Team:Harvard/Parts/LacI

From 2008.igem.org

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=lacI Inducible System=
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=LacI Inducible System=
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In this system, the lac repressor (LacI) is controlled by a strong constitutive promoter, and is upstream of mtrB under the control of pLac, a LacI regulated promoter. In the default state, LacI is expressed, and inhibits transcription at pLac. Thus, in the default state, mtrB is not expressed. IPTG (an analog of allolactose) induces mtrB expression by binding to LacI, thereby preventing it from inhibiting transcription at pLac.
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In this system, the lac repressor (LacI) is controlled by a strong constitutive promoter, and is upstream of mtrB under the control of pLac, a LacI regulated promoter. In the default state, LacI is expressed, and inhibits transcription at pLac. This should allow us to control the expression of mtrB. In the default state, mtrB is not expressed. IPTG (an analog of allolactose) induces mtrB expression by binding to LacI, thereby preventing it from inhibiting transcription at pLac.
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<div style="text-indent:0pt;color:black">[[Image:BBa_K098984.png|thumb|650px|center|BBa_K098984 with BioBrick Prefix and Suffix]]</div>
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<div style="text-indent:0pt;color:black">[[Image:BBa_K098984.png|thumb|650px|center|[http://partsregistry.org/Part:BBa_K098984 BBa_K098984] with BioBrick Prefix and Suffix, an example of the circuity we engineered. [http://partsregistry.org/Part:BBa_K098983 BBa_K098983] is similar, with a weaker promoter driving lacI expression.]]</div>
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==Inducibility Test for Lac System with GFP Reporter and Weak Promoter==
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We didn't have enough time to finish thoroughly testing the above system, as the cloning of mtrB (toxic in ''E. coli'') was rather difficult. Our preliminary findings are quite interesting, though, so check out our [[Team:Harvard/Project| Project page]].
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An induction test was designed to test the inducibility of the lac system by IPTG when the repressor (LacI) is driven by a weak promoter.
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==Induction Test for LacI System with GFP==
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In preparing to make the lacI system for mtrB, we tested the IPTG inducibility of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098982 BBa_K098982] in ''E. coli''. In this system, the repressor is driven by a weak promoter.
===Method===
===Method===
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<div style="text-indent:0pt;color:black">[[Image:Lac.png|thumb|150px|Click for diagram of induction experimental method]]</div><br>
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Starter cultures of E. coli with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098982| BBa_K098982] were grown overnight.  They were then diluted and grown to OD 0.2 before separation into induced (+IPTG, 1mM) and uninduced cultures (-IPTG).  OD and GFP readings were taken at time 0, 2, and 4 hours.  Additionally, after diluting T=2hrs samples to OD 0.2 for accurate GFP measurements, samples were further diluted 1000x, induced (or not induced) again, and placed back in the incubator until the end of the experiment, when OD and GFP readings were taken.
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Our test method is diagrammed below.
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<div style="text-indent:0pt;color:black">[[Image:Lac.png|thumb|800px|center|]]</div><br>
===Results===
===Results===
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Induction of GFP expression was observed at both 2 and 4 hours after adding IPTG.  Levels of GFP expression in uninduced samples, however, remained relatively the same throughout the 4 hours.  Meanwhile, IPTG induction was not observed in either the negative control ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K098981| BBa_K098981]) or the constitutive GFP control ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K098991| BBa_K098991]).
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Induction of GFP expression was observed at both 2 and 4 hours after adding IPTG.  Levels of GFP expression in uninduced samples, however, remained relatively the same throughout the 4 hours.  Meanwhile, IPTG induction was not observed in either the negative control ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K098981 BBa_K098981]) or the constitutive GFP control ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K098991 BBa_K098991]). Note that in the graph below, it appears that such an induction method is not reliable at 6 hours.
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<div style="text-indent:0pt;color:black">[[Image:Baseline-corrected_of_lac_system.png|720px|thumb|center|IPTG successfully induces higher levels of GFP expression in cells containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098982 BBa_K098982]<br>This data is also available in [https://static.igem.org/mediawiki/2008/f/fa/Baseline-corrected_of_lac_system.pdf PDF format].]]</div>
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PDF version: [https://static.igem.org/mediawiki/2008/c/ce/CIts_system.pdf]
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Additionally, it appears that even the uninduced state of such a system has significant expression of GFP. Tighter control may require higher levels of lacI expression. This leakiness may explain the difficulty we had in cloning mtrB (a gene toxic to ''E. coli'').
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<div style="text-indent:0pt;color:black">[[Image:Lac.jpg|650px|thumb|center|IPTG induction raw data]]</div>
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<div style="text-indent:0pt;color:black">[[Image:Lac.jpg|720px|thumb|center|Lac Inducibility Results]]</div>
 
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The 1000x dilutions of samples at 2 hours after induction produced similar results after 5-6 hours.  These cells are presumably at a different growth phase from cells that have been growing at much higher concentration for longer, and are thus induced in different conditions.
 
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<div style="text-indent:0pt;color:black">[[Image:Lac1000.jpg|720px|thumb|center|Lac Inducibility 1000x Results]]
 
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Latest revision as of 04:53, 30 October 2008



LacI Inducible System

In this system, the lac repressor (LacI) is controlled by a strong constitutive promoter, and is upstream of mtrB under the control of pLac, a LacI regulated promoter. In the default state, LacI is expressed, and inhibits transcription at pLac. This should allow us to control the expression of mtrB. In the default state, mtrB is not expressed. IPTG (an analog of allolactose) induces mtrB expression by binding to LacI, thereby preventing it from inhibiting transcription at pLac.

[http://partsregistry.org/Part:BBa_K098984 BBa_K098984] with BioBrick Prefix and Suffix, an example of the circuity we engineered. [http://partsregistry.org/Part:BBa_K098983 BBa_K098983] is similar, with a weaker promoter driving lacI expression.

We didn't have enough time to finish thoroughly testing the above system, as the cloning of mtrB (toxic in E. coli) was rather difficult. Our preliminary findings are quite interesting, though, so check out our Project page.

Induction Test for LacI System with GFP

In preparing to make the lacI system for mtrB, we tested the IPTG inducibility of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098982 BBa_K098982] in E. coli. In this system, the repressor is driven by a weak promoter.

Method

Our test method is diagrammed below.

Lac.png

Results

Induction of GFP expression was observed at both 2 and 4 hours after adding IPTG. Levels of GFP expression in uninduced samples, however, remained relatively the same throughout the 4 hours. Meanwhile, IPTG induction was not observed in either the negative control ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K098981 BBa_K098981]) or the constitutive GFP control ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K098991 BBa_K098991]). Note that in the graph below, it appears that such an induction method is not reliable at 6 hours.


IPTG successfully induces higher levels of GFP expression in cells containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098982 BBa_K098982]
This data is also available in PDF format.


Additionally, it appears that even the uninduced state of such a system has significant expression of GFP. Tighter control may require higher levels of lacI expression. This leakiness may explain the difficulty we had in cloning mtrB (a gene toxic to E. coli).

IPTG induction raw data




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