Team:WashU StLouis/Project/nif

From 2014.igem.org

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cluster from <span style="font-style: italic;">Cyanothece </span>sp.
cluster from <span style="font-style: italic;">Cyanothece </span>sp.
51142 into <span style="font-style: italic;">Escherichia coli</span></h1>
51142 into <span style="font-style: italic;">Escherichia coli</span></h1>
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Caroline Focht, Richard Hongyi Li<br>
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Caroline Focht, Richard Hongyi Li<br><a name="1"></a>
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<a name="1"></a><h3>Introduction</h3>
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<h3>Introduction</h3>
</div>
</div>
Nitrogen is abundant in the earth’s atmosphere but, unlike carbon,
Nitrogen is abundant in the earth’s atmosphere but, unlike carbon,
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of our team. <br>
of our team. <br>
<br>
<br>
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<div style="text-align: center;">
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<div style="text-align: center;"><a name="2"></a>
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<a name="2"></a><h3>Overview</h3>
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<h3>Overview</h3>
</div>
</div>
Our project consisted of three different phases. <br>
Our project consisted of three different phases. <br>
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style="font-style: italic;">E. coli</span> strains<br>
style="font-style: italic;">E. coli</span> strains<br>
Phase 2: Determine the optimal conditions for cell survival with
Phase 2: Determine the optimal conditions for cell survival with
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plasmid pNif51142 in <span style="font-style: italic;">E. coli</span><br>
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plasmid pNif51142 in <span style="font-style: italic;">E. coli</span><br><
Phase 3: Measure nitrogen fixation activity under determined optimal
Phase 3: Measure nitrogen fixation activity under determined optimal
conditions in <span style="font-style: italic;">E. coli</span> strains<br>
conditions in <span style="font-style: italic;">E. coli</span> strains<br>
<br>
<br>
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<br>
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<br><a name="3"></a>
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<a name="3"></a><div style="text-align: center; font-weight: bold;">Phase 1:
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<div style="text-align: center; font-weight: bold;">Phase 1:
Electro-Transformation of plasmid pNif51142 into <span
Electro-Transformation of plasmid pNif51142 into <span
style="font-style: italic;">E. coli </span>strains<br>
style="font-style: italic;">E. coli </span>strains<br>
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</tbody>
</tbody>
</table>
</table>
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<br>
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<br><a name="4"></a>
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<a name="4"></a><div style="text-align: center;"><span style="font-weight: bold;">Phase
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<div style="text-align: center;"><span style="font-weight: bold;">Phase
2: Determine the optimal conditions for cell survival with plasmid
2: Determine the optimal conditions for cell survival with plasmid
pNif51142 in </span><span
pNif51142 in </span><span
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</tbody>
</tbody>
</table>
</table>
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<br>
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<div style="text-align: center;">
<table
<table
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style="text-align: left; width: 100%; height: 433px; margin-left: auto; margin-right: auto;"
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style="text-align: left; width: 100%; margin-left: auto; margin-right: auto;"
border="0" cellpadding="5" cellspacing="5">
border="0" cellpadding="5" cellspacing="5">
<tbody>
<tbody>
<tr>
<tr>
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<td style="vertical-align: top; text-align: justify;"><img
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<td style="vertical-align: top; width: 70%;"><img
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style="width: 628px; height: 389px;" alt="nif genes"
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style="width: 100%;" alt="Nitrogenase Poisoning"
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src="https://static.igem.org/mediawiki/2014/e/e4/WashU_nifgenes.png"><br>
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src="https://static.igem.org/mediawiki/2014/e/e1/WashU_Nitrogenase_Poisoning.jpg">
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Figure above modified from [5]: The nitrogenase enzyme is deactivated
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in the presence of oxygen</td>
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<td style="vertical-align: top; width: 30%;"><img
 +
style="width: 100%;"
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alt="Bottles of LB media used for culturing strains"
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src="https://static.igem.org/mediawiki/2014/f/f5/WashU_Culturing_Ecoli.jpg">Figure:
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Bottles of LB used for innoculating <i>E.coli</i> strains</td>
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</tr>
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</tbody>
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</table>
<br>
<br>
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</td>
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</div>
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<td style="vertical-align: top; text-align: justify;">Due
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Due
to the
to the
oxidative properties of oxygen, most nitrogenases are
oxidative properties of oxygen, most nitrogenases are
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Anaerobic were both determined not for cell growth but for nitrogenase
Anaerobic were both determined not for cell growth but for nitrogenase
activity testing, which is the next phase.<br>
activity testing, which is the next phase.<br>
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</td>
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<br><a name="5"></a>
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</tr>
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<div style="text-align: center; font-weight: bold;">Phase 3:
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</tbody>
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-
</table>
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-
<br>
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-
<a name="5"></a><div style="text-align: center; font-weight: bold;">Phase 3:
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Measure
Measure
nitrogen fixation activity under determined optimal conditions in E.
nitrogen fixation activity under determined optimal conditions in E.
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<tbody>
<tbody>
<tr align="justify">
<tr align="justify">
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<td colspan="1" rowspan="1" style="vertical-align: top;">We
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<td colspan="1" rowspan="1" style="vertical-align: middle;">We
used&nbsp; an Acetylene Reduction Assay to examine the nitrogenase
used&nbsp; an Acetylene Reduction Assay to examine the nitrogenase
activity
activity
for JM109, BL21(DE3), Top10, DH5α at different cell density referred by
for JM109, BL21(DE3), Top10, DH5α at different cell density referred by
OD600 values.<br>
OD600 values.<br>
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<br style="font-weight: bold;">
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<br>
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<span style="font-weight: bold;">Introduction of Acetylene
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Acetylene (C2H2) has an triple bond similar to that of atmospheric
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Reduction Assay:</span> <br>
