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.
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
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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.
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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 |
 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.
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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
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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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