Latest revision as of 22:31, 17 October 2014

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

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

Figure above modified from [5]: The nitrogenase enzyme is deactivated in the presence of oxygen

Bottles of LB media used for culturing strains

Figure: 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.

Figure above: This is the apparatus for Acetylene Proeduction

The injection ports of Gas phase GC machine used for Acetylene Reduction Assay

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

Procedure

Place small amount of calcium carbide in sealed flask
Fill the adjacent test tube with water
Inject a small amount (<1 mL) of water into the flask with a syringe
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
Using a gas chromatograph, determine the initial ethylene peak
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.

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.

Team:WashU StLouis/Project/nif

From 2014.igem.org

Latest revision as of 22:31, 17 October 2014

Home

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

Introduction

Objectives

Overview

Citation

Special thanks to our 2014 Sponsors:

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@@ Line 5: / Line 5: @@
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+div.floating-menu {position:fixed;background:#fff4c8;border:1px solid #ffcc00;width:150px;z-index:100;}
+div.floating-menu a, div.floating-menu h3 {display:block;margin:0 0.5em;}
 </style>
+<div class="floating-menu">
+     <h3>Home</h3>
+     <a href="#1">Introduction</a>
+     <a href="#2">Overview</a>
+     <a href="#3">Phase 1</a>
+     <a href="#4">Phase 2</a>
+     <a href="#5">Phase 3</a>
+     <a href="#6">Results</a>
+     <a href="#7">Citation</a>
+</div>
 <!--  here ends the section that changes the default wiki template to a white full width background -->
-<table  style="text-align: left;  width: 80%; margin-left: auto; margin-right: auto;"
+<!-- beginning of your page -->
+<!--float-menu -->
+<table
+style="text-align: left; width: 80%; margin-left: auto; margin-right: auto;"
 border="0" cellpadding="5" cellspacing="5">
+<tbody>
 <tr>
-<td bgColor="#FFF" >
+<td style="vertical-align: top;">
-<body>
 <div style="text-align: center;">
-<h1>Introduction</h1>
+<h1>Transferring the <span style="font-style: italic;">nif</span>
+cluster from <span style="font-style: italic;">Cyanothece </span>sp.
+into <span style="font-style: italic;">Escherichia coli</span></h1>
+Caroline Focht, Richard Hongyi Li<br><a name="1"></a>
+<h3>Introduction</h3>
 </div>
 Nitrogen is abundant in the earth’s atmosphere but, unlike carbon,
@@ Line 37: / Line 58: @@
 <br>
 <div style="text-align: center;">
-<h1>Objectives</h1>
+<h3>Objectives</h3>
 </div>
 Rapidly growing, with high survival rate in environment, <span
@@ Line 61: / Line 82: @@
 of our team. <br>
 <br>
-<div style="text-align: center;">
+<div style="text-align: center;"><a name="2"></a>
-<h1>Overview</h1>
+<h3>Overview</h3>
 </div>
 Our project consisted of three different phases. <br>
@@ Line 68: / Line 89: @@
 style="font-style: italic;">E. coli</span> strains<br>
 Phase 2: Determine the optimal conditions for cell survival with
-plasmid pNif51142 in <span style="font-style: italic;">E. coli</span><br>
+plasmid pNif51142 in <span style="font-style: italic;">E. coli</span><br><
 Phase 3: Measure nitrogen fixation activity under determined optimal
 conditions in <span style="font-style: italic;">E. coli</span> strains<br>
 <br>
-<br>
+<br><a name="3"></a>
 <div style="text-align: center; font-weight: bold;">Phase 1:
 Electro-Transformation of plasmid pNif51142 into <span
@@ Line 79: / Line 100: @@
 <br>
 <table
-style="text-align: left; width: 1348px; margin-left: auto; margin-right: auto; height: 551px;"
+style="text-align: left; width: 100%; margin-left: auto; margin-right: auto; height: 551px;"
 border="0" cellpadding="5" cellspacing="5">
 <tbody>
@@ Line 90: / Line 111: @@
 all the necessary genes for nitrogen fixation<br>
 </td>
-<td style="vertical-align: middle; text-align: justify;">Due to
+<td style="vertical-align: middle; text-align: justify;">Due
+to
 the large size of the plasmid pNif51142, 37,630bp, it was very
 challenging to transform it into E. coli strains. <br>
