Team:NTU-Singapore/Wetlab/Experimental Results

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=Testing our Lysis system=
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='''Verification of Detection & Lysis system'''=
As stated before, we wish to verify that the cells with the pLsrA-Lysis system would lyse upon Ai-2 inoculation. We used the OD of the cells as a form of verification in this experiment.
As stated before, we wish to verify that the cells with the pLsrA-Lysis system would lyse upon Ai-2 inoculation. We used the OD of the cells as a form of verification in this experiment.
-
==OD600 measurement of pLsrA-Lysis-containing LuxS mutant upon addition of AI-2==
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=='''OD600 measurement of pLsrA-Lysis-containing LuxS mutant upon addition of AI-2'''==
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===Initial Phase===
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[[Image:OD time.png|thumb|center|800px|Changing OD]]<br/>
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The following picture shows when AI-2 solution is added at initial time
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[[Image:OD_vs_AI2.JPG|thumb|center|600px|Changing OD]]<br>
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Note that, cell only means there is only 200 µL cell in the well, AI-2, 2 means the AI-2 solution is collected at 2hrs.
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For this experiment, samples of LuxS mutants carrying pLsrA-lysis plasmid were introduced with supernatants containing AI-2 and the optical density was measured. As pure AI-2 are very hard to obtain, we harvested them indirectly by taking the supernatants of the wildtype bacteria after centrifuged and filtering through 0.2µm pore size filters (refer to [https://2008.igem.org/Team:NTU-Singapore/Wetlab/Materials_and_Equipment Materials and Equipment] for more details). So the supernatants that we used for testing do not contain only AI-2 but also LB broth. Therefore, for all our testing experiments, we used bacteria added with LB as negative control.  <br/>
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The results shows that with LB or water the cell culture reach higher steady state comparing to that with AI-2 solution as we can see the divergence at around 4hrs. all AI-2 solution (AI-2 3, 4, 5, 6, 8) except AI-2, 2 shows depressed steady state, indicating that, AI-2 is actually working
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We can observe that initially, the curves of cell samples with LB and AI-2 have slightly higher slopes than that of sample with cells only. This can be easily understood because the supernatants also contain considerable amounts of LB, which promotes cell growth.<br/>
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However, after about 3 hours, the cell induced with AI-2 reached steady state while the control samples were still growing. This can be well explained by the effect of lysis, which counters cell growth at the same time.
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Therefore, the cells in fact lyzed under the induction by AI-2. Or in other words, '''our detection system works as expected!!'''
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<br><br>
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<html>
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<script language=Javascript1.2>
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<!--
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===After 1 hr===
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var tags_before_clock = "<b>It is now "
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The following picture shows when AI-2 solution is added at 1hr later
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var tags_middle_clock = "on"
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[[Image:OD_vs_AI2_1hour.JPG|800px|thumb|center|Changing OD]]<br>
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var tags_after_clock  = "</b>"
-
This also shows the expected result, with AI-2 present, the cell culture shows a lower steady state.  
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if(navigator.appName == "Netscape") {
-
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document.write('<layer id="clock"></layer><br>');
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[[Image:AI2_on_LsrA-Lysis.JPG|800px|thumb|center|Bar Chart]]<br>
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}
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This error bar graph shows OD600 of each sample at different time intervals together with the standard deviation. It is clear from the graph that the variance of data is much smaller than the absolute value. Hence, the data obtained from the absorbance-meter is highly reliable.
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if (navigator.appVersion.indexOf("MSIE") != -1){
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document.write('<span id="clock"></span>');
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}
 +
 
 +
DaysofWeek = new Array()
 +
  DaysofWeek[0]="Sunday"
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  DaysofWeek[1]="Monday"
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  DaysofWeek[2]="Tuesday"
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  DaysofWeek[3]="Wednesday"
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  DaysofWeek[4]="Thursday"
 +
  DaysofWeek[5]="Friday"
 +
  DaysofWeek[6]="Saturday"
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 +
Months = new Array()
 +
  Months[0]="January"
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  Months[1]="February"
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  Months[2]="March"
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  Months[3]="April"
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  Months[4]="May"
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  Months[5]="June"
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  Months[6]="July"
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  Months[7]="August"
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  Months[8]="September"
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  Months[9]="October"
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  Months[10]="November"
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  Months[11]="December"
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 +
function upclock(){
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var dte = new Date();
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var hrs = dte.getHours();
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var min = dte.getMinutes();
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var sec = dte.getSeconds();
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var day = DaysofWeek[dte.getDay()]
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var date = dte.getDate()
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var month = Months[dte.getMonth()]
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var year = dte.getFullYear()
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var col = ":";
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var spc = " ";
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var com = ",";
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var apm;
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if (date == 1 || date == 21 || date == 31)
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  {ender = "<sup>st</sup>"}
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else
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if (date == 2 || date == 22)
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  {ender = "<sup>nd</sup>"}
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else
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if (date == 3 || date == 23)
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  {ender = "<sup>rd</sup>"}
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else
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  {ender = "<sup>th</sup>"}
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if (12 < hrs) {
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apm="<font size='-1'>pm</font>";
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hrs-=12;
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}
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else {
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apm="<font size='-1'>am</font>";
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}
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if (hrs == 0) hrs=12;
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if (hrs<=9) hrs="0"+hrs;
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if (min<=9) min="0"+min;
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if (sec<=9) sec="0"+sec;
 +
 
 +
if(navigator.appName == "Netscape") {
 +
document.clock.document.write(tags_before_clock+hrs+col+min+col+sec+apm+spc+tags_middle_clock+spc+day+com+spc+date+ender+spc+month+com+spc+year+tags_after_clock);
 +
document.clock.document.close();
 +
}
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 +
if (navigator.appVersion.indexOf("MSIE") != -1){
 +
clock.innerHTML = tags_before_clock+hrs+col+min+col+sec+apm+spc+tags_middle_clock+spc+day+com+spc+date+ender+spc+month+com+spc+year+tags_after_clock;
 +
}
 +
}
 +
 
 +
setInterval("upclock()",1000);
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//-->
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</script>
 +
</html>

Latest revision as of 23:54, 28 October 2008



Verification of Detection & Lysis system

As stated before, we wish to verify that the cells with the pLsrA-Lysis system would lyse upon Ai-2 inoculation. We used the OD of the cells as a form of verification in this experiment.

OD600 measurement of pLsrA-Lysis-containing LuxS mutant upon addition of AI-2

Changing OD

For this experiment, samples of LuxS mutants carrying pLsrA-lysis plasmid were introduced with supernatants containing AI-2 and the optical density was measured. As pure AI-2 are very hard to obtain, we harvested them indirectly by taking the supernatants of the wildtype bacteria after centrifuged and filtering through 0.2µm pore size filters (refer to Materials and Equipment for more details). So the supernatants that we used for testing do not contain only AI-2 but also LB broth. Therefore, for all our testing experiments, we used bacteria added with LB as negative control.
We can observe that initially, the curves of cell samples with LB and AI-2 have slightly higher slopes than that of sample with cells only. This can be easily understood because the supernatants also contain considerable amounts of LB, which promotes cell growth.
However, after about 3 hours, the cell induced with AI-2 reached steady state while the control samples were still growing. This can be well explained by the effect of lysis, which counters cell growth at the same time. Therefore, the cells in fact lyzed under the induction by AI-2. Or in other words, our detection system works as expected!!

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