Kate/Alex

• Add references
• expand/revise results and discussion section
• Revise intro/explanation of the kit

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.

Motivation

In order to recode UAG, a synthetase must be mutated to effectively "charge" a ncAA onto the corresponding tRNA FIGURE?. Various methods of directed evolution REFERENCE?are typically used to modify a synthetase such that it can interact with and then charge a specific ncAA. The ncAA synthetases available have ranging levels of reported efficiency and are not well characterized. Many of the ncAA are not widely used, they are published in short articles lacking full documentation, and our own unpublished experiences of working with a number of ncAAs indicate that not all ncAA synthetases are created equal. Thus, we created a standard kit designed to characterize the properties of any ncAA synthetase/tRNA pair. Our goal is to produce a standardized kit that is cheap, reproducible, easy to use, AND easily portable (i.e. you don't need a lot of advanced equipment).

A total of seven ncAAs were used in addition to tyrosine. The full list of amino acids used in this study can be found here.

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

Plasmids

Figure 1. pStG contains the specific ncAA 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 or the presence of ncAA. pFRY is a nearly identical plasmid with a single difference, the linker between the GFP and RFP sequences contains an amber codon (star) 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.

The kit consists of a three plasmid system: pStG, pFRYC, and pFRY (Figure 1).

• pStG contains the ncAA Synthetase/tRNA pair (St) to be tested as well as a Gentamicin (G) resistance gene.
• pFRYC is the control plasmid and contains an 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 reporter plasmid and is nearly identical to pFRYC with the exception that its linker sequence contains an amber codon (star in figure 1) in the middle of the linker, whereas pFRYC contains a tyrosine codon in the same location.

Using The Kit

To use the kit properly, each culture must have two of the three plasmids from above. There must always be a pStG plasmid as well as either pFRYC or pFRY. However, additional controls can be conducted using only one of the three plasmids.

Figure 2 Schematic demonstrating the gene expression of the kit plasmids under different growth conditions in the presence of IPTG.
A) pFRYC (+/-) pStG, (+/-) ncAA.
B) pFRY (-) pStG, (+/-) ncAA.
C) pFRY (+) pStG, (-) ncAA.
D) pFRY (+) pStG, (+) ncAA.
The above results represent the "perfect" tRNA synthetase/tRNA pair: one that has 100% fidelity and 100% efficiency. Actual synthetase/tRNA pairs will occasionally misincorporate an incorrect amino acid, and these pairs will not always be perfectly efficient at conducting every step in the process of reading through an amber codon.

• A) In a cell containing pStG and pFRYC, the ribosome will translate the RFP reporter, linker, and sfGFP, producing red and green fluorescent proteins that result in visible yellow fluorescence (Figure 2A). This happens because the reporter contains no amber codon, and thus does not require a ncAA synthetase/tRNA pair.

• B) If a cell contained only pFRY, without pStG, then the ribosome will translate the RFP and terminate at the amber codon on the linker producing a red fluorescence (Figure 2B). This occurs because without the pStG, the amber codon is not recoded to allow for ncAA incorporation.

• C) In a cell containing pStG and pFRY, there are multiple possible outcomes, depending on what is present in the culture. In the absence of ncAA, the ribosome will translate the RFP and terminate at the amber stop codon on the linker producing a red fluorescence (Figure 2C). This occurs because while the codon has been recoded, there is no ncAA and without ncAA there can be no incorporation at the amber codon.

• D) In a cell containing pStG and pFRY, there are multiple possible outcomes. In the PRESENCE of ncAA, the ribosome will translate the RFP, then it incorporates the ncAA at the amber codon in the linker, and finally it proceeds to translate the downstream sfGFP reporter, producing a visible yellow fluorescence (Figure 2D). This occurs because the synthetase found in pStG can covalently attach the ncAA to the tRNA gene present in pStG. This "charged tRNA" is then present during translation, allowing incorporation of the ncAA into the reporter protein.

However, the above scenarios assume that the synthetase/tRNA pair function efficiently and with high fidelity.

• Actual synthetase/tRNA pairs sometimes have low fidelity, leading to the incorporation of an incorrect amino acid. This "misincorporation" means that such a synthetase/tRNA pair would not give results that mimic Figure 2C in the absence of ncAA. Rather, in the absence of ncAA, such a pair would give results similar to Figure 2D, full expression of RFP and sfGFP. High rates of misincorporation are equivalent to low fidelity, and tRNA synthetases with low fidelity are not ideal.

• Actual synthetase/tRNA pairs can also sometimes have low efficiency. As these are artificially selected pairs, sometimes they do not function as well as natural synthetase/tRNA pairs. In such a case, the amount of sfGFP translated in Figure 2D might be significantly lower than expected. low efficiency is also not ideal as it may limit levels of protein expression.

Experimental Preparation

Protection of light-sensitive ncAAs using a foil-wrapped incubator.

An ncAA synthetase/tRNA pair was cloned into pStG 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 (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 the four test tubes were as follows:

• -IPTG,-ncAA
• -IPTG,+ncAA
• +IPTG, -ncAA
• +IPTG, +ncAA

IPTG stock solution was made at 1000X concentration (why is this here?) 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.

Preparation for the ncAA Kit Test

A picture showing culture with or without IPTG. Without IPTG, there is no induction of GFP (left). With IPTG, strong fluorescence is easily seen (right).

Results and Data

Fidelity of Incorporation

Figure 3. Graph showing the level of GFP fluorescence relative to RFP fluorescence for each condition. Each pStG plasmid is referred to based on the tRNA synthetase/tRNA pair present in the specific plasmid. Each of these plasmids was then paired with either pFRY or pFRYC and grown in the presence or absence of a specific ncAA. For example, the "3-AminoY-FRYC" and the "3-AminoY-FRY" samples both contain the 3-AminoY synthetase/tRNA pair and both samples were grown in the absence or presence of the ncAA "3-AminoY". Data are presented as the average of three independent cultures. Error bars denote standard deviation.

