Noncanonical amino acids (ncAAs) are an exciting new tool in the synthetic biologist's toolbox. Incorporating ncAAs into proteins will allow scientists to create bacteria that can perform novel functions. Unfortunately, these ncAAs are often difficult to effectively employ due to poor characterization of the synthetase/tRNA pair, which may mean that the pair have low fidelity or low efficiency. Our project aims to create a cheap, easy-to-use universal kit that can quickly measure the fidelity and incorporation efficiency of any synthetase/tRNA pair. The kit is a simple two-plasmid system. The first plasmid contains an IPTG-inducible RFP-sfGFP reporter. The two domains are connected by a linker sequence containing either a tyrosine codon (as a control) or a recoded amber stop codon (where the ncAA will be incorporated). The other plasmid contains the synthetase/tRNA pair. After the RFP-linker-GFP protein is expressed, the fluorescence of each fluorescent domain of the fusion protein is measured and compared. Depending on the relative intensities of the RFP and GFP fluorescence under different conditions, we can determine both the fidelity of the synthetase/tRNA pair as well as the efficiency of the pair at incorporating the ncAA. We plan to equip researchers with a standardized, quick and easy “plug and play” system. Researchers will be able to insert any plasmid containing a new synthetase/tRNA pair into our Expanded Genetic Code Measurement kit to quickly characterize the synthetase/tRNA pair.
Project: Photocaged T7 RNA Polymerase
Amino acids not included in the native 20 are commonly known as noncanonical amino acids (ncAAs). These ncAAs have unique chemistry that can provide very useful functionality not normally present in an organism. For our purposes, ncAAs were used to prevent protein functionality until spatially and/or temporally triggered.
This summer our team has been working on recreating a light-activatable T7 RNA Polymerase (RNAP) for a method of noninvasive, spaciotemporal control of protein expression. T7 RNAP was our enzyme of choice for this project due to the presence of a tyrosine residue in the active site of the polymerase. By recoding this tyrosine residue, orthonitrobenzyl tyrosine was incorporated into the active site thus acting as a molecular cage. T7 RNAP is only functional when exposed to a certain wavelength of light that cleaves a molecular cage from the polymerase’s active site. In our experiments, orthonitrobenzyl tyrosine (ONBY) was used as our photocaged ncAA. ONBY was used because once the ONB group is cleaved off, the ncAA functions as a normal tyrosine. This proved to be particularly useful because T7 polymerase has a tyrosine residue in its active site that is necessary for proper function of the protein. Once decaged, the polymerase is free to transcribe sequences that are preceded by a T7 promoter. GFP was used as a reporter to analyze and optimize each construct for spaciotemporal specificity. In addition, GFP was used to examine the effect of a certain noncanonical amino acid on fluorescence when placed in the fluorophore.
Human Practices: Caffeinated Coli @ SXSW Create
In the spring of 2014, the UT iGEM team was invited to create a booth at the [http://sxsw.com/exhibitions/sxsw-create/participants-2014 SXSW Interactive Create Event.] Team members took the opportunity to educate the public on the applications and potential benefits of genetically modified organisms. In a concerted effort between past and present team members, the group banded together for the first time to create an informative, and fun presentation relating to the previous iGEM 2012 team project, the Caffeinated Coli. Our new project though went further, using caffeinated coli to calculate the amount of caffeine found in coffee, an organic beverage that is not formulaic. This allowed us to reach out to the wider Austin community and talk with them about science and synthetic biology.
"
