Team:Austin Texas

From 2014.igem.org

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<h1>Project: Expanded Genetic Code Measurement Kit</h1>
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<h1>Project: Expanded Genetic Code Measurement Kit</h1><div align=justify>
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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. '''With this 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.'''</div>
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Noncanonical amino acids (ncAAs) are an exciting new tool in the biological researchers toolbox. Incorporating ncAAs into proteins will allow scientists to create bacteria that can perform novel or new functions. The amberless E. coli used in this kit have had all the amber stop codons in its genome recoded and removed to allow the amber codon to be used for ncAAs. Unfortunately, these noncanonical amino acids are often difficult to incorporate into proteins due to a low fidelity of the synthetase/tRNA pair. Our project aims to create a kit that can measure the fidelity of the synthetase/tRNA pair and incorporation of several different noncanonical amino acids into fluorescent proteins. The kit is a simple two-plasmid system. The first plasmid contains an IPTG-inducible RFP, followed by a linker sequence containing a recoded amber stop codon (where the ncAA will be incorporated) and sfGFP. The other plasmid contains the synthetase/tRNA pair. When the RFP-linker-GFP protein is induced and exposed to the excitation wavelengths for RFP and GFP, the fluorescence of both parts of the fusion protein can be measured and compared. Depending on the relative intensities of the RFP and GFP fluorescent proteins, we can determine how efficient the synthetase/tRNA pairs are at incorporating the ncAA. We plan to equip researchers with a quick and easy “plug and play” system that contains easily interchangeable parts. Researchers will be able to insert any plasmid containing a new synthetase/tRNA pair into our pre-made cells to test the fidelity and incorporation of various ncAAs.
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| [[Image:Austin_Texas_Photocage_Card.png|link=Team:Austin_Texas/photocage]]
| [[Image:Austin_Texas_Photocage_Card.png|link=Team:Austin_Texas/photocage]]
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| <h1>Project: Photocaged T7 RNA Polymerase</h1>
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| <h1>Project: Photocaged T7 RNA Polymerase</h1><div align=justify>
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Noncanonical amino acids (ncAAs) may have unique chemical groups that can provide very useful functionality not normally present in an organism. This summer we used a photocaged ncAA, ortho-nitrobenzyl tyrosine (ONBY), to prevent protein functionality until spatially and/or temporally triggered by light. '''To this end, our team successfully re­created a light-­activatable T7 RNA Polymerase (RNAP) for a method of non­invasive, spacio­temporal control of protein expression.''' We chose T7 RNAP because there is a tyrosine residue in the active site of the polymerase. By changing this codon to an amber codon, we were able to incorporate (ONBY), a photocaged tyrosine, into the active site. The presence of the extra ONB group inhibits T7 RNAP activity.  When exposed to 365 nm light, the molecular cage (the ONB group) is released, leaving tyrosine. Thus, T7 RNAP is only functional when exposed to 365 light, which restores the native T7 polymerase active site that is necessary for proper function of the enzyme. To test this system, we used a GFP reporter with a T7 promoter. '''We were able to see a significant increase in GFP expression upon photodecaging T7 RNAP, as expected.'''</div>
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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.
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This summer our team has been working on re­creating a light­activatable 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.
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| [[file:caffeinecolisxsw.png|200px| link=https://2014.igem.org/Team:Austin_Texas/human_practices]]
| [[file:caffeinecolisxsw.png|200px| link=https://2014.igem.org/Team:Austin_Texas/human_practices]]
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|<h1>Human Practices: Caffeinated Coli @ SXSW Create</h1>
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|<h1>Human Practices: Caffeinated Coli @ SXSW Create</h1><div align=justify>
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In the spring of 2014, the UT iGEM team was invited to create a booth at the 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 gang banded together for the first time to create an informative, and fun presentation on the previous iGEM 2012 team project, the [[link:https://2012.igem.org/Team:Austin_Texas/Caffeinated_coli| ''Caffeinated coli.'']]
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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, [https://2012.igem.org/Team:Austin_Texas/Caffeinated_coli| ''Caffeinated Coli.'']  '''Our new project also went further scientifically, 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 by using something that many of them consume on a daily basis.</div>
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| rowspan="2" |  [[Image:UT_Austin_CSSB.png|link=http://cssb.utexas.edu]]
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| rowspan="2" |  [[Image:UT_Austin_BEACON.png|180px|link=http://beacon-center.org]]
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| [[Image:UT_Austin_ICMB.png|link=http://icmb.utexas.edu|400px]]
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| [[Image:UT_Austin_MBS.png|link=http://molecularbiosci.utexas.edu|400px]]
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Latest revision as of 03:55, 18 October 2014