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nitrogen (N2). Because of this structural similarity, the nitrogenase
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The discovery that the nitrogenase enzyme responsible for N2-fixation
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enzyme can cleave the triple bond in acetylene just as it would cleave
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also reduced C2H2 (acetylene) to C2H4 (ethylene) (Dilworth, 1966)
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the triple bond in N2. Ethylene (C2H4) is produced from this enzymatic
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provided a useful assay for the quantification of the N2-fixation
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activity, so a gas chromatograph can be used to detect the presence of
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process. For quantitative determinations of N2-fixation, many N2
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ethylene and, consequently, nitrogenase activity.<br>
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techniques should be used, however, the acetylene reduction assay is
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<br>
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still used widely because it provides a highly sensitive and
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<table
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inexpensive way to quantify relative nitrogenase enzyme activity in N2
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style="text-align: left; width: 100%; margin-left: auto; margin-right: auto;"
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fixing samples. The nitrogenase enzyme reduces acetylene gas to
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border="0" cellpadding="5" cellspacing="5">
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ethylene. Both gases can easily be quantified using gas
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<tbody>
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chromatography. <br>
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<tr>
 +
<td style="vertical-align: top; width: 60%;"><br>
 +
<br>
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<div style="text-align: center;"><img
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style="width: 100%;" alt="Acetylene Production Apparatus"
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src="https://static.igem.org/mediawiki/2014/7/7d/WashU_Acetylene_Production_Apparatus.jpg">Figure
 +
above: This is the apparatus for Acetylene Proeduction</div>
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</td>
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<td
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style="vertical-align: top; width: 40%; text-align: center;"><img
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style="width: 100%;" alt="GC Machine"
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src="https://static.igem.org/mediawiki/2014/2/28/WashU_GC_Machine.jpg">The
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injection ports of Gas phase GC machine used for Acetylene Reduction
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Assay</td>
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</tr>
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</tbody>
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</table>
<br>
<br>
Materials: <br>
Materials: <br>
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1. Calcium Carbide (CaC2) (1 gram CaC2= 130 mL of C2H2 gas) <br>
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1-2 small fragments of calcium carbide (CaC2)<br>
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2. Deionized water <br>
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Water<br>
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3. Two 10mL Syringes with needles<br>
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<br>
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4. 125 mL volumetric flask with armhole and appropriate sized rubber
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Procedure<br>
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stopper<br>
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<ol>
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5. Large test tube and stopper with two holes <br>
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<li>Place small amount of calcium carbide in sealed flask</li>
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&nbsp;<br>
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<li>Fill the adjacent test tube with water</li>
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Making acetylene gas: <br>
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<li>Inject a small amount (&lt;1 mL) of water into the flask with
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Combine appropriate number of rocks of calcium carbide and water
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a syringe</li>
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(injected by one of the syringes through stopper) in a 125mL flask
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<li>After the water level in the test tube becomes
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(CaC2 +H2O →C2H2). Place flask in hood or allow flask to vent out the
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constant, draw some of the gas in the headspace of the flask
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window until reaction is complete.<br>
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(acetylene) out and inject it into the sealed bottle with the culture</li>
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When the reaction is completed, take another syringe to draw out gas
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<li>Using a gas chromatograph, determine the initial
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from stopper on the armhole of 125 mL flask. The gas in the syringe
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ethylene peak</li>
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would be the pure Acetylene (C2H2).<br>
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<li>Test the bottle again after 3 hours</li>
 +
</ol>
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<br>
<br>
<br>
Growing cultures for Acetylene Reduction Assay:<br>
Growing cultures for Acetylene Reduction Assay:<br>
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proceeded with the JM109, BL21, and WM1788 wild types and mutants. <br>
proceeded with the JM109, BL21, and WM1788 wild types and mutants. <br>
</td>
</td>
 +
</tr>
<tr align="center">
<tr align="center">
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</tr><a name="6"></a><td colspan="1" rowspan="1" style="vertical-align: top;"><img
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<td colspan="1" rowspan="1" style="vertical-align: top;"><img
style="width: 800px; height: 476px;"
style="width: 800px; height: 476px;"
alt="Acetylene reduction assay results"
alt="Acetylene reduction assay results"
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<tbody>
<tbody>
<tr>
<tr>
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<td style="vertical-align: top;"><br>
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<td style="vertical-align: top;"><br><a name="6"></a>
<div style="text-align: justify;"><span
<div style="text-align: justify;"><span
style="font-weight: bold;">Results:</span><br>
style="font-weight: bold;">Results:</span><br>
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32(1):303-306<br>
32(1):303-306<br>
3: Liying Wang, Ray Dixon. PLOS Genetics., 2013 Oct 17; 9(10): 3865–3876<br>
3: Liying Wang, Ray Dixon. PLOS Genetics., 2013 Oct 17; 9(10): 3865–3876<br>
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4: Temme, Zhao, Voigt. PNAS., 2012 March 23; 109(18): 7085–7090</td>
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4: Temme, Zhao, Voigt. PNAS., 2012 March 23; 109(18): 7085–7090<br>
 +
5: Dixon, Kahn. <span style="font-style: italic;">Genetic Regulation
 +
of Biological Nitrogen Fixation. </span>Nature Reviews. August 2004,
 +
Vol 2. P. 621-631.<br>
 +
</td>
</tr>
</tr>
</tbody>
</tbody>