@@ Line 112: / Line 134: @@
 </tbody>
 </table>
-<br>
+<br><a name="4"></a>
 <div style="text-align: center;"><span style="font-weight: bold;">Phase
 : Determine the optimal conditions for cell survival with plasmid
@@ Line 136: / Line 158: @@
 &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; <span style="font-weight: bold;">Strains
 of <span style="font-style: italic;">E. coli</span></span>: JM109,
-BL21(DE3), WM1788, DH5α&nbsp;&nbsp;&nbsp; <br>
+BL21(DE3), WM1788, Top 10 DH5α&nbsp;&nbsp;&nbsp; <br>
 <br>
 <table
@@ Line 143: / Line 165: @@
 <tbody>
 <tr>
-<td style="text-align: center;">Target<br>
+<td>Target<br>
 Strain <br>
 </td>
-<td style="text-align: center;">JM109&nbsp; <br>
+<td>JM109&nbsp; <br>
 </td>
-<td style="text-align: center;">BL21(DE3) <br>
+<td>BL21(DE3) <br>
 </td>
-<td style="text-align: center;">WM1788</td>
+<td>WM1788</td>
-<td style="text-align: center;">DH5α</td>
+<td>Top 10<br>
+</td>
+<td>DH5α</td>
 </tr>
 <tr>
-<td style="text-align: center;">Experimental Plates <br>
+<td>Experimental Plates <br>
 </td>
-<td style="text-align: center;">JM 109 strain w/ plasmid<br>
+<td>JM 109 strain w/ plasmid<br>
 <br>
 Antibiotic <br>
 </td>
-<td style="text-align: center;">BL21(DE3) strain w/ plasmid<br>
+<td>BL21(DE3) strain w/ plasmid<br>
 <br>
 Antibiotic</td>
-<td style="text-align: center;">WM1788 strain w/ plasmid<br>
+<td>WM1788 strain w/ plasmid<br>
 <br>
 Antibiotic <br>
 </td>
-<td style="text-align: center;">DH5α strain w/ plasmid<br>
+<td>Top 10 strain w/ plasmid<br>
+<br>
+Antibiotic<br>
+</td>
+<td>DH5α strain w/ plasmid<br>
 <br>
 Antibiotic</td>
 </tr>
 <tr>
-<td style="text-align: center;">Positive Control</td>
+<td>Positive Control</td>
-<td style="text-align: center;">JM 109 strain w/o plasmid<br>
+<td>JM 109 strain w/o plasmid<br>
 <br>
 No antibiotic</td>
-<td style="text-align: center;">BL21(DE3) strain w/o plasmid<br>
+<td>BL21(DE3) strain w/o plasmid<br>
 <br>
 No antibiotic</td>
-<td style="text-align: center;"> WM1788 strain w/o plasmid<br>
+<td> WM1788 strain w/o plasmid<br>
 <br>
 No antibiotic</td>
-<td style="text-align: center;">DH5α strain w/o plasmid<br>
+<td>Top 10 straing w/o plasmid<br>
+<br>
+No Antibiotic<br>
+</td>
+<td>DH5α strain w/o plasmid<br>
 <br>
 No antibiotic</td>
 </tr>
 <tr>
-<td style="text-align: center;">Negative Control <br>
+<td>Negative Control <br>
 </td>
-<td style="text-align: center;"> JM 109 strain w/o plasmid<br>
+<td> JM 109 strain w/o plasmid<br>
 <br>
 Antibiotic</td>
-<td style="text-align: center;">BL21(DE3) strain w/o plasmid <br>
+<td>BL21(DE3) strain w/o
+plasmid <br>
 <br>
 Antibiotic</td>
-<td style="text-align: center;">WM1788 strain w/o plasmid<br>
+<td>WM1788 strain w/o plasmid<br>
 <br>
 Antibiotic</td>
-<td style="text-align: center;">DH5α strain w/out plasmid<br>
+<td>Top 10 strain w/o plasmid<br>
+<br>
+Antibiotic<br>
+</td>
+<td>DH5α strain w/out plasmid<br>
 <br>
 Antibiotic</td>
@@ Line 204: / Line 241: @@
 </tbody>
 </table>
-<br>
+<div style="text-align: center;">
 <table
-style="text-align: left; width: 1348px; height: 418px; margin-left: auto; margin-right: auto;"
+style="text-align: left; width: 100%; margin-left: auto; margin-right: auto;"
 border="0" cellpadding="5" cellspacing="5">
 <tbody>
 <tr>
-<td style="vertical-align: top; text-align: justify;"><img
+<td style="vertical-align: top; width: 70%;"><img
-style="width: 628px; height: 389px;" alt="nif genes"
+style="width: 100%;" alt="Nitrogenase Poisoning"
-src="https://static.igem.org/mediawiki/2014/e/e4/WashU_nifgenes.png"><br>
+src="https://static.igem.org/mediawiki/2014/e/e1/WashU_Nitrogenase_Poisoning.jpg">
+Figure above modified from [5]: The nitrogenase enzyme is deactivated
+in the presence of oxygen</td>
+<td style="vertical-align: top; width: 30%;"><img
+style="width: 100%;"
+alt="Bottles of LB media used for culturing strains"
+src="https://static.igem.org/mediawiki/2014/f/f5/WashU_Culturing_Ecoli.jpg">Figure:
+Bottles of LB used for innoculating <i>E.coli</i> strains</td>
+</tr>
+</tbody>
+</table>
 <br>
-</td>
+</div>
-<td style="vertical-align: top; text-align: justify;">Due to the
+Due
+to the
 oxidative properties of oxygen, most nitrogenases are
 irreversibly inhibited by dioxygen, which degradatively oxidizes the
