PCR Amplification and Cloning of Staph. aureus purL Gene Into A Vector

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PCR Amplification and Cloning of Staph. aureus
purL Gene Into A Vector

• Jason Catanzaro
• Brian Jubeck
• April 15, 1999

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Wed first like to apologize for dimming the
lights. We know it interferes with your ability
to do your homework, but it is necessary for the
professors to see the screen. Once again, we
apologize for the inconvenience
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Purine Biosynthetic Pathway
TPP
purF purD purN purL(QY) purM
PRPP
AIR
FGAR
FGAM
purEK purC purB
AICAR
GMP
purH purH
guaA guaB
IMP
purB purA
AMP
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Previous Work With FGAR Amidotransferase

• GOAL Clone and express the genes and purify FGAR
  Amidotransferase proteins
• Laura Singer E. coli purL gene
• Lori Schultz S. aureus purY gene
• Rachel Lawton R. etli purY gene
• Kim Mistiszyn R. etli purQ gene

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Purine Biosynthetic Pathway
TPP
purF purD purN purL(QY) purM
PRPP
AIR
FGAR
FGAM
purEK purC purB
AICAR
GMP
purH purH
guaA guaB
IMP
purB purA
AMP
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Enzymatic Action of the PurL Protein
glutamine
5'-phosphoribosyl-N- formylglycinamide
5'-phosphoribosyl-N- formylglycinamidine
FGAR
FGAM
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FGAR Amidotransferase Organization
glutamine
amide transfer
ATP-binding, cleavage
Type I
Single subunit
PurL
- 1295 aa (E.coli)
Escherichia coli, Salmonella typhimurium,
Haemophilus influenzae, Pseudomonas aeruginosa,
Vibrio cholerae, Neisseria gonnorheae, Neisseria
meningitidis, Saccharomyces cerevisiae,
Schizosaccharomyces pombe, Caenorabditis elegans,
Drosophila melanogaster, Gallus gallus, Homo
sapiens.

Type II
PurL
- 742 aa (B.subtilis )
Multi-subunit
Rhizobium etli, Bacillus subtilis, Lactobacillus
caseii, Staphylococcus aureus, Mycobacterium
tuberculosis, Mycobacterium leprae, Streptococcus
pneumoniae, Enterococcus faecaelis,
Synechocystis spp., Deinococcus radiodurans,
Thermotoga maritima, Aquifex aeolicus,
Archaeoglobus fulgidus, Methanococcus jannaschii,
Methanobacterium thermoautotrophicum.
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Target for Antibiotics

• The Big Picture Identify an inhibitor of Type II
  enzyme for use as an antimicrobial agent
• Type II - inhibitor target
• Staphylococcus aureus recently resistant to
  vancomycin and methicillin
• Rhizobium etli - historical value, mutants
  available
• Type I - for comparison
• E.coli - can use entire gene
• Homo sapiens - need cDNA

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Infection by Staphylococcus aureus.

• Scalded Skin Syndrome.
• Caused by production of exotoxins referred to as
  exfoliatins.
• Toxins distributed through the blood.
• Cause formation of vesicles in skin.
• Bursting of vesicles causes outer layer of skin
  to peel off.

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Bioinformatics - What is it?

• The application of computer technology to the
  management of biological information.
• Computers are used to gather, store, analyze, and
  integrate the immense amounts of genetic
  information presently being gathered.

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Bioinformatics - Why do we need it?

• Essential to the use of genomic information in
  understanding human diseases.
• Full assimilation and exploitation of data from
  the Human Genome Project and other genome
  research and its conversion into useful knowledge.

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Bioinformatics in actionWhat is about to unfold
before your very eyes...

• Two brave students will now attempt to replicate
  the research in colloquium.
• Recap of the process by which we found the
  sequence for the purL.
• It is necessary to find the purL sequence to
  design the primers that will be used in the
  traditional lab work

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Primers
NdeI
.CATATG.
purL

• EcoRI and EcoRV restriction endonuclease sites
  were incorporated for start and stop primers.
• TAA, a stop codon, was not incorporated since it
  would prevent our ultimate goal of creating a
  fusion protein.

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Polymerase Chain Reaction

• Staphylococcus aureus purL gene was amplified by
  Polymerase Chain Reaction
• PCR product was purified using QIAquick
  Purification Membranes

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Multiple Cloning Site (MCS)

• Want to incorporate PCR product into EcoRI and
  SmaI restriction sites of pTyb2 vector

NdeI
.CATATG.
purL
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Restriction Sites

• pTyb2 vector cut with EcoRI and SmaI
• Ligation
• Transformation into E. coli
• Plasmid minipreps
• Restriction digests to detect possible clones

pTyb2
EcoRI
SmaI
EcoRI
EcoRV
PCR product
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Results of E. coli purL Cloning

• We were unsuccessful in cloning the E. coli purL
  gene into the pTyb2bummer.
• Possible Reasons
• large size of purL gene consists of 2.5kb
  pairs
• potentially due to restriction enzymes
• initially tried EcoRV and Sal?, and then EcoRI
  and EcoRV
• neither pairing produced significant results

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Thank You

• We would like to give a shout out to
• Dr. Newman, Rachel, Kim, Lori, and Laura without
  whom none of this would have been possible
  Darwin Mendel our parents - the original gene
  donors that kid in the tenth row with the bad
  haircut Watson and Crick and Dr. Fisher.