Louie

Hi I'm Louie! I am 15 an d have lived in London, England for my whole life but all my family is here so this is where I spend my summers. I have a cat called Slinky who stays in London and two brothers who (sadly) do not. Biology has always been something that I have loved. From a young age I wanted to be a naturalist but since I have been introduced to more human biology in school that has been my biggest interest. What I enjoy most about Biology is that there is always another layer of complexity beneath what you are learning about to try and understand and each one makes it all make more sense. In school I often find (especially as we have to stick to a rather dry syllabus for our GCSE's in England) that we only really scratch the surface of each topic which is why I am so excited to come to camp and hopefully go a bit further! As well as Biology, I also love Ballet (both doing and watching it) and acting. I have also recently been taking part in a lot of poetry workshops at my school which I am still unsure about whether I enjoy! In the future I hope to do research in either oncology, immunology or virology but I also find genetics and epigenetics fascinating, having just finished reading The Epigenetics Revolution, so I would definitely like to incorporate some of that into what I do. I'm really looking forward to starting camp on Monday!! :D



**__Unnatural Amino Acids __**

There are 20 naturally occurring amino acids which have a limited number of functions. If we could create more amino acids, either through the use of nonsense stop codons or quadruplet codons, we could increase the productivity of cells, as well as adding entirely new functionalities to amino acids. By using the 4 base codons alone, we could incorporate another 200 unnatural amino acids into the genetic code.

 Amino acids are often considered the most important building block of life despite not being the only aspect essential to it. They make up proteins which make up everything from our hair to our heart and are what really make us living beings. Amino acids are present in every cell and make up nearly every component of the cell. Altering one aspect of an amino acid’s genetic make-up could be catastrophic but, with improving tools and knowledge, we can harness the structure and efficient mechanisms that life has evolved over billions of years to create entirely new amino acids with new and exciting functions. This can help us to better understand the functions of cells and possibly even direct us towards more efficient therapies. Amino acids are made up of 3 main parts: The amino group, the carboxyl group and a side chain which is what causes variation which are all based around a central carbon. The 20 major amino acids found in living organisms are usually split up into 4 groups based on the properties of the variable ‘R’ group. Amino acids can be acidic (polar), basic (polar), positively charged or negatively charged but they can be classified in many other ways as well. The 20 most commonly studied amino acids are not the only ones. There are also many ‘minor’ amino acids that are simply slight alterations of major ones. There are also around 5 unnatural amino acids found in the human body. To alter the structure of amino acids, all that is needed is to alter the structure of the R group. Amino acids are synthesised through a process called Strecker synthesis. This creates an alpha-amino acid from ammonia (the amine precursor), cyanide (the carboxyl precursor), and an aldehyde. Acid or base catalysis can then take place to hydrolyze the then amino nitrile and form an amino acid.
 * Amino acids **

Stop codons are the codons that come at the end of every gene and are what stops mRNA synthesis. There are 3 of them in total and none of them code for an amino acid so they offer exciting new prospects in synthetic biology. Harnessing the uselessness of stop codons has been a new and popular area of research because they can be manipulated so easily without affecting the test subject. One of the most well-known areas of work has been in replacing one stop codon with another throughout an organism through the CAGE or MAGE methods.   This technique can make an organism resistant to viral infection because the stop codons that the virus is used to when replicating in a host cell would be changed and so the proteins of the virus would be made ineffective. Another useful application for stop codons, and the one that I am focused on is the attachment of an amino acid to the stop codon which could then be placed into genes to alter protein structure or serve functions that I will address.
 * Stop codon use **

The amber codon was first successfully used to code for an artificial amino acid in E coli in 1998 by Furter. It took many modifications and positive and negative selection to reach the desired outcome, making sure that the AARS is specific to the amino acid (using site-directed mutagenesis and other selective processes) but there are now over 100 UAAs across a wide range of organisms. One example was the use of the amber stop suppressor from E coli being placed into yeast. This prevented the crossing of the orthogonal pair with the native tRNA/ tRNA aminoacylase pair. The amber suppressor is the tRNA that binds to the amber stop codon and so is its anticodon. It usually carries tyrosine so a mutation library of aminoacylases can be built from tyrosine aminoayclase. The most efficient aminoacylase can then be paired with the tRNA and the AARS, which attaches the artificial amino acid onto the tRNA. Many different AARS and tRNA/ tRNA aminoacylase pairs can be created  to form new UAAs.


 * Quadruplet codons **

Another method that scientists have experimented with is creating codons which are 4 letters long instead of the natural 3. If mastered, this would dramatically expand the genetic code, adding a potential of 200 new amino acids that could serve a variety of purposes. Using quadruplet codons allows us to start with a completely blank canvas, rather than trying to work with what we have when it comes to the stop codons. The process could also be quicker and more efficient because you could potentially have more than one artificial amino acid being produced at once. There are downsides however. Unlike using the stop codon, orthogonal ribosomes are required to prevent the reading of 3 letter combinations within the 4 letter codons which is what could happen if we were to try carrying out the process in a natural ribosome. The amino acids cannot easily be incorporated into a regular protein because orthogonal ribosomes do not recognise natural, 3 letter codons other than the amber stop codon which is what the ribosome codes the amino acid in response to. There are still many functions for these amino acids because artificial peptides can be formed to have specific functions from strings of 4 letter codons and, in future, the technology could be improved to incorporate them into the genetic code itself.



