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						<div class="heading-title"><h1 style="padding:0px">GROWTH CURVES</h1></div>
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									<p>INTRO</p>
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									<p>LB</p>
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									<p>M9</p>
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									<p>DEFINED</p>
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                        <h2>Introduction and Aims</h2>
			<p>Our proposed reporter systems required an <i>E. coli</i> strain that allows the manipulation of intracellular iron levels via <button class="btn btn-lg btn-danger" data-placement="top" data-toggle="popover" title="Siderophores" data-content="Small organic molecules produced by some bacteria and fungi capable of binding iron with extremely high affinity, these can then be taken up by the organism where they release their bound iron.">siderophore</button> uptake. We chose to use the <button class="btn btn-lg btn-danger" data-placement="top" data-toggle="popover" title="JC28" data-content="A mutant <i>E. coli</i> strain with the genotype: W3110 <i>∆fecABCDE ∆zupT ∆mntH ∆entC ∆feoABC</i>. Deficient in siderophore production.">JC28</button> strain that has an <button class="btn btn-lg btn-danger" data-placement="top" data-toggle="popover" title="<i>entC</i>" data-content="An <i>E. coli</i> gene coding for Isochorismate synthase, a key enzyme in the siderophore synthesis pathway. Knocked out in the JC28 mutant strain."><i>entC</i></button> knockout mutation, meaning that it is unable
to produce siderophores. In order to keep intracellular iron-levels of JC28 low while the strain still grows, we needed to test a range of media and varying iron concentrations. </p>

                         <p>We carried out growth curve experiments to form an understanding of the growth model of both the <button class="btn btn-lg btn-danger" data-placement="top" data-toggle="popover" title="W3110" data-content="A common lab strain of <i>E. coli</i> K12. Parent strain of the JC28 mutant type.">W3110</button>
 (<button class="btn btn-lg btn-danger" data-placement="top" data-toggle="popover" title="Wild Type" data-content="A strain, gene, or characteristic which prevails among individuals in natural conditions, as distinct from an atypical mutant type">Wild Type</button>) and JC28 (<button class="btn btn-lg btn-danger" data-placement="top" data-toggle="popover" title="Mutant" data-content="A strain, gene, or characteristic that is distinct from the typical “wild type”
">mutant</button>) <i>E. coli</i> strains. As part of this, we cultured the wild type  and mutant strains and measured the growth of the cultures both indirectly, by measuring optical density (fig. 1), and directly, by counting the number of colony forming units (<button class="btn btn-lg btn-danger" data-placement="top" data-toggle="popover" title="CFU" data-content="Colony Forming Units, a single viable cell forming a colony on an agar plate.
">CFU</button>) plated on agar plates (fig. 2).</p>

                          <p>The bacteria were grown in three different growth media; Lysogeny broth (LB), M9 minimal media and EZ defined media. This was necessary because we wanted to characterise the growth of our mutant over a range of iron conditions. It was hypothesised that the mutant would grow nearly as well as the wild type  when iron is plentiful and it would not have to rely on siderophores for iron uptake, but would be significantly limited by low-iron conditions.</p>

                           <p>The growth curves behaving as predicted would confirm that our mutant is impaired in iron acquisition, which is to be expected if the mutant is unable to produce siderophores.</p>
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                                <h2>LB liquid media</h2>
                                <p>It was hypothesised that when both strains were grown in the LB liquid media there would be very little difference in the growth curves. This was expected due to the LB media containing a sufficient source of iron, ensuring that iron availability is not a limiting factor to growth. As predicted, there is only a slight difference in the growth curves of the two strains cultured in LB liquid media.</p>