Latest revision as of 22:31, 17 October 2014



Home

Introduction Overview Phase 1 Phase 2 Phase 3 Results Citation

Transferring the nif cluster from Cyanothece sp. 51142 into Escherichia coli

Caroline Focht, Richard Hongyi Li

Introduction

Nitrogen is abundant in the earth’s atmosphere but, unlike carbon, cannot be directly assimilated by plants.[1] Nitrogen can be directly fixed from the atmosphere by some Cyanobacteria such as Cyanothece 51142, which possesses the enzyme nitrogenase translated from nif genes. Some plant species have evolved close symbiotic associations with nitrogen-fixing bacteria. Engineering crops with the capability to fix their own nitrogen could one day address the problems created by the abuse of fertilizers in agriculture. This could be achieved either by expression of a functional nitrogenase enzyme in the cells of the crop or through transferring the capability to form a symbiotic association with nitrogen-fixing bacteria. Our project mainly focuses on expressing the nif clusters in E. coli strains under various conditions in order to study the nif system in simpler internal environment of prokaryotic cells.

Objectives

Rapidly growing, with high survival rate in environment, E. coli has many attributes that make it an ideal candidate for use as a model organism, which is a species that is extensively studied to understand a specific phenomenon—we expect that the knowledge gained can be applied to other species as well in future.
The genomes of many strains of E. coli have been sequenced. These sequences have been scrutinized so heavily that the way the cell works is very well understood, and changing and manipulating the DNA sequence will lead to predictable results.  Thus, from the previous research of internal energy management and nutritional capability on various strains of E. coli, we have proposed to selected four strains[2], JM109, BL21(DE3), WM1788, and DH5α to carry plasmid pNif51142, which insert the nif cluster from Cyanothece sp. 51142, therefore expressing the nitrogenase activity. The general objective is to adjust parameters of environmental conditions to show nitrogen-fixing activity in E. coli strains and eventually adapt to the light-controlling promoter system from the side of our team.