To create an amino acid for a codon made up of 4 letters a certain type of ribosome is needed. The ribosomes used are called orthogonal ribosomes and detect only certain types of heterologous mRNAs, not the ones coming from the cell itself. The ribosome found to be the most productive in this process is the Ribo-Q orthogonal ribosome, which was created by a process of artificial evolution by saturation mutagenesis. This means that researchers created all possible mutations within a small stretch of DNA. In this case the 16s rRNA of Ribo-X was used as the starting point because it had already been evolved to efficiently decode the amber stop codon which is what is necessary in quadruplet decoding and they created the most efficient ribosome possible through the use of the reporter construct AAGA. The resulting orthogonal ribosome was of a similar efficiency to the natural triplet-decoding ribosomes. It included two main mutations: A1196G and A1197G. These enabled the ribosome to more efficiently accept and translate tRNA with a 4-letter anticodon. <span style="font-family: 'Times New Roman',serif;">For amino acid synthesis to occur in these orthogonal ribosomes the orthogonal set is also required. This is composed of the tRNA, the aminoacyl-tRNA synthetase and the codon itself. In this case, the tRNA/ tRNA aminoacyl pair for lysine from the archaebacterium P. //horikoshii// can be (and has been) used. The pair is only in contact with lysine at two points: Glu 41 and Tyr 268 so these could simply be mutated to stop incorporating lysine and instead take up an artificial amino acid and transport it to the orthogonal ribosome to be added in response to orthogonal mRNAs or a codon inserted into the organism on a plasmid. This has been done with the codon AGGA, which was placed in position 24 on the gene for myoglobin in yeast and coded successfully for the required amino acid: homoglutamine.

<span style="font-family: 'Times New Roman',serif;">There are many ways that scientists hope to harness unnatural amino acids in the future. These vary from posttranslational modifications to spectroscopic probes and, although the technology is still very new, is certainly a viable option when considering certain questions faced by the scientific community. __<span style="font-family: 'Times New Roman',serif;">Posttranslational modifications __ <span style="font-family: 'Times New Roman',serif;">Artificial amino acids can be incorporated into proteins following translation. This has been successfully tried in E coli where they inserted the unnatural amino acid 3-axidotyrosine using the amber stop codon and the tRNA/ mutated tyrosyl-tRNA synthetase pair. With the unnatural amino acid placed at certain points, modifications could take place. __<span style="background-color: #ffffff; font-family: 'Times New Roman',serif;">Photo reactive probes __ <span style="background-color: #ffffff; font-family: 'Times New Roman',serif;">Amino acids can now be used to regulate cellular activity and the activity of individual proteins and pathways within that cell. This has been done in various different ways. Photodynamic regulation of proteins such as the proapoptotic cysteine protease, caspase 3, has been achieved through the site-specific incorporation of photocaged amino acids (o-nitrobenzyl cysteine in this case). This has also been done with the Pho4 transcription factor in yeast, which had its phosphorylation blocked by a photocaged serine. Irradiation with blue light uncaged the serine and allowed the phosphorylated Pho4 to be followed by way of its nuclear export. Proteins can now also be cross-linked so they can be linked to their interacting pair within the cell /and can be cleaved in the presence of light. O- nitrophenylaline can site-specifically cleave a protein through a light-induced mechanism. __<span style="font-family: 'Times New Roman',serif;">Protein evolution __ <span style="color: #403838; font-family: 'Times New Roman',serif;">Unnatural amino acids have been found to actually increase the productivity of proteins in some situations, in particular in the example of CDR3H (complementary determiningregion3heavy chain). This is an antibody and, in in vitro selection, the strains containing boronate or sulfotyrosine actually performed better in their respective tasks. These amino acids could incorporate whole new sites and aspects into cells that could increase both efficiency and functionality. __<span style="font-family: 'Times New Roman',serif;">Metal Chelation __ <span style="font-family: 'Times New Roman',serif;">Amino acids can be altered to chelate metals. These could be used in a new generation of metallaproteins (proteins that can bind to metal ions) which could be used in chelation therapy and in transportation and storage of metals such as copper. One process that has been successfully attempted was to introduce a Cu2+ -chelating amino acid that could site specifically cleave DNA.
 * <span style="font-family: 'Times New Roman',serif;">Functions and applications for unnatural amino acids **

<span style="font-family: 'Times New Roman',serif;">There is an ongoing effort among the scientific community to continue the genetic code expansion. This requires both new amino acids and new orthogonal sets to allow synthesis to occur. Work is also being done to try and extend this expansion beyond E coli and other simple organisms into C Elegans and eventually even mice.
 * <span style="font-family: 'Times New Roman',serif; line-height: 1.5;">Where next? **

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[|http://www2.chemistry.msu.edu/faculty/reusch/virttxtjml/proteins.htm#aacd5]

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