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        <h1 class="blog-title">Flight iGEM</h1>
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            <p class="blog-post-meta"> by <a href="#">Susu Chen</a></p>
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            <p style="text-align: center;">AI-2&nbsp;Quantification</p>
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<p>&nbsp;&nbsp;&nbsp;&nbsp;E.coli&nbsp;and&nbsp;Bacillus&nbsp;sp.&nbsp;are&nbsp;the&nbsp;main&nbsp;pathogens&nbsp;in&nbsp;spoiled&nbsp;yogurt.&nbsp;Detection&nbsp;of&nbsp;these&nbsp;pathogens&nbsp;can&nbsp;be&nbsp;used&nbsp;to&nbsp;identify&nbsp;whether&nbsp;yogurt&nbsp;has&nbsp;got&nbsp;bacterial&nbsp;contamination.&nbsp;The&nbsp;traditional&nbsp;detecting&nbsp;methods&nbsp;contain&nbsp;some&nbsp;cumbersome&nbsp;steps,&nbsp;need&nbsp;some&nbsp;expensive&nbsp;reagents&nbsp;and&nbsp;still&nbsp;have&nbsp;high&nbsp;inaccuracy.&nbsp;We&nbsp;checking&nbsp;the&nbsp;references,&nbsp;we&nbsp;found&nbsp;that&nbsp;autoinducer&nbsp;2&nbsp;(AI-2),&nbsp;a&nbsp;signal&nbsp;molecule&nbsp;in&nbsp;the&nbsp;quorum&nbsp;sensing&nbsp;system&nbsp;in&nbsp;bacteria,&nbsp;can&nbsp;also&nbsp;be&nbsp;used&nbsp;as&nbsp;a&nbsp;signal&nbsp;detected&nbsp;by&nbsp;our&nbsp;engineered&nbsp;Lactobacillus.&nbsp;Thus,&nbsp;we&nbsp;wanted&nbsp;to&nbsp;measure&nbsp;AI-2&nbsp;produced&nbsp;by&nbsp;pathogens&nbsp;in&nbsp;spoiled&nbsp;yogurt&nbsp;and&nbsp;then&nbsp;analyzed&nbsp;the&nbsp;relationship&nbsp;between&nbsp;AI-2&nbsp;output&nbsp;and&nbsp;bacterial&nbsp;growth.&nbsp;</p>
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<p>The&nbsp;molecular&nbsp;structure&nbsp;of&nbsp;AI-2&nbsp;is&nbsp;somewhat&nbsp;similar&nbsp;to&nbsp;ascorbic&nbsp;acid.&nbsp;It&nbsp;was&nbsp;expected&nbsp;that&nbsp;AI-2&nbsp;would&nbsp;also&nbsp;act&nbsp;as&nbsp;a&nbsp;reducing&nbsp;agent&nbsp;and&nbsp;reduce&nbsp;Fe(III)&nbsp;ions&nbsp;in&nbsp;the&nbsp;presence&nbsp;of&nbsp;1,10-phenanthroline&nbsp;to&nbsp;form&nbsp;the&nbsp;orange-red&nbsp;colored&nbsp;[(o-phen)3&nbsp;Fe(II)]SO4&nbsp;ferroin&nbsp;complex&nbsp;that&nbsp;could&nbsp;be&nbsp;quantified&nbsp;colorimetrically.</p>
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<p>Protocol:</p>
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<p>1&nbsp;Inoculating</p>
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<p>E.coli&nbsp;and&nbsp;Bacillus&nbsp;were&nbsp;inoculated&nbsp;into&nbsp;LB&nbsp;media&nbsp;(2%,&nbsp;v/v),&nbsp;respectively,&nbsp;and&nbsp;shaken&nbsp;overnight&nbsp;at&nbsp;37&deg;C.</p>
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<p>2&nbsp;Sampling</p>
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<p>After&nbsp;incubation,&nbsp;the&nbsp;media&nbsp;were&nbsp;centrifuged&nbsp;at&nbsp;6000&nbsp;g&nbsp;for&nbsp;3&nbsp;min.&nbsp;the&nbsp;supernatant&nbsp;was&nbsp;collected&nbsp;and&nbsp;was&nbsp;filtered&nbsp;through&nbsp;a&nbsp;0.22&mu;m&nbsp;membrane</p>
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<p>3&nbsp;AI-2&nbsp;Quantification</p>