<p>1 ml from an overnight culture of <button class="btn btn-lg btn-danger" data-placement="top" data-toggle="popover" title="W3110" data-content="A common lab strain of <i>E. coli</i> K12. Parent strain of the JC28 mutant type.">W3110</button> and <button class="btn btn-lg btn-danger" data-placement="top" data-toggle="popover" title="JC28" data-content="A mutant <i>E. coli</i> strain with the genotype: W3110 <i>∆fecABCDE ∆zupT ∆mntH ∆entC ∆feoABC</i>. Deficient in siderophore production.">JC28</button> was diluted into 50 ml of fresh LB media at an <button class="btn btn-lg btn-danger" data-placement="top" data-toggle="popover" title="OD<sub>600</sub>" data-content="The optical density of a sample, which is indicative of the concentration of cells. This is determined by measuring absorbance at 600nm using a spectrophotometer.
">OD<sub>600</sub></button> of around 0.01 and incubated at 37 °C and 200 rpm. Growth kinetics were monitored taking the OD<sub>600</sub> in 30 min intervals from the time of inoculation using 1 ml samples (see Fig. 1).</p>

<p><button class="btn btn-lg btn-danger" data-placement="top" data-toggle="popover" title="CFU" data-content="Colony Forming Units, a single viable cell forming a colony on an agar plate.
">CFU</button> counts were performed from the same growth curve experiment. 1 ml samples were taken at 30 min intervals and 3x 10 µl of dilutions from 10-1 to 10-6 were spotted on a LB agar plate ad incubated at 37 °C overnight. The average CFU of these spots were taken for each dilution (Fig. 2).</p>

<p>As predicted, only slight differences in growth between both strains were observed in liquid LB media (Fig. 1 and 2). Interestingly, at the same time point (T4.5) the size of the <button class="btn btn-lg btn-danger" data-placement="top" data-toggle="popover" title="Mutant" data-content="A strain, gene, or characteristic that is distinct from the typical “wild type”
">mutant</button> JC28 colonies were significantly smaller than the <button class="btn btn-lg btn-danger" data-toggle="popover" data-placement="top" title="Wild Type" data-content="A strain, gene, or characteristic which prevails among individuals in natural conditions, as distinct from an atypical mutant type">Wild Type</button>  W3110 colonies (Fig. 3). </p>

 
				<img src="https://static.igem.org/mediawiki/2016/b/b8/T--Sheffield--Growth-graph4.png">
<p>Figure 1. Growth curves of <i>E. coli</i> W3110 (wild type) and JC28 (mutant) in liquid LB media from OD<sub>600</sub></p>
                                <img src="https://static.igem.org/mediawiki/2016/8/84/T--Sheffield--Growth-graph3.png">
<p>Figure 2. CFU counts of W3110 (wild type) and JC28 (mutant) grown in liquid LB media.</p>
                                <img src="https://static.igem.org/mediawiki/2016/d/dd/T--Sheffield--Growth-colony-size.png">
<p>Figure 3. Comparison of wild type and mutant colony sizes from T4.5 on LB agar plates after being incubated overnight at 37 °C.</p>

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                                        <h2>M9 media</h2>
					<p>We measured the growth of <button class="btn btn-lg btn-danger" data-placement="top" data-toggle="popover" title="W3110" data-content="A common lab strain of <i>E. coli</i> K12. Parent strain of the JC28 mutant type.">W3110</button> (<button class="btn btn-lg btn-danger" data-toggle="popover" title="Wild Type" data-placement="top" data-content="A strain, gene, or characteristic which prevails among individuals in natural conditions, as distinct from an atypical mutant type">Wild Type</button> ) and <button class="btn btn-lg btn-danger" data-placement="top" data-toggle="popover" title="JC28" data-content="A mutant <i>E. coli</i> strain with the genotype: W3110 <i>∆fecABCDE ∆zupT ∆mntH ∆entC ∆feoABC</i>. Deficient in siderophore production.">JC28</button> (<button class="btn btn-lg btn-danger" data-placement="top" data-toggle="popover" title="Mutant" data-content="A strain, gene, or characteristic that is distinct from the typical “wild type”
">mutant</button>) in M9 minimal media with a range of added iron concentrations. It was hypothesised that JC28 would grow at a similar rate to W3110 in the high-iron media, but be unable to grow as rapidly in low-iron media. 
					</p>
					<p>1 ml from an overnight culture of W3110 and JC28 was diluted into 50 ml of fresh M9 minimal media at an <button class="btn btn-lg btn-danger" data-placement="top" data-toggle="popover" title="OD<sub>600</sub>" data-content="The optical density of a sample, which is indicative of the concentration of cells. This is determined by measuring absorbance at 600nm using a spectrophotometer.
">OD<sub>600</sub></button> of around 0.01 and incubated at 37 °C and 200 rpm. Growth kinetics were monitored taking the OD<sub>600</sub> in 30 or 60 min intervals from the time of inoculation using 1 ml samples (Fig. 4).. 
					</p>
                                        <p>No significant growth of W3110 or JC28 was observed after 5 hours at any of the tested iron concentrations. This experiment showed that M9 media was missing some nutrients that our strains require, leading us to investigate a richer defined medium where we could still control the level of iron. </p>

