Difference between revisions of "Part:BBa K606018:Experience"

 
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<p>We characterized T7 autoloop (receiver part of the construct, <a href="https://parts.igem.org/wiki/index.php/Part:BBa_K606036">BBa_K606036</a>) in E.coli, when hosted in the plasmid pSB1C3.</p>
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<p>We know that, due to stochastic leakage, some cells should express T7 RNA polymerase even without induction. Our <a href="http://2011.igem.org/Team:Paris_Bettencourt/Modeling/T7_diffusion">modeling</a> suggests that only a few polymerases are required to activate the T7 autoloop. Without any induction, we therefore <em>expected to have a few very bight cells</em> (autoloop activated) while the other remain dark or only marginally fluorescent (bit of leakage on the GFP gene only).</p>
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<p>We tried two configurations: one with a terminator before the <i>pT7</i> promoter and one without. This was to see if we could reduce leakage with one extra terminator.</p>
  
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
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{| border="1" class="wikitable" style="text-align: center;"
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how you used this part and how it worked out.
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|+T7 autoloop in E.coli at 37°C
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===Applications of BBa_K606018===
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|[[Image:T7 autoloop ctrl-25.jpg|450px|thumb|center|E.coli T7 autoloop without terminator at 37°C  (trans image)]]
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|[[Image:T7 autoloop ctrl-26.jpg|450px|thumb|center|E.coli T7 autoloop without terminator at 37°C  (gfp image)]]
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===User Reviews===
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|[[Image:T7_loop_1.jpg|450px|thumb|center|E.coli T7 autoloop with terminator  at 37°C  (trans image)]]
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|[[Image:T7 loop 1 gfp.jpg|450px|thumb|center|E.coli T7 autoloop with terminator at 37°C  (gfp image)]]
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|[[Image:T7 autoloop ctrl-10.jpg|450px|thumb|center|E.coli negative control  at 37°C  (trans image)]]
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|[[Image:T7 autoloop ctrl-11.jpg|450px|thumb|center|E.coli negative control at 37°C  (gfp image)]]
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<p>These pictures show that the T7 GFP autoloop system is efficient since some cells are glowing with GFP fluorescence. Thus, we can conclude that the T7 autoloop is activated because of stochastic leakage.</p>
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<p>Finally, we notice there is no difference between the GFP autoloop with and without terminator before the T7 promoter. It could be due to that terminator which is a <i>B.subtilis</i> terminator. Moreover, we know that this <i>E.coli</i> plasmid has 4 terminators before our construct, pretty much nullifying the effect of our extra terminator in <i>E.coli</i>.</p>
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Revision as of 12:41, 21 October 2011

We characterized T7 autoloop (receiver part of the construct, BBa_K606036) in E.coli, when hosted in the plasmid pSB1C3.

We know that, due to stochastic leakage, some cells should express T7 RNA polymerase even without induction. Our modeling suggests that only a few polymerases are required to activate the T7 autoloop. Without any induction, we therefore expected to have a few very bight cells (autoloop activated) while the other remain dark or only marginally fluorescent (bit of leakage on the GFP gene only).

We tried two configurations: one with a terminator before the pT7 promoter and one without. This was to see if we could reduce leakage with one extra terminator.

T7 autoloop in E.coli at 37°C
E.coli T7 autoloop without terminator at 37°C (trans image)
E.coli T7 autoloop without terminator at 37°C (gfp image)
E.coli T7 autoloop with terminator at 37°C (trans image)
E.coli T7 autoloop with terminator at 37°C (gfp image)
E.coli negative control at 37°C (trans image)
E.coli negative control at 37°C (gfp image)

These pictures show that the T7 GFP autoloop system is efficient since some cells are glowing with GFP fluorescence. Thus, we can conclude that the T7 autoloop is activated because of stochastic leakage.

Finally, we notice there is no difference between the GFP autoloop with and without terminator before the T7 promoter. It could be due to that terminator which is a B.subtilis terminator. Moreover, we know that this E.coli plasmid has 4 terminators before our construct, pretty much nullifying the effect of our extra terminator in E.coli.