Overview

Our project consisted of three different phases.
Phase 1: Electro-Transformation of plasmid pNif51142 into E. coli strains
Phase 2: Determine the optimal conditions for cell survival with plasmid pNif51142 in E. coli
< Phase 3: Measure nitrogen fixation activity under determined optimal conditions in E. coli strains


Phase 1: Electro-Transformation of plasmid pNif51142 into E. coli strains

nif cluster
Figure above: nif cluster of Cyanothece sp. 51142 containing all the necessary genes for nitrogen fixation
 Due to the large size of the plasmid pNif51142, 37,630bp, it was very challenging to transform it into E. coli strains.
 
To successfully transform pNif51142, we used Electro-Transformation, also known as Electroporation.  Electroporation provides a method of transforming E. coli to efficiencies greater than are possible with the best chemical methods. By subjecting mixtures of cells and DNA to exponentially decaying fields of very high initial amplitude, we were able to deliver the plasmid into all of E. coli strains that were tested in the project.

Results:
According to the gel running and antibiotic testing, bands in the gel and the survival of all strains transformed in antibiotic Kanamycin (there was an cluster of Kanamycin-resistant marker gene in sequence of pNif51142) both prove that the Electro-Transformation was successful.

Phase 2: Determine the optimal conditions for cell survival with plasmid pNif51142 in E. coli
Testing Media: Minimal M9

Conditions/Parameters Tested:
        Carbon Source: Glucose (1mM, 10mM, 100mM)
        Nitrogen Source: Glutamine (1mM, 10mM, 100mM), Glutamate (1mM, 10mM, 100mM), NH4Cl (1mM, 10mM,100mM) All Concentration range determined by [3]
        Temperature: 30°C, 37°C, 40°C
        pH: 6, 7, 8
        O2 Level: Anaerobic or Aerobic
        Strains of E. coli: JM109, BL21(DE3), WM1788, Top 10 DH5α   

Target
Strain
JM109 
BL21(DE3)
WM1788 Top 10
 DH5α
Experimental Plates
JM 109 strain w/ plasmid

Antibiotic
BL21(DE3) strain w/ plasmid

Antibiotic		 WM1788 strain w/ plasmid

Antibiotic
Top 10 strain w/ plasmid

Antibiotic
 DH5α strain w/ plasmid

Antibiotic
Positive Control JM 109 strain w/o plasmid

No antibiotic		 BL21(DE3) strain w/o plasmid

No antibiotic		 WM1788 strain w/o plasmid

No antibiotic		 Top 10 straing w/o plasmid

No Antibiotic
 DH5α strain w/o plasmid

No antibiotic
Negative Control
JM 109 strain w/o plasmid

Antibiotic		 BL21(DE3) strain w/o plasmid

Antibiotic		 WM1788 strain w/o plasmid

Antibiotic		 Top 10 strain w/o plasmid

Antibiotic
 DH5α strain w/out plasmid

Antibiotic
Nitrogenase Poisoning Figure above modified from [5]: The nitrogenase enzyme is deactivated in the presence of oxygen Bottles of LB media used for culturing strainsFigure: Bottles of LB used for innoculating E.coli strains

Due to the oxidative properties of oxygen, most nitrogenases are irreversibly inhibited by dioxygen, which degradatively oxidizes the Fe-S cofactors. This requires mechanisms for nitrogen fixers to protect nitrogenase from oxygen in vivo. Hence in our experiment, we firstly selected anaerobic condition as part of preparation step for the nitrogenase activity testing.


Results:
In the minimal M9 media, all possible combinations of parameters listed above were tested.
None of the concentrations of glucose had any affect on the growth of E. coli. It was expected that as the concentration of glucose increased, the growth of E. coli also increased. However, the variation between the concentrations of glucose may have been too small for a noticeable increase in E. coli growth as the concentration of glucose increased. Also, even the maximum concentration of glucose tested, 100mM, may have been too low to affect the growth of E. coli to an observable extent. Eventually, 10mM was determined to be the optimal glucose concentration for the purpose of least interference possible in solution.
NH4Cl as minimal nitrogen source was proven to be not suitable for E. coli growth at any concentration as the OD600 testing results showed that cell density didn’t change throughout the time. It was probably due to the permeability of cell membrane was limited for NH4+ and Cl- ions. Eventually Glutamate at concentration of 10mM supported cell growth the best and thus chosen as part of optimal environment condition.