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<p>(1)&nbsp;Preparing&nbsp;the&nbsp;working&nbsp;solution:&nbsp;A&nbsp;working&nbsp;solution&nbsp;of&nbsp;10&nbsp;mM&nbsp;1,&nbsp;10-phenanthroline/3.32&nbsp;mM&nbsp;Fe&nbsp;(III)&nbsp;was&nbsp;prepared&nbsp;by&nbsp;dissolving&nbsp;0.198&nbsp;g&nbsp;of&nbsp;1,10-phenanthroline&nbsp;in&nbsp;50&nbsp;ml&nbsp;of&nbsp;deionized&nbsp;distilled&nbsp;water.&nbsp;The&nbsp;solution&nbsp;was&nbsp;adjusted&nbsp;to&nbsp;pH&nbsp;2&nbsp;using&nbsp;1M&nbsp;HCl.&nbsp;Ferric&nbsp;ammonium&nbsp;sulphate&nbsp;(0.16g)&nbsp;was&nbsp;added&nbsp;and&nbsp;the&nbsp;solution&nbsp;was&nbsp;brought&nbsp;to&nbsp;100&nbsp;ml&nbsp;using&nbsp;deionized&nbsp;distilled&nbsp;water.</p>
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<p>(2)&nbsp;For&nbsp;the&nbsp;Fe&nbsp;(III)&nbsp;ion&nbsp;reduction&nbsp;test,&nbsp;1ml&nbsp;of&nbsp;the&nbsp;cell&nbsp;free&nbsp;supernatant&nbsp;was&nbsp;mixed&nbsp;with&nbsp;1&nbsp;ml&nbsp;to&nbsp;develop&nbsp;the&nbsp;full&nbsp;color.&nbsp;The&nbsp;solution&nbsp;was&nbsp;then&nbsp;diluted&nbsp;to&nbsp;5ml&nbsp;and&nbsp;was&nbsp;filtered&nbsp;through&nbsp;a&nbsp;0.22&nbsp;&mu;m&nbsp;membrane，and&nbsp;scanned&nbsp;for&nbsp;the&nbsp;absorption&nbsp;spectrum&nbsp;against&nbsp;a&nbsp;blank&nbsp;solution&nbsp;within&nbsp;3&nbsp;min&nbsp;using&nbsp;a&nbsp;Lambda&nbsp;25&nbsp;UV/VIS&nbsp;spectrometer.&nbsp;</p>
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<p>Results</p>
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<p>&nbsp;</p>
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<p>E.coli&nbsp;and&nbsp;Bacillus&nbsp;sp.&nbsp;are&nbsp;the&nbsp;representative&nbsp;strains&nbsp;of&nbsp;Gram-negative&nbsp;and&nbsp;Gram-positive&nbsp;bacteria.&nbsp;We&nbsp;verified&nbsp;that&nbsp;both&nbsp;of&nbsp;them&nbsp;can&nbsp;produce&nbsp;AI-2&nbsp;during&nbsp;their&nbsp;growth&nbsp;process.&nbsp;Additionally,&nbsp;AI-2&nbsp;production&nbsp;by&nbsp;both&nbsp;of&nbsp;the&nbsp;two&nbsp;strains&nbsp;is&nbsp;significantly&nbsp;positive&nbsp;correlated&nbsp;with&nbsp;bacterial&nbsp;growth&nbsp;in&nbsp;the&nbsp;first&nbsp;two&nbsp;hours.&nbsp;All&nbsp;the&nbsp;results&nbsp;showed&nbsp;that&nbsp;AI-2&nbsp;could&nbsp;be&nbsp;produced&nbsp;by&nbsp;both&nbsp;Gram-negative&nbsp;and&nbsp;Gram-positive&nbsp;strains.&nbsp;Thus,&nbsp;our&nbsp;bio-detecting&nbsp;system&nbsp;might&nbsp;not&nbsp;only&nbsp;be&nbsp;used&nbsp;in&nbsp;Gram-negative&nbsp;bacteria&nbsp;detection,&nbsp;but&nbsp;also&nbsp;Gram-positive&nbsp;bacteria&nbsp;detection.</p>
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            <h4>About</h4>
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            <p>Etiam porta <em>sem malesuada magna</em> mollis euismod. Cras mattis consectetur purus sit amet fermentum. Aenean lacinia bibendum nulla sed consectetur.</p>
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Revision as of 07:42, 16 September 2015

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