Team:WashU StLouis/Project/nif
From 2014.igem.org
(Difference between revisions)
Line 16: | Line 16: | ||
<td style="vertical-align: top;"> | <td style="vertical-align: top;"> | ||
<div style="text-align: center;"> | <div style="text-align: center;"> | ||
- | <h1> | + | <h1>Transferring the <span style="font-style: italic;">nif</span> |
+ | cluster from <span style="font-style: italic;">Cyanothece </span>sp. | ||
+ | 51142 into <span style="font-style: italic;">Escherichia coli</span></h1> | ||
+ | Caroline Focht, Richard Hongyi Li<br> | ||
+ | <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, | ||
Line 37: | Line 41: | ||
<br> | <br> | ||
<div style="text-align: center;"> | <div style="text-align: center;"> | ||
- | < | + | <h3>Objectives</h3> |
</div> | </div> | ||
Rapidly growing, with high survival rate in environment, <span | Rapidly growing, with high survival rate in environment, <span | ||
Line 62: | Line 66: | ||
<br> | <br> | ||
<div style="text-align: center;"> | <div style="text-align: center;"> | ||
- | < | + | <h3>Overview</h3> |
</div> | </div> | ||
Our project consisted of three different phases. <br> | Our project consisted of three different phases. <br> | ||
Line 137: | Line 141: | ||
<span style="font-weight: bold;">Strains | <span style="font-weight: bold;">Strains | ||
of <span style="font-style: italic;">E. coli</span></span>: JM109, | of <span style="font-style: italic;">E. coli</span></span>: JM109, | ||
- | BL21(DE3), WM1788, DH5α <br> | + | BL21(DE3), WM1788, Top 10 DH5α <br> |
<br> | <br> | ||
<table | <table | ||
Line 144: | Line 148: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
- | <td | + | <td>Target<br> |
Strain <br> | Strain <br> | ||
</td> | </td> | ||
- | <td | + | <td>JM109 <br> |
</td> | </td> | ||
- | <td | + | <td>BL21(DE3) <br> |
</td> | </td> | ||
- | <td | + | <td>WM1788</td> |
- | <td | + | <td>Top 10<br> |
+ | </td> | ||
+ | <td>DH5α</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td | + | <td>Experimental Plates <br> |
</td> | </td> | ||
- | <td | + | <td>JM 109 strain w/ plasmid<br> |
<br> | <br> | ||
Antibiotic <br> | Antibiotic <br> | ||
</td> | </td> | ||
- | <td | + | <td>BL21(DE3) strain w/ plasmid<br> |
<br> | <br> | ||
Antibiotic</td> | Antibiotic</td> | ||
- | <td | + | <td>WM1788 strain w/ plasmid<br> |
<br> | <br> | ||
Antibiotic <br> | Antibiotic <br> | ||
</td> | </td> | ||
- | <td | + | <td>Top 10 strain w/ plasmid<br> |
+ | <br> | ||
+ | Antibiotic<br> | ||
+ | </td> | ||
+ | <td>DH5α strain w/ plasmid<br> | ||
<br> | <br> | ||
Antibiotic</td> | Antibiotic</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td | + | <td>Positive Control</td> |
- | <td | + | <td>JM 109 strain w/o plasmid<br> |
<br> | <br> | ||
No antibiotic</td> | No antibiotic</td> | ||
- | <td | + | <td>BL21(DE3) strain w/o plasmid<br> |
<br> | <br> | ||
No antibiotic</td> | No antibiotic</td> | ||
- | <td | + | <td> WM1788 strain w/o plasmid<br> |
<br> | <br> | ||
No antibiotic</td> | No antibiotic</td> | ||
- | <td | + | <td>Top 10 straing w/o plasmid<br> |
+ | <br> | ||
+ | No Antibiotic<br> | ||
+ | </td> | ||
+ | <td>DH5α strain w/o plasmid<br> | ||
<br> | <br> | ||
No antibiotic</td> | No antibiotic</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td | + | <td>Negative Control <br> |
</td> | </td> | ||
- | <td | + | <td> JM 109 strain w/o plasmid<br> |
<br> | <br> | ||
Antibiotic</td> | Antibiotic</td> | ||
- | <td | + | <td>BL21(DE3) strain w/o |
plasmid <br> | plasmid <br> | ||
<br> | <br> | ||
Antibiotic</td> | Antibiotic</td> | ||
- | <td | + | <td>WM1788 strain w/o plasmid<br> |
<br> | <br> | ||
Antibiotic</td> | Antibiotic</td> | ||
- | <td | + | <td>Top 10 strain w/o plasmid<br> |
+ | <br> | ||
+ | Antibiotic<br> | ||
+ | </td> | ||
+ | <td>DH5α strain w/out plasmid<br> | ||
<br> | <br> | ||
Antibiotic</td> | Antibiotic</td> | ||
Line 273: | Line 291: | ||
border="0" cellpadding="5" cellspacing="5"> | border="0" cellpadding="5" cellspacing="5"> | ||
<tbody> | <tbody> | ||
- | <tr> | + | <tr align="justify"> |
