Team:Lethbridge/ACK Production

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<li><a href="https://2012.igem.org/Team:Lethbridge"><img src="http://reamsbottom.net/igem/images/homeicon.png"/></a></li>
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<li><a class="active" href="https://2012.igem.org/Team:Lethbridge/projectoverview">The Project</a></li>
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<li><a href="https://2012.igem.org/Team:Lethbridge/projectoverview">The Project</a></li>
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<li><a href="#">Results</a></li>
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<li><a class="active" href="#">Results</a></li>
<li><a href="https://2012.igem.org/Team:Lethbridge/notebook">Notebook</a></li>
<li><a href="https://2012.igem.org/Team:Lethbridge/notebook">Notebook</a></li>
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<h2 id="pagetitle">Results</h2>
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<a href="https://2012.igem.org/Team:Lethbridge/Acetic Acid Production">Acetic Acid Production</a>
<a href="https://2012.igem.org/Team:Lethbridge/Acetic Acid Production">Acetic Acid Production</a>
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<a href="https://2012.igem.org/Team:Lethbridge/ACK Production">ACK Production</a>
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<a href="https://2012.igem.org/Team:Lethbridge/Overexpression of ACK">ACK Production</a>
<a href="https://2012.igem.org/Team:Lethbridge/Cyanobacteria Growth">Cyanobacteria Growth</a>
<a href="https://2012.igem.org/Team:Lethbridge/Cyanobacteria Growth">Cyanobacteria Growth</a>
<a href="https://2012.igem.org/Team:Lethbridge/InvA and GalU">InvA and GalU</a>
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<h2 class="pagetitle">ACK Production (BBa_K901015)</h2>
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<h2 class="pagetitle">Overexpression of ACK (BBa_K901015)</h2>
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<p>Increasing global oil demands require new, innovative technologies for the extraction of unconventional oil sources such as those found in Alberta’s Carbonate Triangle. Carbonate oil deposits account for almost 50% of the world’s oil reserves and approximately 26% of the bitumen found in Alberta 1. Due to unstable oil prices in Western Canada, these vast reserves have historically been set aside in favour of less time consuming, more economical sites. Microbial enhanced oil recovery (MEOR) has been utilized across the world to increase the productivity of difficult resources including carbonate oil deposits. Using a synthetic biology approach, we have designed the CAB (CO2, acetic acid, and biosurfactant) extraction method that demonstrates a modified MEOR method for extracting carbonate oil deposits. CAB extraction will utilize the natural carbon fixation machinery in the cyanobacteria Synechococcus elongatus to convert CO2 into sugars to fuel acetic acid and biosurfactant production in Escherichia coli. Acetic acid applied to carbonate rock increases the pore sizes and allows for enhanced oil recovery. The reaction produces gases that will help pressurize the well site to facilitate extraction. The natural biosurfactant rhamnolipid will also be applied to the carbonate rock to further enhance extraction yields.</p>  
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<p>Overview</p>
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<p>To test whether the construct for the acetogenesis protein ACK was working as expected, we tested the overexpression of ACK in E. coli after induction by IPTG.</p><br>
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<p>Experimental Setup</p>
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<p>Overnight cultures of E. coli were used to inoculate fresh LB media to a starting optical density (OD) of 0.1. At an OD600 of 0.6, the expression of the constructs was induced by the addition of 1 mM IPTG. After induction, 1 OD600 of cells were taken at 0.5, 1, 2, and 3 h for analysis by SDS-PAGE.</p><br>
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<p>Results</p>
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<p>The expected size of ACK is 43 kDa. The samples from the overexpression were analyzed by 12% SDS-PAGE (Fig. 1). When compared to the protein molecular weight marker, there is a band that appears around the 45 kDa mark. The protein EF-Tu is of a similar size and could be contributing to the band seen at this size. However, there appears to be an increase in intensity when comparing the sample from 0 h after induction to 3 h after induction. This suggests that ACK was overexpressed and the expression of the construct is working as expected.</p>
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<p><img src="https://static.igem.org/mediawiki/2012/0/03/Ack.JPG"></p>
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<p>Figure 1. 12% SDS-PAGE analysis of overexpression of ACK by E. coli. Lane numbers indicate time in hours after induction with IPTG; L indicates ladder. The black arrow indicates the band showing overexpression of ACK.
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<p>By coupling carbon capture with acetic acid and biosurfactant production, carbonate oil deposits can be mined with reduced greenhouse gas emissions. The use of carbon fixation to feed downstream systems can be tailored for use as a module in many applications requiring inexpensive methods for fueling biological systems. CAB extraction will be suitable for large-scale bioreactors, providing an alternative, inexpensive, and environmentally sustainable method for MEOR from Alberta’s oil deposits. Furthermore, developing the carbon capture module will be of interest in oil extraction strategies using steam, as it will help with the mitigation of CO2 release caused by steam production using for example natural gas. </p>
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Latest revision as of 03:46, 4 October 2012

2012 iGEM - University of Lethbridge

Results

Overexpression of ACK (BBa_K901015)

Overview

To test whether the construct for the acetogenesis protein ACK was working as expected, we tested the overexpression of ACK in E. coli after induction by IPTG.


Experimental Setup

Overnight cultures of E. coli were used to inoculate fresh LB media to a starting optical density (OD) of 0.1. At an OD600 of 0.6, the expression of the constructs was induced by the addition of 1 mM IPTG. After induction, 1 OD600 of cells were taken at 0.5, 1, 2, and 3 h for analysis by SDS-PAGE.


Results

The expected size of ACK is 43 kDa. The samples from the overexpression were analyzed by 12% SDS-PAGE (Fig. 1). When compared to the protein molecular weight marker, there is a band that appears around the 45 kDa mark. The protein EF-Tu is of a similar size and could be contributing to the band seen at this size. However, there appears to be an increase in intensity when comparing the sample from 0 h after induction to 3 h after induction. This suggests that ACK was overexpressed and the expression of the construct is working as expected.

Figure 1. 12% SDS-PAGE analysis of overexpression of ACK by E. coli. Lane numbers indicate time in hours after induction with IPTG; L indicates ladder. The black arrow indicates the band showing overexpression of ACK.

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