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.

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