Team:Harvard/Parts/LacI

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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.
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.
===Method===
===Method===
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[[Image:Lac.png|thumb|150px|Lac Induction Experimental Design]]<br>
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<div style="text-indent:0pt">[[Image:Lac.png|thumb|150px|Lac Induction Experimental Design]]</div><br>
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.
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.

Revision as of 22:31, 29 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. 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.

BBa_K098984 with BioBrick Prefix and Suffix

Inducibility Test for Lac System with GFP Reporter and Weak Promoter

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.

Method

Lac Induction Experimental Design

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.

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]).

PDF version: [1]


Lac Inducibility Results

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.

Lac Inducibility 1000x Results



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