Team:Austin Texas/kit

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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, ATC, when both are present in the cell.

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.

Figure 1

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.

Figure 2

An ncAA synthetase/tRNA pair was cloned into 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 was grown in LB with the appropriate antibiotics at 37 degrees Celsius and 225rpm. 10 milliliters of media with the appropriate antibiotics was inoculated with 100 microliter of overnight culture and allowed to grown under the same environmental conditions until the culture density was 0.2~0.3 OD, approximately 3 hours. The 10 milliliter culture was split between 4 different sterile test-tubes, 2 milliliters 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 1 mM concentration. Sterile DI water was added in the place of ncAA and IPTG as a control. Once the controls, IPTG, and ncAA were added appropriately, the cultures were allowed to grow to 0.5 OD. 70 micro liters of each culture condition and control culture was added to a separate transparent wells for fluorescence and OD readings.

[ADD MORE DETAILS LATER?]

Results and Data

Discussion

Conclusion

ncAA Table

ncAA	Molecular Weight	Melting Point	Soluble in	Other Notes
Tyrosine	181.19	343 °C	Soluble in water; heat to 70C 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 70C 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 70C and vortex to dissolve completely	Stock Concentration: 10mM; Concentration in Culture: 1mM
Iodotyrosine	307.09	210 °C	Soluble in water; heat to 70C 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 70C 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 70C 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 70C and vortex to dissolve completely	Stock Concentration: 10mM; Concentration in Culture: 1mM

ncAA Synthetases

Name of Synthetase	Parent Synthetase	Synthetase Mutations	tRNA	Synthetase Reference	Sequence confirmed?	Biobrick?
Tyrosine RS	M. jannaschii Tyrosine aaRS (Mj-TyrRS)	Wild Type	Mj-tRNA Tyr (CUA)	Wang, L., Brock, A., Herberich, B., Schultz, P.G. Science (2001).	Yes	[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1416001 Yes], 2014
Aminotyrosine RS	Mj-TyrRS	Y32Q, L65D, F108G, Q109L, D158S, L162Y	Mj-tRNA Tyr (CUA)	Seyedsayamdost, M. R., et al. JACS (2007).		No
3-Nitrotyrosine RS	Mj-TyrRS	Y32R, H70L, Q155M, D158G, I159L, L162H	Mj-tRNA Tyr (CUA)	Neumann, H., et al. JACS (2008).		No
3-Iodotyrosine RS	Mj-TyrRS	H70A, D158T, I159S, D286Y	Mj-tRNA Tyr (CUA)	Sakamoto, K., et al. Structure (2009).		No
L-Dopa RS	Mj-TyrRS	Y32L, A67S, H70N, A167Q	Mj-tRNA Tyr (CUA)	Alfonta, L., et al. JACS (2003).	Yes	[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1178000 Yes], 2013
ONBY RS	Mj-TyrRS	Y32G, L65G, F108E, D158S, L162E	Mj-tRNA Tyr (CUA)	Deiters, A., et al. Angewandte Chemie (2006).	Yes	[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1416000 Yes], 2014
Azidophenylalanine RS	Mj-TyrRS	Y32T, E107N, D158P, I159L, L162Q?	Mj-tRNA Tyr (CUA)	Chin, J.W., et al. JACS (2002).		No
Cyanophenylalanine RS	Mj-TyrRS	Y32L, L65W, F108W, Q109M, D158G, I159A	Mj-tRNA Tyr (CUA)	Schultz, K.C., et al. JACS (2006).		No

@@ Line 81: / Line 81: @@
 ==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.
+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, ATC, when both are present 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==