Genetic Encoding of 4-Nitro-L-Phenylalanine in Eukaryotes

Maximillian Pearson Author

07/29/2021 Added

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Adding 4-Nitrophenylalanine to the genetic code of yeast.

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[00:00:00.780]Hello. My name is Max Pearson,
[00:00:02.910]and I am doing my research on genetic encoding of 4-Nitro-L-phenylalanine
[00:00:07.080]in Eukaryotes.
[00:00:10.260]Almost all organisms utilize the same genetic code consisting of 64 codons
[00:00:15.540]which encode the 20 natural amino acids and three stop codons. However,
[00:00:20.490]protein function may be improved by expanding the genetic code via the
[00:00:24.030]incorporation of unnatural amino acids. In this work,
[00:00:28.260]we seek to develop a method to incorporate the unnatural amino acid 4-Nitro-
[00:00:32.910]L-phenylalanine,
[00:00:34.290]which has potential biomedical use in triggering autoimmune responses.
[00:00:38.400]Biochemical use as a floor for quencher.
[00:00:42.210]Into
[00:00:43.140]the cells of Saccharomyces cerevisiae,
[00:00:47.880]a model Eukaryotic organism.
[00:00:50.640]We hypothesized that by mutating four active site residues of Escheria
[00:00:55.460]coli tyrosyl tRNA synthetase
[00:00:58.620]and stop codon on tRNA pare the tRNA synthetase could be reprogrammed to
[00:01:03.120]incorporate 4-Nitrophenylalanine in place of tyrosine in response to the
[00:01:07.500]amber stop codon UAG.
[00:01:10.650]This system would be orthogonal to the S cerevisiae cellular
[00:01:15.390]machinery,
[00:01:16.260]providing a reliable method of adding 4-Nitrophenylalanine to the genome
[00:01:20.070]of eukaryotes.
[00:01:23.010]To find a mutant capable of incorporating four nitro phenylalanine.
[00:01:26.670]We mutated the four codons of the active site of wild-type E coli
[00:01:30.750]tRNA synthetase via inverse PCR to randomize to
[00:01:35.580]N N K, where N is any base and K is
[00:01:38.680]cytosine or thymine, resulting in a library of approximately 10 to the sixth
[00:01:42.360]possible tRNA synthetase mutants.
[00:01:47.630]The above library was then ligated to a plasmid vector designated
[00:01:52.070]pEcYRS-lib, which also contained a gene for tryptophan synthesis,
[00:01:56.810]ampicillin resistance, and the stop codon tRNA, we also used another plasmid,
[00:02:01.790]which contained a copy of the GAL4 regulatory gene with two stop codon
[00:02:05.960]mutations included, a site for leucine synthesis, and ampicillin
[00:02:10.220]resistance.
[00:02:11.630]Both plasmids were transformed into yeast cells and the library was ready for
[00:02:15.350]screening.
[00:02:18.230]Shown here is the methodology used to select mutant tRNA synthetases which
[00:02:22.670]selectively encoded for 4-NO2F. Ten to the seventh cells
[00:02:27.650]from the library were plated on media
[00:02:29.360]lacking leucine and tryptophan to maintain both plasmids and lacking Uracil.
[00:02:33.530]Only those cells,
[00:02:35.870]which incorporate an amino acid in place of the Amber stop code on can produce
[00:02:40.340]full length GAL4, which activates the native URA3 gene for
[00:02:45.260]Uracil synthesis allowing only cells with a functional tRNA
[00:02:49.790]synthetase and tRNA pair to survive.
[00:02:54.500]The surviving clones from the
[00:02:55.880]positive selection were then amplified overnight and ten to the fourth cells
[00:03:00.490]were plated on media lacking 4-Nitrophenylalanine,
[00:03:04.180]but including Uracil and 5-FOA, a chemical,
[00:03:08.530]which gets converted to a toxic byproduct by an activated URA3 gene,
[00:03:12.860]Thus, cells
[00:03:15.750]which only incorporate 4-Nitrophenylalanine
[00:03:18.420]don't produce GAL4 and do not activate the URA3 gene and survive.
[00:03:25.230]Then the surviving colonies were selected at random and screened by growing them
