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Lecture 1

BSCI 223 Lecture Notes - Lecture 1: Main Source, Planctomycetes, Gammaproteobacteria


Department
Biological Sciences Program
Course Code
BSCI 223
Professor
mciver
Lecture
1

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http://2012.igem.org/wiki/index.php?title=Team:JUIT-
India/Project&oldid=210135
ABSTRACT
“Global warming is too serious for the world any longer to ignore its danger
or split into opposing factions on it”, quoted Tony Blair back in 2005. Well
how much concerning this appears, since then, a lot more similar quotes
can be added in its reference. We have come together as a team to have a
different insight in dealing with this problem through genetic engineering.
Rice, which is the staple diet of India and many other countries around the
world, is believed to engender many potential green house gases or global
warming gases per se like carbon dioxide, methane and nitrous oxide.
Nitrous oxide, again, is released due to the inevitable use of nitrogen
fertilizers which are added in the paddy fields. We are dealing with the
conversion of this highly potential global warming gas, nitrous oxide into
nitrate form using synthetic biology tools to insert two genes into a
bacterial cassette along with its detection systems. This nitrate, as we
know, can in turn, be utilized by the plant itself, solving our purpose and
adding a new dimension to this diversion and in turn being beneficial for
the farmers reducing the compromise factor that would, otherwise, have
been done.
Overall project
Synthetic biology aims to design and construct new biological functions
and system that are not found in nature. We have given the name ‘Captain
Green’ to our organism M.capsulatus who plays ‘hero’ like figure in
providing the greener and healthy environment. Global warming has
become an alarming issue in 21st century and we aim to take action
against it. The tremendous increase in methane, nitrous oxide and carbon
dioxide emission has become a great concern. While rice has the third-
highest worldwide production and a staple crop for nearly half the world's
population with the worldwide consumption of ~367 million metric ton per
year but anoxic conditions in the wetland soils of rice paddies are ideal for
microbes that produce methane, which trails only carbon dioxide in terms
of its greenhouse effect. Rice agriculture is a big source of atmospheric
methane, possibly the biggest of man-made methane sources. With an
increasing world population, reductions in rice agriculture remain largely
untenable as on Methane emission reduction strategy. Methane emission
from paddy field makes up 29% of the total of Methane and Nitrous Oxide
emission from agricultural land makes up 55%. So, greenhouse gas

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emissions from rice paddy fields are considered as one of the most
important emission sources. The average concentration of nitrous oxide in
the atmosphere is now increasing at a rate of 0.2 to 0.3% per year.
Methane and nitrous oxide are both potent greenhouse gasses, with global
warming potentials approximately 25 and 298 times that of carbon dioxide.
Complete Description
Project Description
We are going to use the nif (From M.capsulatus) and nos (from
Pseudomonas) genes. The mmo genes are present in to forms in
M.capsualatus i.e sMMO(soluble MMO) and pMMO(particulate MMO).
MMO enzyme catalyzes the conversion of methane to methanol. Once
methanol is converted into formaldehyde it enters various biochemical
cycles in the cell. Our system involves the utilization of the methanol as an
inducer for MxaF promoter. The nosz gene is used to convert nitrous oxide
into nitrogen. NosZ is a gene derived from P.aeruginosa and is used for
nitrogen fixation. NifA is a specific transcriptional activator of the nif genes
and acts in conjunction with RNA polymerase. Nif genes then converts the
nitrogen into nitrate, which can be easily be taken up by the plant. The
main reason for addition of fertilizer is to increase the inorganic nutrients in
the soil. By utilizing NosZ and Nif genes we are converting the nitrous
oxide into nitrate, thus reducing the need of fertilizers in the soil by
increasing the nitrogen available to the plants. This is a small step towards
a more environment friendly future.
Details:
MxaF:
Methylotrophic bacteria are a diverse group of microorganisms with the
ability to utilize single-carbon (C1) substrates more reduced than carbon
dioxide as their sole source of carbon and energy.Methanotrophs
possesses native methanol-inducible promoters, notably promoters which
are located upstream of genes that encode methanol dehydrogenase and
other proteins required for its activity and enzymes required for the
synthesis of the methanol dehydrogenase prosthetic group,
pyrroloquinoline quinone. Of these, the promoter PmxaF has been
thoroughly scrutinized both biochemically and in expression studies. In its
native form in the chromosome, this strong promoter is methanol inducible.
However, when this promoter is cloned in expression vectors, it acts
essentially in a constitutive mode. The mxaF gene is approximately 1.8 kb
in size and encodes a 66-kDa polypeptide. Our system involves the
utilization of the methanol as an inducer for MxaF promoter. This would
result in the diversion of the flux thus leading to a faster degradation of

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methane for the cell to survive.
NosZ:
The complete denitrification of nitrate by bacteria to dinitrogen (N,) is
generally an anaerobic respiratory process. The last step involves the
dissimilatory reduction of nitrous oxide (N,O), the free energy change of
which can be coupled to phosphorylation (Zumft,1992; Zumft & Kroneck,
1990). nosZ is the structural gene for the periplasmic N,O reductase which
is required to the conversion of nitrous oxide into free nitrogen. . The
nitrous oxide that is emitted in the paddy fields is caused due to the
excessive use of fertilizers. The microbes naturally present in the paddy
field lead to the emission of nitrous oxide by utilizing these chemical
fertilizers. . NosZ is a gene derived from P.aeruginosa and is used for
denitrification.
NifA:
The biological nitrogen fixation reaction is catalyzed by a complex
metalloenzyme called nitrogenase (6, 14). Nitrogenase is composed of two
separately purified proteins, both of which are extremely oxygen sensitive.
Expression of the nif genes is regulated at the transcriptional level by the
products of nifA in response to molecular oxygen or ammonia. NifA is a
specific transcriptional activator of the nif genes and acts in conjunction
with RNA polymerase holoenzyme containing the alternative sigma factor,
sigma 54. NifA binds to a characteristic palindromic motif, TGT-N10-ACA,
also known as upstream activation sequence (UAS), that is located more
than 100 bp upstream of nif promoters. The NifA protein has three
arbitrarily designated domains (20): an amino-terminal domain which is
implicated in regulatory function, a catalytic domain that interacts with the
sigma-RNA polymerase holoenzyme, and a C-terminal helixturn-helix motif
which recognizes the UAS on the nif promoters.
SacB:
Expression from sacB confers sensitivity to sucrose in a wide variety of
gram positive and negative bacteria and thus has been used extensively
for the last twenty years as a negative selectable marker. The Bacillus
subtilis sacB gene encodes the enzyme levansucrase (EC 2.4.1.10), which
is secreted by B. subtilis cells into the culture medium. Levansucrase is a
transfructosylase catalyzing sucrose hydrolysis and levan synthesis. In
addition, this enzyme is capable of adding fructosyl residues to a wide
range of acceptor molecules. In Escherichia coli and other gram-negative
bacteria such as Rhizobium, Agrobacterium, or Cyanobacterium species,
expression of sacB is lethal in the presence of sucrose
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