DNA is the Flash but Proteins are the Cash of Biotech

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DNA is the Flash but Proteins are the Cash of
Biotech

• pAmylase Restriction Digestion (Lab 8b)

40 bp
Ellyn Daugherty SMBCP, San Mateo
CA www.BiotechEd.com www.SMBiotech.com www.emcp.co
m/biotech www.sargentwelch.com/biotech
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The Amylase Project Model of rDNA/protein
Business
lt Chapters 1-5 Basic SLOP lt Lab 5f Amylase PAGE
(revised) lt Lab 6e Amylase Producing Bacteria lt
Lab 6c Amylase Activity Assay lt Lab 6d Amylase
ELISA and W Blot (new) lt Labs 7a/7b/7f/7g
Amylase Spectrophotometry lt Lab 8b Restriction
Digestion of pAmylase lt Lab 4j/8b/8g DNA Gel
Electrophoresis (revised) lt Lab 8c pAmy
Transformation of E. coli lt Lab 9c/9d Amy Ion-Ex
Chromatography lt Lab 13i Amylase Gene PCR (new) lt
Lab 8g/4h Genomic plasmid DNA Isolation
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We use lots of ASSAYS in Biotech

• DNA
• Presence gel electrophoresis (RE Digests
  PCR), dot blot tests
• Concentration UV spectrophotometry, dot blot
  tests

• Protein
• Presence protein indicators, spectrophotometry,
  PAGE, Western blot
• Activity/Function - protein indicators, enzyme
  activity assays, spectrophotometry
• Concentration protein indicators,
  spectrophotometry, ELISA
• Stability/Shelf-life or other characteristics

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Genetic Engineering uses Recombinant Plasmids
For Amylase Project (Ch 6-9) we trick E. coli
cells to take up the amylase gene (on a pAmy
plasmid) and express it to make large amounts
of amylase. PCR and Restriction Digestion each
can confirm the recombinant plasmid is right.
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The pAmylase Plasmid (Lab 8b Restriction
Digestion)

• pAmy is about 6700 bp long and contains
• The amylase gene
• (from Geobacillus stearrnothermophilus)
• gtgtgt can PCR and run a gel to recognize the gene
• Several restriction enzyme recognition sites
  including
• 1 for HindIII 3 for BamHI,
• gtgtgt see evidence of this using restriction
  digestion and gel electrophoresis.

40 bp

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Agarose Gel Preparation (Lab 4i)

• Determine amount of agarose (g) to use
• Mix it with 1X TAE (or other electrophoresis
  buffer)
• Heat on medium power in microwave until
  completely dissolved
• Cool to 65C
• Place well comb
• Pour agarose to about 7 mm thick
• Allow 10-15 min to cool
• Add electrophoresis buffer before removing comb

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The pAmylase Plasmid after RE Digestion
Draw in the expected DNA fragments produced
during restriction digestion by HinDIII and Bam HI
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Get even more help!
Ellyn Daugherty SM Biotech Career
Pathway www.SMBiotech.com www.BiotechEd.com www.em
cp.com/biotech www.sargentwelch.com/biotech Ellyn_at_
BiotechEd.com
Amy Naum VWR Ed/Sargent Welch www.sargentwelch.com
/biotech Amy_Naum_at_vwreducation.com 585-321-9422
Holly Ahern VWR Ed/Sargent Welch www.sargentwelch.
com/biotech Holly_ahern_at_vwreducation.com
512-789-3155