[00:03:29.430]in media with and without 4-Nitrophenylalanine, out of those
[00:03:33.750]which grew better with 4-Nitrophenyl alanine were selected for quantitative
[00:03:38.070]analysis.
[00:03:40.680]The
[00:03:40.830]plasmids from four potential hits (colonies 2, 4, 5, and 13
[00:03:45.000]circled above in red) were isolated and transformed into chemically
[00:03:49.200]competent GeneHog E coli cells,
[00:03:51.540]which were plated on ampicillin 100 media and streaked to produce single
[00:03:56.190]colonies. Three colonies from each plate designated A, B,
[00:04:00.090]and C were then selected for the isolation of plasmids. Purified,
[00:04:04.470]pEcYRS plasmids from colonies 2A,
[00:04:07.350]4B, 5A, and 13A were then mixed with pGAD-pTDH3,
[00:04:12.330]which contained a gene for super folder
[00:04:16.620]glowing fluorescent protein,
[00:04:19.530]with a stop codon mutation at residue 149. Mixtures of the two plasmids
[00:04:24.390]were then transformed into chemically competent Mav203 S cerevisiae cells
[00:04:29.280]and three colonies from each transformation were selected for quantitative
[00:04:32.550]analysis. The above colonies were cultured for 24 hours,
[00:04:36.870]diluted 20 times and to media either containing or lacking their respective
[00:04:40.770]unnatural amino acid and assayed for sfGFP expression
[00:04:45.300]via the emission at 528 nanometers due to
[00:04:49.770]excitation at 485 nanometers.
[00:04:53.190]The results were then normalized to the cell density via OD values measured at
[00:04:57.930]600 nanometers.
[00:05:00.570]These results
[00:05:01.320]were compared to the incorporation of p-carboxymethyl-L-phenylalanine (CMF),
[00:05:06.120]another unnatural amino acid,
[00:05:08.100]which had been successfully incorporated into the S cerevisiae genome previously
[00:05:12.420]via its respective tRNA synthetase-tRNA pair.
[00:05:16.050]This was to act as a control to confirm the overall quantitative experimental
[00:05:19.740]protocol was carried out successfully.
[00:05:23.970]The data in figure three indicates that 4-Nitrophenylalanine was not
[00:05:27.450]successfully incorporated at a high efficiency by any one of the
[00:05:32.550]four selected mutants.
[00:05:34.500]As there was no statistical difference in sfGFP expression between the cells
[00:05:38.490]grown with or without the natural amino acid.
[00:05:42.240]The successful incorporation of CMF confirmed that the experiment was carried
[00:05:46.170]out correctly.
[00:05:48.150]The tRNA synthetase gene was then also sequenced for this for selected mutants
[00:05:52.740]and changes at the DNA and amino acid levels are shown above the lack of
[00:05:57.380]consensus mutation between the mutants indicates the selected tRNA synthetase
[00:06:01.850]mutants are false positives that bypassed the selection process.
[00:06:07.670]Future work for us includes repeating the selection process under more stringent
[00:06:11.750]conditions and testing known tRNA synthetase mutants that have
[00:06:16.370]promiscuous activity towards 4-Nitrophenylalinine.
[00:06:20.600]If 4-Nitropheynalanine
[00:06:21.470]is able to be successfully incorporated into yeast cells.
[00:06:25.040]the next step is to conduct experiments in mammalian cells for potential
[00:06:28.400]biochemical uses as a
[00:06:32.150]fluorophore quencher or biomedical uses as an autoimmune
[00:06:36.710]response trigger.
[00:06:41.640]Here's my poster. Thank you for listening. I'd like to say thank you to my PI.
[00:06:45.840]Dr. Niu, my mentoring Xinyuan,
[00:06:48.390]the University of Nebraska Lincoln and the National Science Foundation Research
[00:06:52.470]Experience for Undergrads.

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Genetic Encoding of 4-Nitro-L-Phenylalanine in Eukaryotes

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