With CASAmino solution’s buffering utility, the pH was controlled little bit below 7 but close to 7.

To protect the iron core of the nitrogenase, temperature of 30°C and Anaerobic were both determined not for cell growth but for nitrogenase activity testing, which is the next phase.

Phase 3: Measure nitrogen fixation activity under determined optimal conditions in E. coli strains

We used  an Acetylene Reduction Assay to examine the nitrogenase activity for JM109, BL21(DE3), Top10, DH5α at different cell density referred by OD600 values.

Acetylene (C2H2) has an triple bond similar to that of atmospheric nitrogen (N2). Because of this structural similarity, the nitrogenase enzyme can cleave the triple bond in acetylene just as it would cleave the triple bond in N2. Ethylene (C2H4) is produced from this enzymatic activity, so a gas chromatograph can be used to detect the presence of ethylene and, consequently, nitrogenase activity.



Acetylene Production ApparatusFigure above: This is the apparatus for Acetylene Proeduction
 GC MachineThe injection ports of Gas phase GC machine used for Acetylene Reduction Assay

Materials:
1-2 small fragments of calcium carbide (CaC2)
Water

Procedure
  1. Place small amount of calcium carbide in sealed flask
  2. Fill the adjacent test tube with water
  3. Inject a small amount (<1 mL) of water into the flask with a syringe
  4. After the water level in the test tube becomes constant, draw some of the gas in the headspace of the flask (acetylene) out and inject it into the sealed bottle with the culture
  5. Using a gas chromatograph, determine the initial ethylene peak
  6. Test the bottle again after 3 hours


Growing cultures for Acetylene Reduction Assay:
After deciding to culture the strains in an M9 medium before performing the assay, the experimenters encountered a few setbacks in the preparation of that medium. After a couple of days of trial and error, a protocol was established that produced a viable M9 medium. To create the 100 mL of 10X M9 stock solution, 0.026 g CaCl2·H2O, 0.030 g MgSO4,10.4 g Na2HPO4, 3.4 g KH2PO4, and 4 g glucose were dissolved in the appropriate volume of water. The resulting solution was then filtered for sterility. 100 mL of a 1000X supplemental stock solution was prepared by dissolving 0.3 g MnSO4, 7.6 g Na2MoO4*2H2O, 0.010 g p-aminobenzoic acid, and 0.005 g biotin in the appropriate volume of water. A 100X ferric citrate solution was also prepared by dissolving 0.36 g Ferric citrate in water to create 100 mL of solution. The viable M9 medium was prepared by mixing the appropriate volumes of these solutions together along with a glutamine solution for all cultures as a nitrogen source and kanamycin for the experimental tubes and bottles. After observing their growth, DH5α and Top 10 were eliminated due to their inability to grow well in the medium, and all experiments proceeded with the JM109, BL21, and WM1788 wild types and mutants.
Acetylene reduction assay results

Results:
1) Of the five E. coli strains tested, JM109 and WM1788 showed strongest nitrogenase activity.
 
2) The linear relationship between nitrogen fixation activity and time matches that seen in nature.

3) Optimal conditions determined:
  • glucose as carbon-source
  • glutamate as nitrogen-source
  • LB as inoculating media
  • minimal M9 as testing media for GC assay
  • anaerobic environment
  • 30 °C during the overnight preparation

Citation

1: Christian Rogers, Giles E. D. Oldroyd. Journal of Experimental Botany Advance Access., 2014 May; 65(8):1939-46
2: Heladia Salgado, Socorro Gama, César Bonavides‐Martínez and Julio Collado‐Vides. Oxford Journals of Nucleic Acid Research., 2003 October; 32(1):303-306
3: Liying Wang, Ray Dixon. PLOS Genetics., 2013 Oct 17; 9(10): 3865–3876
4: Temme, Zhao, Voigt. PNAS., 2012 March 23; 109(18): 7085–7090
5: Dixon, Kahn. Genetic Regulation of Biological Nitrogen Fixation. Nature Reviews. August 2004, Vol 2. P. 621-631.

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