Set the experiment up - What do we want to test? How are we going to test it (keep it simple)? You may just need to rearrange the paragraph. The fidelity of each ncAA sythetase/tRNA pair was measured by comparing the production of GFP in cultures containing pStG/pFRY with or without ncAAs. In the absence of an ncAA only RFP should be translated, as translation is expected to terminate between RFP and GFP at the amber stop codon (UAG) on the linker sequence in pFRY. Alternatively, if the corresponding ncAA for pStG is present or if the synthetase/tRNA pair has low fidelity and can misincorporate a different amino acid, translation should continue through the UAG. In this case, RFP and GFP should both be translated. The amount of fluorescence from RFP and GFP is detectable by fluorometer.I don't think you need to mention the fluorometer here... methods We also tested pStG/pFRYC strains in (+/-) ncAA conditions as a control, control for what? for how fluorescence/growth changes with ncAA which should express RFP and GFP in all conditions since pFRYC does not have an amber stop codon in the linker (Figure 1). However, if the presence of ncAA affects cell growth or fluorescence activity, we will need these controls to determine the extent of the effect.

italics = methods Strains containing pFRY and pStG plasmids were grown in the presence and absence of the corresponding non-canonical amino acid. The fluorescence of RFP and GFP readings of each culture were recorded at a culture density of 0.5 OD 600.

• This part below is confusing. I would setup the paragraph... "to deterimine _____, the GFP values of each culture were..."

GFP values of each culture were normalized to RFP values for analysis. The normalized GFP values were compared between cultures grown in the presence of ncAA and cultures grown in the absence of ncAA. Assuming (+) ncAA cultures showed the higher GFP fluorescence, the greater difference in GFP fluorescence between (+/-) ncAA cultures, the more accurate the ncAA synthetase/tRNA pair.

• Then, you can go into this paragraph, and describe what we actually see... which synthetases work well/poorly. You will need to explicitly say what bars you are comparing and how/why you are making your assessment.

Most ncAA synthetase/tRNA pairs resulted in higher GFP fluorescence in the presence of ncAA than in the absence of ncAA, with the exception of 3-aminotyrosine. This suggests that 3-aminotyrosine synthetase/tRNA pair is the least accurate pair from the library tested.

Synthetase efficiency

Can we determine how well the synthetases incorporate the ncAA??? yes/no?

Incorporation Value

Figure 4.
May need to consider a different naming convention or the cultures. Possibly AY-C and AY-E for Control and Experimental?? The fluorescence and OD600 readings of each culture were used to calculate a value for incorporation efficiency of each synthetase. Need to add an explanation of how these values were calculated

Start by explaining the incorporation value... what does it mean? To determine the incorporation value for each synthetase/tRNA pair, ______________.

• specifically, what values did we look at and compare? NOT OD600.

Shouldn't we use "the highest incorporation values..."? The synthetase/tRNA pairs that showed the highest level of ncAA incorporation include 3-iodotyrosine, 4-azidophenylalanine, 3-nitrotyrosine, and ortho-nitrobenzyltyrosine in decreasing order Is this order correct?. Among the ncAAs tested, ONBY consistently slowed the growth rate of the culture significantly (as seen by OD600 readings, data not shown) suggesting possible toxicity to the cell. However, ncAA incorporation remained high. Synthetase/tRNA pairs that showed the lowest levels of ncAA incorporation include 3-aminotyrosine, L-DOPA, tyrosine, and 4-cyanophenylalanine.

Discussion

ADD MASS SPEC COMMENTARY Due to the nature of the ncAA Kit Test, in vivo influences will affect certain data and results. This knowledge is very useful by providing a broader and biological perspective of in vivo perspective of cellular health.

In general, the presence of ncAAs slowed the growth of amberless E. coli. Among the library of tested non-cannonicals, ONBY and 3-iodotyrosine slowed growth the most, especially ONBY. For consistency, cultures grown with ONBY required nearly twice the amount of time before reaching the appropriate OD 600 for measurement. Despite these inhibitions, the ONBY synthetase/tRNA pair proved to be one of the most efficient pairs tested.

Influences that resulted from ncAA addition were accounted for and should be noted. These include: light-sensitivity, oxidation, and interference with fluorescence readings. Certain ncAAs were protected from light due to their light-sensitive molecular structures such as ONBY and AzF. These ncAAs were prepared in a dark room and wrapped in foil. All cultures were grown in a foil-wrapped incubator for consistency. Some ncAAs are more prone to oxidation specifically as L-dopa. When oxidized, the solution turns black. To prevent oxidation, each ncAA was prepared the day of the test for consistency. By this method, the oxidation of L-dopa did not occur quickly enough to have an affect on the data. Most non-canonical solutions were transparent once prepared with the exception of 3-nitrotyrosine. The yellow-orange tint of 3-nitrotyrosine solution was accounted for by measuring the fluorescence of 1mM 3-nitrotyrosine in media and subtracting any possible background fluorescence from culture fluorescence grown in 1mM 3-nitrotyrosine. With these provisions, the ncAA Test Kit data is unbiased.

Conclusion

This year the UT Austin iGEM Team has chosen to take part in the Measurement Track of the iGEM competition by contributing to ncAA methods of analysis. Our team has designed a unique testing kit for ncAA incorporation in vivo using common reporter parts to characterize very uncommon synthetase/tRNA machinery. This method of testing was designed to be undergraduate administrable and more affordable that other methods of characterization and analysis.

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.