Team:Austin Texas/kit

ncAA Kit Project

Kit Introduction

In recent years the ability to expand the genetic code has been made possible by re-coding the Amber stop codon UAG via the use of modified tRNA synthetase/tRNA pairs. These synthetase/tRNA pairs act together to charge the tRNA with a non-canonical amino acid (ncAA), an amino acid that is not one of the 20 amino acids normally encoded by a codon. While the library of ncAA synthetase/tRNA pairs continues to grow, the properties of each pairing have yet to be systematically characterized using a standardized methodology. The 2014 University of Texas at Austin iGEM Team has developed a method that allows for efficient in vivo characterization, both qualitative and quantitative, of these novel synthetase/tRNA pairs.

Desired properties??? High fidelity? High efficiency of incorporation? We should indicate what we want our kit to test for.

Background

The genetic code is a composition of 20 highly conserved amino acids that are essential to all organisms on Earth. While the genetic code is specific, it is also degenerate, meaning that more than one codon can encode for the incorporation of a specific amino acid. For example, there are six serine codons and three stop codons (called amber, ochre, and opal). By recoding one of the redundant codons, the recoded codon can signal for the incorporation of a non-canonical amino acid (ncAA) rather than the codon's original usage. Of the three stop codons, the amber codon is the least abundant and thus, the easiest and most efficient to recode.

[Maybe a paragraph on the first recoding??? what amino acid tRNA synthetase and tRNA was used...? This could then transition to something from the following paragraph... but the following paragraph need to be shortened. We would rather talk more about ncAA than amberless e. coli. -dennis]

Complications arise when the genetic code is recoded. In a normal bacterium, release factor RF1 is responsible for terminating translation when the ribosome reaches the amber stop codon. To avoid termination at a UAG amber codon, a strain of E. coli was engineered by the Church and Isaacs groups using MAGE and CAGE (ref) to remove all of the amber codons from the genome and knock out the RF1 gene. The resulting strain, called "amberless" E. coli, has its amber codon free to code for any ncAA. During translation, a synthetase with mutations that allow the acceptance of a different amino acid than the wild type charges that ncAA onto a tRNA with the amber codon's anticodon, CUA, when both are present in the cell.

Experimental Design and Method

In order to recode UAG, a synthetase must be mutated to effectively grab onto an ncAA. Various methods of directed evolution are typically used to modify a synthetase such that it can accept and charge a non-canonical amino acid. The library of ncAA synthetases available have ranging levels of reported efficiency and are not well characterized. This year the UT iGEM Team created a test kit designed to characterize the efficiency of any ncAA synthetase/tRNA pair.

A total of seven ncAA were used along with Tyrosine. The full list of amino acids used in this study can be found here.

Figure 1 Schematic demonstrating the gene expression of the kit plasmids under different growth conditions. Need to revise this figure. The current text in the image, and how it is positioned is not ideal.

The kit consists of a three plasmid system: pBLG, pFRYC, and pFRY. pBLG contains the ncAA synthetase/tRNA pair to be tested as well as a gentamicin resistance gene. pFRYC is the control plasmid and contains the IPTG-induced reporter system and a kanamycin resistance gene. The reporter system is composed of RFP and sfGFP fused via a linker sequence between the two. pFRY is the experimental plasmid and is nearly identical to pFRYC with the exception that its linker sequence contains an amber stop codon in the middle of the linker whereas pFRYC contains a codon for tyrosine in the same location. In a cell containing pFRYC, the ribosome will translate the RFP reporter, linker, and finally sfGFP, producing red and green fluorescent proteins that result in visible yellow fluorescence. In a cell containing pFRY, the ribosome will translate the sfGFP and terminate at the amber stop codon on the linker producing a green fluorescence. When pBLG and pFRY are present in the cell, the ribosome will incorporate an ncAA at the amber codon in the linker and continue translation producing both RFP and sfGFP reporters if the synthetase/tRNA pair encoded on pBLG effectively incorporate the ncAA.

Figure 2. Plasmids used in the ncAA kit.KATE, FIX the "pBIG" ALSO NO STOP CODON???
pBLG contains the specific synthetase/tRNA pair being tested. pFRYC and pFRY are the ncAA kit reporter plasmids. pFRYC is the control plasmid that yields GFP and RFP expression regardless of synthetase/tRNA pair. pFRY is a nearly identical plasmid with a single difference, the linker between the GFP and RFP sequences contains an amber codon in place of the tyrosine codon found in pFRYC. Thus, the level of GFP expression for pFRY is directly dependent on the efficiency and fidelity of the synthetase/tRNA pair being tested.

