Metalloprotease's Presentation
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Transcript of 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