Difference between revisions of "Team:Exeter/Interlab"

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<h2>Chapter 1: New Beginnings</h2>
 
<h2>Chapter 1: New Beginnings</h2>
  
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<h4>The Basics of DNA:</h4>
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<p>
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Once upon a time, there was an iGEM team from the University of Exeter.
  
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<div style="float:left; width:590px; min-height:400px">
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Determined to contribute to the field of synthetic biology, we decided to participate in the Second International Interlab Measurement Study. While determining fluorescence levels for the three Interlab devices, we hoped to gain some experience in the lab before starting our iGEM project, Ribonostics.  
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  <div class="slide"><img src="https://static.igem.org/mediawiki/2015/c/c8/Exeter_adenine.png" title="Figure 1: Adenine deoxynucleotide."></div>
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  <div class="slide"><img src="https://static.igem.org/mediawiki/2015/a/af/Exeter_cytosine.png" title="Figure 1: Cytosine deoxynucleotide."></div>
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  <div class="slide"><img src="https://static.igem.org/mediawiki/2015/d/d6/Exeter_guanine.png" title="Figure 1: Guanine deoxynucleotide."></div>
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  <div class="slide"><img src="https://static.igem.org/mediawiki/2015/9/95/Exeter_thymine.png" title="Figure 1: Thymine deoxynucleotide."></div>
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  <div class="slide"><img src="https://static.igem.org/mediawiki/2015/d/d5/Exeter_ssDNA_structure.png" title="Figure 1: Single stranded DNA structure."></div>
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We aimed to build and measure the fluorescence of three BioBrick devices:
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DNA (deoxyribonucleic acid) is a biological molecule found in all forms of life, excepting some types of viruses. DNA is known as a polymer molecule, which means that it is made up of many subunits. In DNA, these subunits are known as nucleotides/bases, of which there are four types; adenine (A), thymine (T), guanine (G), and cytosine (C). Each nucleotide in DNA has three main sections; a phosphate group, a deoxyribose sugar, and the nucleotide (either A, T, C or G). These nucleotides are joined together via phosphodiester bonds between the phosphate group of one nucleotide's phosphate group and another nucleotide's deoxyribose sugar to form a phosphate backbone, which makes up the backbone of DNA. The DNA molecule also has direction (i.e. it has a beginning and an end). The beginning is known as the 5' (five prime) end, and the end is known as the 3' (three prime). New DNA bases (nucleotides) join on to the 3' end of the existing DNA molecule. (figure 1).</br>
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</br>
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As well as nucleotides being able to join to adjacent nucleotides via phosphodiester bonds, each type of nucleotide is able to bond to another specific nucleotide perpendicular (at right angles to) the phosphate backbone via H-bonds in a process known as base pairing. In DNA, adenine (A) is able to base pair to thymine (T), and guanine (G) to cytosine (C). Nucleotides which base pair are called complementary, therefore A & T are complementary, as are G & C. In nature, DNA is rarely found as a single strand, instead it is found as a complex of two DNA strands, one wrapped around the other to give the familiar double stranded helix structure associated with DNA. Each DNA strand is anti-parallel and complementary to the other (i.e. their directions are opposite and where one strand has, for example, an A, the other will have a T). The DNA strand which is in the 5' to 3' orientation is called the sense strand, and the strand which runs from 3' to 5' is known as the antisense strand (figure 2).</br>
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DNA's primary role in cells is to store genetic information. The information stored on DNA molecules refers to the characteristics and functions of a cell, and therefore the entire organism. In multicellular organisms (e.g. animals), each cell contains identical genetic information, however the information which is used depends on the type of cell. For example, cells which make up the eyes will use information corresponding to sight and eye colour, while muscle cells will use information which corresponds to contraction and relaxation of the cells during use.</br>
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The genetic information on DNA is stored in discrete units called genes. Each gene contains information which corresponds to at least one characteristic/function of the cell (And therefore the organism), and is encoded in the language of nucleotides. The sequence of nucleotides within a gene (e.g. ATTCTGCTA) is used to produce a specific molecule (normally a protein). This process is described in more detail in the next section; 'Translation and Transcription'. (Figure 3).
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1.J23101 + I13504
  
