Difference between revisions of "Team:Exeter/Proof"

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<p id="pp">Our own growth curve was performed to determine the maximum specific growth rate of <i>E. coli </i> BL21 (DE3) in our lab, but could not be conducted for a sufficient length of time to be accurate. A maximum specific growth rate value of 1.730 was used (Cox, 2004). The ministat must be set to a flow rate at which dilution rate is less than maximum specific growth rate. This prevents the culture being washed out of the growth chambers. The dilution rate of the culture was calculated by measuring flow rate at a setting of 7.5 rpm on the peristaltic pump. For practical reasons the pump could not be run faster than this due to the amount of media needed. The dilution rate was set at 0.2 which produced cultures that grew at an average OD of 3.47 for KillerRed samples, 3.64 for KillerOrange samples and 3.17 for lysozyme samples. The ministat must be set to flow rate at which dilution rate is below the maximum specific growth rate. This prevents the culture being washed out of the chamber. OD was measured daily with a Bug Lab OD scanner. When the same sample was measured in a tecan infinite 200 pro plate reader the Bug Lab showed reading approximately three times higher. The difference between the samples was consistent regardless of the method used to measure OD.
 
<p id="pp">Our own growth curve was performed to determine the maximum specific growth rate of <i>E. coli </i> BL21 (DE3) in our lab, but could not be conducted for a sufficient length of time to be accurate. A maximum specific growth rate value of 1.730 was used (Cox, 2004). The ministat must be set to a flow rate at which dilution rate is less than maximum specific growth rate. This prevents the culture being washed out of the growth chambers. The dilution rate of the culture was calculated by measuring flow rate at a setting of 7.5 rpm on the peristaltic pump. For practical reasons the pump could not be run faster than this due to the amount of media needed. The dilution rate was set at 0.2 which produced cultures that grew at an average OD of 3.47 for KillerRed samples, 3.64 for KillerOrange samples and 3.17 for lysozyme samples. The ministat must be set to flow rate at which dilution rate is below the maximum specific growth rate. This prevents the culture being washed out of the chamber. OD was measured daily with a Bug Lab OD scanner. When the same sample was measured in a tecan infinite 200 pro plate reader the Bug Lab showed reading approximately three times higher. The difference between the samples was consistent regardless of the method used to measure OD.
 
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<h6><u>Ministat experiment</u></h6>
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<p id="pp">All samples from the ministat were tested using the KillerRed, KillerOrange protocol found <a href="#KRKOProt">
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here</a>. Glycerol stocks were made of the samples taken at each time interval, testing was done using these glycerol stocks.</p>
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<p id="pp"><b>Fig.7,9</b> Average number of colonies after 0 h, 24 h, 120 h and 168 h of continuous culture. Values were
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averaged across three biological repeats. A max value of 300 colonies is set as any plate with more than 300 colonies was
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not counted and assigned the max value. All samples were induced to a final concentration of 0.2 nM IPTG. All samples were
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diluted 1000 times in a final volume of 4.5 ml LB. Error bars represent the standard error of the mean</p>
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<p id="pp"> <b>Fig. 9,10</b> Data from Fig.7,8 represented as percentage viable cells over time. 100% viable is given
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when the CFU count for the kill switch condition equaled the control. Error bars represent the standard error of the mean.</p>
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<div class="row">
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    <div class="col-xs-6" style="padding:5px 2% 5px 10%;margin:0;">
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        <img src="https://static.igem.org/mediawiki/2016/7/78/T--Exeter--KRcont.jpg"
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style="max-width:100%;margin:auto;display:block;">
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            <span class="caption">Fig. 7. Comparison of CFUs formed by KillerRed exposed to light and kept in the dark.</span>
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<br>
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<br>
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    </div>
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    <div class="col-xs-6" style="padding:5px 10% 5px 2%;margin:0;">
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        <img src="https://static.igem.org/mediawiki/2016/9/9f/T--Exeter--KRDpercent2.jpg"
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style="max-width:100%;margin:auto;display:block;">
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            <span class="caption">Fig. 8. Percentage viable cells of KillerRed exposed to light.</span>
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<br>
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<br>
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    </div>
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</div>
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<div class="row">
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    <div class="col-xs-6" style="padding:5px 2% 5px 10%;margin:0;">
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        <img src="https://static.igem.org/mediawiki/2016/d/d0/T--Exeter--KOcont.jpg"
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style="max-width:100%;margin:auto;display:block;">
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            <span class="caption">Fig. 9. Comparison of CFUS of KillerOrange exposed to light and in the dark.</span>
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<br>
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<br>
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    </div>
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<div class="row">
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    <div class="col-xs-6" style="padding:5px 10% 5px 2%;margin:0;">
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        <img src="https://static.igem.org/mediawiki/2016/0/01/T--Exeter--KOpercent2.jpg"
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style="max-width:100%;margin:auto;display:block;">
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            <span class="caption">Fig. 10. Percentage viable cells of KillerOrange exposed to light.</span>
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<br>
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<br>
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    </div>
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</div>
  
 
</div>
 
</div>

Revision as of 12:44, 16 October 2016