates a higher signal-to-quantization-noise ratio and requires less memory than for instance convergent BFP. A 2048-point pipeline FFT has been fabricated in a standard-CMOS process from AMI Semiconductor (Lenart and Öwall, 2003), and a field-programmable gate array prototype integrating a 2-D FFT core in a larger design shows that the architecture is suitable for image reconstruction in digital holographic imaging