@@ Line 255: / Line 303: @@
 Anaerobic were both determined not for cell growth but for nitrogenase
 activity testing, which is the next phase.<br>
-</td>
+<br><a name="5"></a>
-</tr>
+<div style="text-align: center; font-weight: bold;">Phase 3:
-</tbody>
+Measure
-</table>
-<br>
-<div style="text-align: center; font-weight: bold;">Phase 3: Measure
 nitrogen fixation activity under determined optimal conditions in E.
 coli strains<br>
@@ Line 269: / Line 314: @@
 border="0" cellpadding="5" cellspacing="5">
 <tbody>
-<tr>
+<tr align="justify">
-<td style="vertical-align: top; text-align: justify;">We
+<td colspan="1" rowspan="1" style="vertical-align: middle;">We
 used&nbsp; an Acetylene Reduction Assay to examine the nitrogenase
 activity
@@ Line 276: / Line 321: @@
 OD600 values.<br>
 <br>
+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.<br>
 <br>
-<span style="font-weight: bold;">Results:</span><br>
+<table
-) Of the four <span style="font-style: italic;">E. coli</span>
+style="text-align: left; width: 100%; margin-left: auto; margin-right: auto;"
+border="0" cellpadding="5" cellspacing="5">
+<tbody>
+<tr>
+<td style="vertical-align: top; width: 60%;"><br>
+<br>
+<div style="text-align: center;"><img
+style="width: 100%;" alt="Acetylene Production Apparatus"
+src="https://static.igem.org/mediawiki/2014/7/7d/WashU_Acetylene_Production_Apparatus.jpg">Figure
+above: This is the apparatus for Acetylene Proeduction</div>
+</td>
+<td
+style="vertical-align: top; width: 40%; text-align: center;"><img
+style="width: 100%;" alt="GC Machine"
+src="https://static.igem.org/mediawiki/2014/2/28/WashU_GC_Machine.jpg">The
+injection ports of Gas phase GC machine used for Acetylene Reduction
+Assay</td>
+</tr>
+</tbody>
+</table>
+<br>
+Materials: <br>
+-2 small fragments of calcium carbide (CaC2)<br>
+Water<br>
+<br>
+Procedure<br>
+<ol>
+<li>Place small amount of calcium carbide in sealed flask</li>
+<li>Fill the adjacent test tube with water</li>
+<li>Inject a small amount (&lt;1 mL) of water into the flask with
+a syringe</li>
+<li>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</li>
+<li>Using a gas chromatograph, determine the initial
+ethylene peak</li>
+<li>Test the bottle again after 3 hours</li>
+</ol>
+<br>
+<br>
+Growing cultures for Acetylene Reduction Assay:<br>
+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
+.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. <br>
+</td>
+</tr>
+<tr align="center">
+<td colspan="1" rowspan="1" style="vertical-align: top;"><img
+style="width: 800px; height: 476px;"
+alt="Acetylene reduction assay results"
+src="https://static.igem.org/mediawiki/2014/f/ff/WashU_ARA_results.png"></td>
+</tr>
+<tr>
+<td colspan="1" rowspan="1" style="vertical-align: top;">
+<table
+style="text-align: left; width: 100%; height: 271px; margin-left: auto; margin-right: auto;"
+border="0" cellpadding="5" cellspacing="5">
+<tbody>
+<tr>
+<td style="vertical-align: top;"><br><a name="6"></a>
+<div style="text-align: justify;"><span
+style="font-weight: bold;">Results:</span><br>
+) Of the five <span style="font-style: italic;">E. coli</span>
 strains tested, JM109 and WM1788 showed strongest nitrogenase activity.<br>
 &nbsp;<br>
@@ Line 285: / Line 413: @@
 <br>
 ) Optimal conditions determined: <br>
-<ul>
+</div>
+<ul style="text-align: justify;">
 <li>glucose as carbon-source</li>
 <li>glutamate as
@@ Line 297: / Line 426: @@
 </ul>
 </td>
-<td style="vertical-align: top;"><img
+</tr>
-style="width: 800px; height: 476px;"
+</tbody>
-alt="Acetylene reduction assay results"
+</table>
-src="https://static.igem.org/mediawiki/2014/f/ff/WashU_ARA_results.png"><br>
 </td>
 </tr>
@@ Line 306: / Line 434: @@
 </table>
 <div style="text-align: center;">
-<h1>Citation</h1>
+<a name="7"></a><h3>Citation</h3>
 </div>
 : Christian Rogers, Giles E. D. Oldroyd. Journal of Experimental
@@ Line 315: / Line 443: @@
 : Liying Wang, Ray Dixon. PLOS Genetics., 2013 Oct 17; 9(10): 3865–3876<br>
 : Temme, Zhao, Voigt. PNAS., 2012 March 23; 109(18): 7085–7090<br>
-<br>
+: Dixon, Kahn. <span style="font-style: italic;">Genetic Regulation
-<br>
+of Biological Nitrogen Fixation. </span>Nature Reviews. August 2004,
-</body>
+Vol 2. P. 621-631.<br>
 </td>
 </tr>
+</tbody>
 </table>
 </html>
 {{:Team:WashU_StLouis/footer}}