- | <td style="vertical-align: top | + | <td colspan="1" rowspan="1" style="vertical-align: top;">We |
used an Acetylene Reduction Assay to examine the nitrogenase | used 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> | ||
+ | <br style="font-weight: bold;"> | ||
+ | <span style="font-weight: bold;">Introduction of Acetylene | ||
+ | Reduction Assay:</span> <br> | ||
+ | The discovery that the nitrogenase enzyme responsible for N2-fixation | ||
+ | also reduced C2H2 (acetylene) to C2H4 (ethylene) (Dilworth, 1966) | ||
+ | provided a useful assay for the quantification of the N2-fixation | ||
+ | process. For quantitative determinations of N2-fixation, many N2 | ||
+ | techniques should be used, however, the acetylene reduction assay is | ||
+ | still used widely because it provides a highly sensitive and | ||
+ | inexpensive way to quantify relative nitrogenase enzyme activity in N2 | ||
+ | fixing samples. The nitrogenase enzyme reduces acetylene gas to | ||
+ | ethylene. Both gases can easily be quantified using gas | ||
+ | chromatography. <br> | ||
<br> | <br> | ||
+ | Materials: <br> | ||
+ | 1. Calcium Carbide (CaC2) (1 gram CaC2= 130 mL of C2H2 gas) <br> | ||
+ | 2. Deionized water <br> | ||
+ | 3. Two 10mL Syringes with needles<br> | ||
+ | 4. 125 mL volumetric flask with armhole and appropriate sized rubber | ||
+ | stopper<br> | ||
+ | 5. Large test tube and stopper with two holes <br> | ||
+ | <br> | ||
+ | Making acetylene gas: <br> | ||
+ | Combine appropriate number of rocks of calcium carbide and water | ||
+ | (injected by one of the syringes through stopper) in a 125mL flask | ||
+ | (CaC2 +H2O →C2H2). Place flask in hood or allow flask to vent out the | ||
+ | window until reaction is complete.<br> | ||
+ | When the reaction is completed, take another syringe to draw out gas | ||
+ | from stopper on the armhole of 125 mL flask. The gas in the syringe | ||
+ | would be the pure Acetylene (C2H2).<br> | ||
<br> | <br> | ||
- | <span style="font-weight: bold;">Results:</span><br> | + | Growing cultures for Acetylene Reduction Assay:<br> |
- | 1) Of the | + | 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. <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> | ||
+ | <div style="text-align: justify;"><span | ||
+ | style="font-weight: bold;">Results:</span><br> | ||
+ | 1) Of the five <span style="font-style: italic;">E. coli</span> | ||
strains tested, JM109 and WM1788 showed strongest nitrogenase activity.<br> | strains tested, JM109 and WM1788 showed strongest nitrogenase activity.<br> | ||
<br> | <br> | ||
Line 289: | Line 372: | ||
<br> | <br> | ||
3) Optimal conditions determined: <br> | 3) Optimal conditions determined: <br> | ||
- | <ul> | + | </div> |
+ | <ul style="text-align: justify;"> | ||
<li>glucose as carbon-source</li> | <li>glucose as carbon-source</li> | ||
<li>glutamate as | <li>glutamate as | ||
Line 301: | Line 385: | ||
</ul> | </ul> | ||
</td> | </td> | ||
- | < | + | </tr> |
- | + | </tbody> | |
- | + | </table> | |
- | + | ||
</td> | </td> | ||
</tr> | </tr> | ||
Line 310: | Line 393: | ||
</table> | </table> | ||
<div style="text-align: center;"> | <div style="text-align: center;"> | ||
- | < | + | <h3>Citation</h3> |
</div> | </div> | ||
1: Christian Rogers, Giles E. D. Oldroyd. Journal of Experimental | 1: Christian Rogers, Giles E. D. Oldroyd. Journal of Experimental |
Revision as of 23:08, 15 October 2014
Transferring the nif cluster from Cyanothece sp. 51142 into Escherichia coliCaroline Focht, Richard Hongyi LiIntroductionObjectivesThe 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. OverviewPhase 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
Phase
2: Determine the optimal conditions for cell survival with plasmid
pNif51142 in E. coli
Testing Media: Minimal M9Conditions/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
Phase 3:
Measure
nitrogen fixation activity under determined optimal conditions in E.
coli strains
Citation2: 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 |