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<h4>The Basics of RNA:</h4>
 
  
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2.J23106 + I13504
  
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RNA (ribonucleic acid) is another polymer molecule which shares some similarity with DNA. The subunits which make up RNA (called ribonucleotides/bases) are similar to those which make up DNA, however they have a few crucial differences. The first is that while both DNA and RNA bases contain a phosphate group and the nucleotide, instead of a deoxyribose sugar, ribonucleotides have a ribose sugar. In addition, in RNA there is no thymine (T) bases, instead there is another type of base called uracil (U). A and U are complementary in RNA. Excepting these differences, the basic structure of RNA is very similar to that of DNA; they both have directions (5' to 3'), and both have their subunits joined by phosphodiester bonds to form a phosphate backbone (figure 4).</br>
 
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Another difference between DNA and RNA is that RNA is often found as a single strand, as opposed to the double stranded helix of DNA. Although this may make it seem like RNA will be found as a linear molecule, it is important to realise that this is not the case; RNA can actually have more complex structures than DNA. As the RNA strand is not bound to another RNA strand, it has all of its ribonucleotides free to base pair, which they do. The ribonucleotides can bind with complementary bases on the same RNA strand, or indeed with those on other RNA strands to form an RNA-RNA complex (although it is rare that this complex will have the double helix structure of DNA). This allows RNA to have a great many structures (figure 5).</br>
 
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As many diferent structures of RNA can be formed within a cell, it is perhaps not surprising that there are many types of RNA, each with different functions within the cell. Three types of RNA are used in the process of utilising the genetic information stored on DNA, and is described in the next section; 'Transcription and Translation'. Other functions of RNA are described in the section 'The Functions of RNA'.
 
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3.J23117 + I13504
  
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  <div class="slide"><img src="https://static.igem.org/mediawiki/2015/f/fb/Exeter_RNA_self_binding.png" title="Figure 1: RNA strand binding to itself."></div>
 
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  <div class="slide"><img src="https://static.igem.org/mediawiki/2015/2/26/Exeter_RNA-RNA_complex.png" title="Figure 1: RNA-RNA complex."></div>
 
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All built into the pSB1C3 plasmid backbone.
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In the first week of our iGEM induction, we started out by hydrating DNA from the kit, after which we followed the standard protocols for BioBrick assembly. The first step was transforming our BioBrick DNA into competent DH5α cells (New England Biolabs), and growing them in overnight cultures. After transforming, we performed our first miniprep to obtain the DNA needed for further experiments, and checked the DNA concentration using Qubit. We then used values obtained from this to calculate the volumes of all of components of the digestion step. After the digestion, we ran a part of each sample on a gel to see if the insert and vector were the correct size. The ligation was the last step; after this, we plated our samples and incubated them overnight. We hoped that the next morning, there would be glowing green colonies on our plates.
  
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<h4 >The Basics of Proteins:</h4>
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Protocols of these experiments can be found on these pages of the lab book: [transformation, miniprep, Qubit, digestion, ligation]
  
