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 re-coding one of the redundancies, a codon can signal for the incorporation of an ncAA rather than the original canonical. The Amber codon is the least abundant stop codon of amber, ochre, and opal and thus perfect for re-coding purposes due to minimization of resulting disruptions in the cell.
Complications arise when the genetic code is re-coded. Release Factor RF1 is responsible for terminating translation when the ribosome reaches the Amber stop codon. To avoid termination at UAG, a strain of E. Coli was engineered to remove the RF1 gene. During translation, the ncAA synthetase charges an ncAA onto the tRNA. Rather than translation termination, the tRNA enters the ribosome for ncAA incorporation and translation continues. Another complication occurs in the RFO strain of E. Coli due to the presence of Amber codons on the F plasmid. The F plasmid contains essential genes to the cell. The functionality of the proteins encoded would be compromised in a genetically expanded cell. To prevent this lethality, a strain of E. Coli with the RF1 knockout was engineered to have alternative stop codons on the F plasmid known as Amberless E. Coli.
Experimental Design and Method
In order to re-code 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 synthase such that it can grab onto and charge a non-canonical. The library of ncAA synthetases available have a ranging levels of reported efficiency and are not well characterized. The This year the UT iGEM Team created a test kit designed to characterize the efficiency of an ncAA synthetase/tRNA system.
The kit consists of a three plasmid system: pBLG, pFRYC, and pFRY. pBLG contains a gentamicin resistance as well as the ncAA synthetase/tRNA pair. pFRYC is the control plasmid and contains a kanamycin resistance gene and an IPTG induced reporter system. The reporter system is composed of sfGFP connected via a linker sequence upstream of mCherry RFP. pFRY is the experimental plasmid and is similar to pFRYC however the pFRY linker sequence contains an amber stop codon whereas the pFRYC linker does not. In a cell containing pFRYC, the ribosome will translate the sfGFP reporter, linker, and finally mCherry producing a fluorescent reporter of green and red resulting in a yellow fluorescent. 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 sfGFP and mCherry reporters if the synthetase/tRNA pair encoded on pBLG effectively incorporate the ncAA.
An ncAA synthetase/tRNA pair should be cloned into the pBLG and transformed into pFPYC Amberless E. Coli. and pFPY Amberless E. Coli. Other necessary control strains include mCherry Amberless E.Coli (RFP control), sfGFP Amberless E. Coli (GFP control), Amberless E. Coli (OD 600 control), and LB with ncAA (media background). An overnight culture of each strain should be grown overnight.
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ncAA Table
ncAA
Molecular Structure
Molecular Weight
Melting Point
Soluble in?
Function
Other Notes
Amino Tyrosine
287.14
158 - 160 °C
Soluble in water; heat to 70C and vortex to dissolve completely
Row 1, Cell 6
Stock Concentration: 10mM; Concentration in Culture: 1mM
Iodotyrosine
307.09
210 °C
Soluble in water; heat to 70C and vortex to dissolve completely
Row 2, Cell 6
Stock Concentration: 10mM (.122g in 10mL H20); Concentration in Culture: 1mM
Tyrosine
181.19
343 °C
Soluble in water; heat to 70C and vortex to dissolve completely; Must stay incubated to remain in solution
Row 3, Cell 6
Stock Concentration: 10mM; Concentration in Culture: 1mM
L-DOPA
197.1879
292 °C
Soluble in water; heat to 70C and vortex to dissolve completely
Row 4, Cell 6
Stock Concentration: 10mM; Concentration in Culture: 1mM
Azidophenyl alanine (AzF)
206.204
322.53 °C
Soluble in 10% DMSO in Water; Requires heating and overnight shaking to dissolve
Row 5, Cell 6
Stock Concentration: <10mM (.02g in 10mL); Concentration in Culture: 1mM; Light Sensitive
Ortho-nitrobenzyl Tyrosine (ONBY)
316.308
row 6, cell 4
Soluble in 50% DMSO in Water; Heat up to 70C and add small amount of NaOH to dissolve
Row 5, Cell 6
Stock Concentration: 50mM; Concentration in Culture: 1mM; Light Sensitive
Nitrotyrosine
226.2
233.00 - 236.00 °C
Soluble in water; heat to 70C and vortex to dissolve completely
Row 7, Cell 6
Stock Concentration: 10mM; Concentration in Culture: 1mM
Cyanophenyl alanine
190.2
190-192 °C
Soluble in water; heat to 70C and vortex to dissolve completely
Row 8, Cell 6
Stock Concentration: 10mM; Concentration in Culture: 1mM
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