An ncAA synthetase/tRNA pair was cloned into pBLG and transformed into pFRYC amberless E. coli. and pFRY amberless E. coli. Other necessary control strains include RFP amberless E. coli (RFP control), sfGFP amberless E. coli (GFP control), amberless E. coli (cell background control), and LB media supplemented with ncAA (was it supplemented?) (media background control). An overnight culture of each strain was grown in LB with the appropriate antibiotics at 37ºC and 225rpm. 10 mL of media with the appropriate antibiotics was inoculated with 100 µL of overnight culture and allowed to grow in the same conditions until the culture density was ~0.2-0.3 OD, or ~3 hours. The 10 mL culture was split between 4 different sterile test-tubes, 2 mL of culture per tube. The conditions of test tubes A through D were as follows: A (-IPTG,-ncAA), B (-IPTG,+ncAA), C (+IPTG, -ncAA), and D (+IPTG, +ncAA). IPTG stock solution was made at 1000X concentration (?) and the ncAA was added to yield a concentration of 1 mM. Sterile deionized water was added in the place of ncAA and IPTG as a control (?). Once the controls, IPTG, and the ncAA were added appropriately, the cultures were allowed to grow to ~0.5 OD. 70 µL of each culture condition and control culture was added to a separate wells in a transparent 96-well plate for fluorescence and OD readings in a microplate reader.

[ADD MORE DETAILS LATER?]

Results and Data

Discussion

Conclusion

ncAA Table

ncAA	Molecular Structure	Molecular Weight (g)	Soluble in	Other Notes
tyrosine (Canonical)		181.19	Soluble in water Heat to 70°C and vortex to dissolve Must stay warm to remain in solution	Stock Concentration: 10mM Concentration in Culture: 1mM
3-aminotyrosine		287.14	Soluble in water Heat to 70°C and vortex to dissolve	Stock Concentration: 10mM Concentration in Culture: 1mM
3-nitrotyrosine		226.2	Soluble in water Heat to 70°C and vortex to dissolve	Stock Concentration: 10mM Concentration in Culture: 1mM
3-iodotyrosine		307.09	Soluble in water Heat to 70°C and vortex to dissolve	Stock Concentration: 10mM (.122g in 10mL H20) Concentration in Culture: 1mM
L-3,4-dihydroxyphenylalanine (L-DOPA)		197.1879	Soluble in water Heat to 70°C and vortex to dissolve	Stock Concentration: 10mM Concentration in Culture: 1mM
o-(2-nitrobenzyl)tyrosine (ONBY)		316.308	Soluble in 50% DMSO in Water Heat up to 70°C and add NaOH to dissolve	Stock Concentration: 50mM Concentration in Culture: 1mM Light Sensitive
4-azidophenylalanine (AzF)		206.204	Soluble in 10% DMSO in Water Requires heating and overnight shaking to dissolve	Stock Concentration: <10mM (.02g in 10mL) Concentration in Culture: 1mM Light Sensitive
4-cyanophenylalanine (CNF)		190.2	Soluble in water Heat to 70°C and vortex to dissolve	Stock Concentration: 10mM Concentration in Culture: 1mM

ncAA Synthetase Table

References

• Seyedsayamdost, M. R., Xie, J., Chan, C. T. Y., Schultz, P. G., Stubbe, J. (2007) Site-specific insertion of 3-aminotyrosine into subunit α-2 of E . coli ribonucleotide reductase: Direct evidence for involvement of Y₇₃₀ and Y₇₃₁ in radical propagation. J. Am. Chem. Soc. 129: 15060–15071.
• Wang, L., Brock, A., Herberich, B., Schultz, P. G. (2001) Expanding the genetic code of Escherichia coli. Science 292: 498–500.
• Neumann, H., Hazen, J. L., Weinstein, J., Mehl, R. A., Chin, J. W. (2008) Genetically encoding protein oxidative damage. J. Am. Chem. Soc. 130: 4028-4033.
• Sakamoto, K., Murayama, K., Oki, K., Iraha, F., Kato-Murayama, M., Takahashi, M., Ohtake, K., Kobayashi, T., Kuramitsu, S., Shirouzu, M., Yokoyama, S. (2009) Genetic encoding of 3-iodo-L-tyrosine in Escherichia coli for single-wavelength anomalous dispersion phasing in protein crystallography. Structure. 17: 335-344.
• Alfonta, L., Zhang, Z., Uryu, S., Loo, J. A., Schultz, P. G. (2003) Site-specific incorporation of a redox-active amino acid into proteins. J. Am. Chem. Soc. 125:14662-14663.
• Deiters, A., Groff, D., Ryu, Y., Xie, J., Schultz, P. G. (2006) A genetically encoded photocaged tyrosine. Angew Chem Int Ed Engl 45:2728-2731.
• Chin, J. W., Santoro, S. W., Martin, A. B., King, D. S., Wang, L., Schultz, P. G. (2002) Addition of p-azido-L-phenylalanine to the genetic code of Escherichia coli. J. Am. Chem. Soc. 1294:9026-9027.
• Schultz, K. C., Supekova, L., Ryu, Y., Xie, J., Perera, R., Schultz, P. G. (2006) A genetically encoded infrared probe. J. Am. Chem. Soc. 129:13984-13985.