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  <div class="slide"><img src="https://static.igem.org/mediawiki/2015/2/21/Exeter_AA.png" title="Figure 5: Amino acid general structure."></div>
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  <div class="slide"><img src="https://static.igem.org/mediawiki/2015/0/04/Exeter_AA_polymer.png" title="Figure 5: Section of an amino acid polymer."></div>
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  <div class="slide"><img src="https://static.igem.org/mediawiki/2015/a/a5/Exeter_protein_structure.png" title="Figure 5: Protein structure of GFP."></div>
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Proteins are another type of biological molecule, which is a polymer like DNA and RNA, however the subunits which make up proteins are known as amino acids. There are 21 types of (natural) amino acid, and all of them share a similar structure; a hydrogen group (H), a carboxylic acid group (COOH), an amino group (NH2), and a functional group (R). The functional group is different for each type of amino acid. Unlike with DNA and RNA, amino acids are no joined by phosphodiester bonds, but by amide bonds between the carboxylic acid group of one amino acid, and the amino group of another.</br>
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The next day, we came into the lab, hoping to see colonies so green they would be blinding. After viewing the plates under blue light to visualise the green fluorescent protein (GFP) in our constructs, we understood that the statement ‘biology sometimes doesn’t work’ is entirely accurate. There were no glowing colonies on our plates. However, this moment of truth inspired us to diagnose what was wrong with our devices, and give the Interlab Study another try in the following week.
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The interactions of the functional groups, both with other functional groups of the same/different proteins, and with other molecules/etc. in its environment, gives the protein its overall function (figure 6). These functions can range from catalytic speed up the rate of a reaction) to structural (shape/strength of a cell), to virulence (causing disease). As has been eluded to before, these proteins are encoded for by DNA and the production of them involves RNA. In the next section we will see how exactly this mechanism works.
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</p>
 
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</div>
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<p>
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Our lab induction and Interlab Study were a good chance to find out where the equipment and resources in the lab were, as well as to meet some of the researchers working there – many of them helped at various stages of our project. Those amongst us with little or no lab experience learned basic techniques, such as pouring agar and making LB broth, but also made an effort to understand the biological concepts behind the Interlab Study. One of our physicists, Todd, said that in the first week, he learned the skills necessary for any budding biologist – pipetting, making, and pouring agar. ‘I thought it was very useful. It was great.’ – a direct quote from Todd. Equipped with these skills, we set out to complete the Interlab Study accurately, efficiently, and most importantly – successfully.
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</p>
  
  

Revision as of 16:26, 26 August 2015

Interlab Study

Chapter 1: New Beginnings

Once upon a time, there was an iGEM team from the University of Exeter. Determined to contribute to the field of synthetic biology, we decided to participate in the Second International Interlab Measurement Study. While determining fluorescence levels for the three Interlab devices, we hoped to gain some experience in the lab before starting our iGEM project, Ribonostics. We aimed to build and measure the fluorescence of three BioBrick devices: 1.J23101 + I13504 2.J23106 + I13504 3.J23117 + I13504 All built into the pSB1C3 plasmid backbone.

In the first week of our iGEM induction, we started out by hydrating DNA from the kit, after which we followed the standard protocols for BioBrick assembly. The first step was transforming our BioBrick DNA into competent DH5α cells (New England Biolabs), and growing them in overnight cultures. After transforming, we performed our first miniprep to obtain the DNA needed for further experiments, and checked the DNA concentration using Qubit. We then used values obtained from this to calculate the volumes of all of components of the digestion step. After the digestion, we ran a part of each sample on a gel to see if the insert and vector were the correct size. The ligation was the last step; after this, we plated our samples and incubated them overnight. We hoped that the next morning, there would be glowing green colonies on our plates. Protocols of these experiments can be found on these pages of the lab book: [transformation, miniprep, Qubit, digestion, ligation]

The next day, we came into the lab, hoping to see colonies so green they would be blinding. After viewing the plates under blue light to visualise the green fluorescent protein (GFP) in our constructs, we understood that the statement ‘biology sometimes doesn’t work’ is entirely accurate. There were no glowing colonies on our plates. However, this moment of truth inspired us to diagnose what was wrong with our devices, and give the Interlab Study another try in the following week.

Our lab induction and Interlab Study were a good chance to find out where the equipment and resources in the lab were, as well as to meet some of the researchers working there – many of them helped at various stages of our project. Those amongst us with little or no lab experience learned basic techniques, such as pouring agar and making LB broth, but also made an effort to understand the biological concepts behind the Interlab Study. One of our physicists, Todd, said that in the first week, he learned the skills necessary for any budding biologist – pipetting, making, and pouring agar. ‘I thought it was very useful. It was great.’ – a direct quote from Todd. Equipped with these skills, we set out to complete the Interlab Study accurately, efficiently, and most importantly – successfully.

  • Contact us:
    exeterigem@gmail.com