Metalloprotease's Presentation

Ana Velázquez and Michael RubinRISE Program

Department of Biology University of Puerto Rico at Cayey

Metalloproteases> enzymes that comprise a diverse super family of proteases.

Metal ion in their catalytic sites.

Shape and modify extracellular matrix (ECM).

Irregularities can cause tumor formations, metastasis, and other diseases.

Human MMPs are a family of 23 enzymes.

Facilitate turnover and breakdown of the ECM in both physiology and pathology processes.

Native from Borneo

Arboreal specie

Length of 1.5 meters aprox.

Threatened specie

Genomic DNA used for this experiment

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 controled.

Analyze metalloprotease gene sequence for recording.

Bring valuable information related to global health issue.

Short term goals: Obtain a purified gene 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 Do a protease detection

Transform plasmid with MP gene to E. coli

Grow cells with plasmid DNA in liquid culture

Purify the plasmid DNA from the culture

Perform a plasmid minipreps

Send purified gene to bioinformatics

Do a protease detection

Transformation> Insert the plasmid into the vector (E. coli)

Plasmid Purification> Extract the replicated plasmid from the vector

Miniprep> Produce small quantities of purified plasmid

Add competent cells to ligation reaction.Ice for 20min

Add competent cells to ligation reaction.Ice for 20min

Heat shockPut on iceHeat shockPut on ice

Add SOC medium to the tubes:-Transformed cells (950µL)

Add SOC medium to the tubes:-Transformed cells (950µL)

IncubateIncubate

Add -900µL -50µLto plates

Add -900µL -50µLto plates

Incubate plates overnightIncubate plates overnight

Take one colony with micropipette tip from 50µL plate and put it in LB stock with 50µg/mL of ampicillin

Take one colony with micropipette tip from 50µL plate and put it in LB stock with 50µg/mL of ampicillin

CultivateCultivate

Transfer LB broth to micro centrifuge tube.

Transfer LB broth to micro centrifuge tube.

Now we have cells with the desired plasmid

Now we have cells with the desired plasmid

CentrifugeCentrifuge

-Add resuspension solution to the cells (200µL)

-Add resuspension solution to the cells (200µL)

-Centrifugate and remove flow-through

-Centrifugate and remove flow-through

-Add matrix solution (200µL)-Add matrix solution (200µL)

-Remove supernatant-Discard the precipitate

-Remove supernatant-Discard the precipitate

-Add neutralization solution (250µL)-Centrifugate

-Add neutralization solution (250µL)-Centrifugate

-Add lysis solution to the cells (250µL).

-Add lysis solution to the cells (250µL).

-Store column with purified plasmid-Store column with purified plasmid

-Add wash buffer (200µL) to the plasmid.-Centrifugate for 30sec

-Add wash buffer (200µL) to the plasmid.-Centrifugate for 30sec

Marker

Pongo pygmaeus PCR

150bp

200bp

300bp

400bp500bp

600bp700bp

1,200bp

1,400bp

Work by: Angiemar Maldonado

Marker

Pongo pygmaeus cloning

Marker

1,200bp

Work by: Angiemar Maldonado

Vector

Colonies in LB/ampicillin/IPTG/X-Gal plate

50µL SOC medium 900µL SOC medium

64 colonies 540 colonies

Melisa MedinaPaola MontesLydia CortezAndrés BetancourtChristopher Quintanal RISE Program

Ana Velázquez and Michael RubinRISE Program

Department of Biology University of Puerto Rico at Cayey