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. As the library of synthetase/tRNA pairs continue to grow for non-canonical incorporation, the characterization of each remains largely vague. Measuring the efficiency of a synthetase can be time consuming and costly when considering all that is necessary for mass spec. The University of Texas iGEM Team has developed a method that allows for in vivo, qualitative, quantitative, and affordable efficiency characterization of synthetase/tRNA pairs.

Background

The genetic code is a composition of 20 highly conserved amino acids that are essential to all organisms on Earth. While specific, the genetic code is degenerate which conveniently adds flexibility to the code. By recoding one of the redundancies, a 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 (amber, opal and ochre), the amber codon is the least abundant of the three and thus, the easiest and most efficient to recode.

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/tRNA pair much be modified 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 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 Text needs to be bigger. I can't read the names on the plasmid. Make the font in the original image bigger, if possible.

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	Melting Point	Soluble in	Other Notes
Tyrosine		181.19	343 °C	Soluble in water; heat to 70°C and vortex to dissolve completely; Must stay warm to remain in solution	Stock Concentration: 10mM; Concentration in Culture: 1mM
Amino Tyrosine		287.14	158 - 160 °C	Soluble in water; heat to 70°C and vortex to dissolve completely	Stock Concentration: 10mM; Concentration in Culture: 1mM
Nitrotyrosine		226.2	233.00 - 236.00 °C	Soluble in water; heat to 70°C and vortex to dissolve completely	Stock Concentration: 10mM; Concentration in Culture: 1mM
Iodotyrosine		307.09	210 °C	Soluble in water; heat to 70°C and vortex to dissolve completely	Stock Concentration: 10mM (.122g in 10mL H20); Concentration in Culture: 1mM
L-DOPA		197.1879	292 °C	Soluble in water; heat to 70°C and vortex to dissolve completely	Stock Concentration: 10mM; Concentration in Culture: 1mM
Ortho-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
Azidophenyl alanine (AzF)		206.204	322.53 °C	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
Cyanophenyl alanine		190.2	190-192 °C	Soluble in water; heat to 70°C and vortex to dissolve completely	Stock Concentration: 10mM; Concentration in Culture: 1mM

ncAA Synthetases

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.