					<img src="https://static.igem.org/mediawiki/2016/2/26/T--Sheffield--Growth-graph5.png">
                                        <p>Figure 4. Growth curves of <i>E. coli</i> W3110 wild type and JC28 (mutant) in liquid M9 minimal media from OD<sub>600</sub></p>

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                                <h2>Defined media</h2>
				
				<p>We investigated the growth of <button class="btn btn-lg btn-danger" data-placement="top" data-toggle="popover" title="W3110" data-content="A common lab strain of <i>E. coli</i> K12. Parent strain of the JC28 mutant type.">W3110</button> (<button class="btn btn-lg btn-danger" data-toggle="popover" data-placement="top" title="Wild Type" data-content="A strain, gene, or characteristic which prevails among individuals in natural conditions, as distinct from an atypical mutant type">Wild Type</button> ) and <button class="btn btn-lg btn-danger" data-placement="top" data-toggle="popover" title="JC28" data-content="A mutant <i>E. coli</i> strain with the genotype: W3110 <i>∆fecABCDE ∆zupT ∆mntH ∆entC ∆feoABC</i>. Deficient in siderophore production.">JC28</button> (<button class="btn btn-lg btn-danger" data-placement="top" data-toggle="popover" title="Mutant" data-content="A strain, gene, or characteristic that is distinct from the typical “wild type”
">mutant</button>) in EZ defined media with two added iron concentrations, 0 µM and 20 µM. It was predicted that JC28 would grow at a similar rate to W3110 in the high-iron media, but be unable to grow as rapidly in low-iron media. 
                                <p>1 ml from an overnight culture of W3110 and JC28 was diluted into 50 ml of fresh defined media, either with or without iron (III) chloride added to 20 µM, at an <button class="btn btn-lg btn-danger" data-placement="top" data-toggle="popover" title="OD<sub>600</sub>" data-content="The optical density of a sample, which is indicative of the concentration of cells. This is determined by measuring absorbance at 600nm using a spectrophotometer.
">OD<sub>600</sub></button> of around 0.01 and incubated at 37 °C and 200 rpm. Growth kinetics were monitored taking the OD<sub>600</sub> in 30 or 60 min intervals from the time of inoculation using 1 ml samples (Fig. 5).</p>
                                <p>The growth curves in defined media (Fig. 5) show that W3110 (wild type )  is not significantly affected by the varying iron concentrations we tested. However, JC28 (mutant) demonstrated significantly reduced growth when grown under iron-limited conditions. This shows that JC28 is deficient in iron uptake.</p>
                                <img src="https://static.igem.org/mediawiki/2016/d/d5/T--Sheffield--Growth-graph2.png" >
                                <p>Figure 5. Growth curves of <i>E. coli</i> W3110 (wild type) and JC28 (mutant) in liquid defined media from OD<sub>600</sub>, with either added iron (III) chloride to 20 µM or without.</p>
				
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