Team:Harvard/Parts/LacI

From 2008.igem.org

(Difference between revisions)
(Project Overview)
(lacI Inducible System)
Line 57: Line 57:
=lacI Inducible System=
=lacI Inducible System=
-
The metabolically versatile bacterium Shewanella oneidensis adapts to anaerobic environments by transporting electrons to its exterior, reducing a variety of environmental substrates. When grown anaerobically and provided with lactate as a carbon source, S. oneidensis transfers electrons to an electrode of a microbial fuel cell. We sought to engineer S. oneidensis to report variations in environmental conditions through changes in current production. A previous study has shown that S. oneidensis mutants deficient in the mtrB gene produce less current than the wildtype strain, and that current production in these mutants can be restored by the addition of exogenous mtrB. We attempted to control current production in mtrB knockouts by introducing mtrB on lactose, tetracycline, and heat inducible systems. These novel biosensors integrate directly with electrical circuits, paving the way for the development of automated, biological measurement and reporter systems.
+
===Lac system (will be moved to separate page)===
 +
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.
 +
 
 +
<div style="text-indent:0pt">[[Image:BBa_K098984.png|thumb|650px|center|BBa_K098984 with BioBrick Prefix and Suffix]]</div>
 +
 
 +
====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.
 +
=====Experimental Design=====
 +
[[Image:Lac.png|thumb|150px|Lac Induction Experimental Design]]<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.
 +
 
 +
=====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: [http://2008.igem.org/wiki/images/c/ce/CIts_system.pdf]
 +
 
 +
<div style="text-indent:0pt">[[Image:CIts.png|thumb|720px|center|//caption..]]</div>
 +
 
 +
old one: [[:Image:Lac.jpg|800px|Lac Inducibility Results]]<br>
 +
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.
 +
[[Image:Lac1000.jpg|800px|Lac Inducibility 1000x Results]]
-
==Experimental overview==
 
-
==Results==
 
-
==Future directions==
 
|}
|}

Revision as of 22:24, 29 October 2008



lacI Inducible System

Lac system (will be moved to separate page)

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.

Experimental Design
Lac Induction Experimental Design

Starter cultures of E. coli with 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 (BBa_K098981) or the constitutive GFP control (BBa_K098991).

PDF version: [1]

//caption..

old one: 800px|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