Metalloprotease's Written Proposal
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Transcript of Metalloprotease's Written Proposal
Sequence determination and Bioinformatics Analyses of
Metalloproteases Domains cloned from Pongo pygmaeus Primate
Genomic DNA
Ana Velazquez and Dr. Michael Rubin
RISE Program
University of Puerto Rico at Cayey
Department of Biology
Abstract
Metalloproteases are a group of proteases collectively responsible for the degradation of the
extracellular matrix. They are important for the shape and modifications needed in the
extracellular matrix. Metalloproteases are important in many aspects of biology, ranging from
cell proliferation, differentiation and remodeling of the extracellular matrix (ECM) to
vascularization and cell migration. Pongo pygmaeus is an orangutan specie native from Borneo.
The genomic DNA of this specie is used for this experiment. The first objective in this
investigation is to transform plasmid with MP gene to E. coli and grow these transformed cells
with plasmid DNA, to purify the plasmid DNA from the culture, and perform plasmid miniprep.
The long term goals are to send purified gene to sequence, determine the sequence of the MP
gene in Pongo pygmaeus genomic DNA, and do a protease detection. The different methods used
in this experiment are transformation, plasmid purification and plasmid miniprep preparation.
Background
Metalloproteases are a group of proteases
collectively responsible for the degradation
of the extracellular matrix. They need a
metal ion on their catalytic site for
functioning. They are important for the
shape and modifications needed in the
extracellular matrix. Metalloproteases are
important in many aspects of biology,
ranging from cell proliferation,
differentiation and remodeling of the
extracellular matrix (ECM) to
vascularization and cell migration. These
events occur several times during
organogenesis in both normal development
and during tumor progression. These
proteins are frequently studied, aimed
specifically to study cell interaction and
tumor formations. They are naturally
regulated by endogenous inhibitors. An
imbalance between the active enzymes and
the natural inhibitors leads to the accelerated
destruction of connective tissue associated
with the pathology of diseases such as
arthritis, cancer, multiple sclerosis, and
cardiovascular diseases.
Pongo pygmaeus is an orangutan specie
native from Borneo. They live in the tree
tops and feed from fruits, bark, bird eggs,
and insects. The genomic DNA of this
specie is used for this experiment because it
is similar to human DNA and the results will
be more accurate to the results it would have
with human beings. By studying the MMP
gene in Pongo pygmaeus genomic DNA we
can control the irregularities in the MMP
production.
Problem
How can we control irregularities in
metalloprotease’s enzymatic activity?
Hypothesis
By the inhibition of the metalloprotease
gene, the irregularities of metalloprotease
enzymes can be controlled.
Significance
This work is important because it would
bring different options to health treatments
for diseases affecting worldwide. It would
also provide the scientific community with
more information about these frequently
studied enzymes.
Specific Aims
The first objective in this investigation is to
transform plasmid with MP gene to E. coli
and grow these transformed cells with
plasmid DNA in liquid culture for the
replication of the plasmid with the cells.
After the replication of the bacteria with the
desired plasmid is finished, the next step is
to purify the plasmid DNA from the culture.
This will remove the plasmid out of the
bacteria and discard the bacteria residues.
The next desired aim after the purification of
the plasmid is to perform plasmid minipreps,
which is to produce small quantities of the
purified plasmid. In a long term aim the
project is going to be focused on sending the
purified desired gene to bioinformatics and
eventually do a protease detection.
Short term goals Obtain a purified plasmid
with desired gene to send for sequencing.
Long term goals Send purified gene to
sequence, determine the sequence of the MP
gene in Pongo pygmaeus genomic DNA,
and do a protease detection.
Procedures
Transformation:
This procedure will be done to insert the
previously ligated plasmid with the desired
gene into the vector used, which is going to
be E. coli. Then these same cells are going
to be cultivated in agar plates, replicating
with them the vector used. The protocol
followed for this procedure was provided by
pGEM®-T and pGEM®-T Easy Vector
Systems Technical Manual.
Plasmid Purification:
After the competent E. coli cells are
transformed and replicated, the replicated
plasmid is purified from the whole cell. By
doing this it will provide only the plasmid
with the metalloprotease gene.
Plasmid Miniprep Preparation:
The objective of this procedure is to produce
small quantities of the purified plasmid. This
samples are the ones used in the
Electrophoresis gel analysis after going
through enzyme digestion. Other miniprep
samples are going to be the ones send for
sequencing. The protocol followed for this
procedure was the Quantum Prep™ Plasmid
Miniprep Kit Instruction Manual. For the
samples that are going to be sequenced the
protocol used was provided by QIAprep®
Miniprep Handbook.
Preliminary Studies
The total of transformed bacteria colonies
incubated in the agar plates at 37°C were 64
colonies in the plate containing 50µl of SOC
medium with bacteria and 540 colonies in
the plate containing 900µl of SOC medium
with bacteria.
References
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