The rAmylase Project
Faster, Better Biotech
Ellyn Daugherty
SMBCP, San Mateo CA
www.BiotechEd.com
www.SMBiotech.com
www.emcp.com/biotech
www.sargentwelch.com/biotech
Tulsa Community College February 22-23, 2013
The rAmylase Project (Faster, Better Biotech)
Friday 2/22/12 5-8 pm
5:00 pm
Amylase collection (before you eat – spit into a tube)
5:15 pm
Welcomes/Light dinner/Intro to The rAmylase Project
5:45 pm
Set up Lab 6c Amylase Assay
6:45 pm
Part I - Amylase ELISA Kit (Prepare/plate samples)
7:45 pm
Plan for Saturday (9 am sharp!)
Sat 2/23/12 9-4 pm
9:00 am
10:30 am
11:30 am
12:30 pm
1:15 pm
3:45 pm
Part II - Amylase ELISA Kit (add Ab, colorimetric visualization)
Lab 8b pAmylase Restriction Digestion (kit)
Prepare samples & set up for gel electrophoresis
Run gels and visualize (1XTAE gels vs 1X LB gels)
Lab 5f Amylase PAGE (set up gel, load, run gels during lunch)
working Lunch (results & Intro to Column Chromatography)
Lab 9c Amylase Chromatography (kit)
"What now?"
In Biotech, companies find a product of interest
(amylase) and make sure to have assays to show it’s
presence, activity, & concentration
Amylase is an enzyme that
catalyzes starch digestion.
It is used commercially in
several ways including:
1) Remove starch in products
2) Produce sugar from starch
3) Processing of cellulostic
biofuel
The rAmylase Project
is the focus of the 2nd semester of the SMBCP
• Learn how to assay for a protein of commercial interest (amylase)
• Transform cells to produce that protein (using pAmylase and C2523 cells)
• Scale-up cells to volumes where amylase can be purified and assayed
Absorbance (a.u)
Ion Exchange Chromatography
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
-0.05 0
5
Fractions (#)
10
The rAmylase Project
Model of rDNA/protein Business
< Chapters 1-5 Basic SLOP
< Lab 5f Amylase PAGE (revised)
< Lab 6e Amylase Producing Bacteria
< Lab 6c Amylase Activity Assay (revised)
< Lab 6d Amylase ELISA and W Blot (new)
< Labs 7a/7b/7f/7g Amy Spectrophotometry
< Lab 8b Restriction Digestion of pAmylase
< Lab 8b DNA Gel Electrophoresis (revised)
< Lab 8c pAmy Transformation of E. coli
< Lab 9c/9d Amy Ion-Ex Chromatography
< Lab 8g/4h Genomic & plasmid DNA Isolation
< Lab 13i Amylase Gene PCR (new)
The San Mateo Biotechnology Career Pathway
ELISAs taught in Biotech 2 (Ch 6) and Biotech 4 (Ch 14)
Lab 6c (modified) Amylase Activity Assay
Basic Procedure:
• 2% (modified) starch is added to wells.
• Amylase known or unknown is added to well. 3 min incubation.
• Add Iodine to determine starch remaining.
Introduction to ELISA
ELISA – short for
enzyme-linked immunosorbant assay
An ELISA uses antibodies with
conjugated enzymes to specifically
identify and measure the amount
(concentration) of a protein in a solution
ELISA
•A specific antibody (Ab)
recognizes a specific
protein (Ag).
•An enzyme bound to
the Ab causes a color
change in a substrate
(so you can “see” the
Ab is there).
•The more color in a
well, the more Ab-enz
complex is bound to
target protein = higher
target protein
concentration.
Di
Lab 6d Amylase Elisa Kit Expected Results
<<<<< The Standards should look like this …
…and the unknowns should
look something like this >>>
Automated ELISA Plate Reader
Lab 8b Restriction Digestion of pAmylase (kit)
Basic Procedure:
•Restriction enzyme is added to pAmylase plasmid tubes.
•Mixture is incubated for 15-30 min at 37°C.
•Samples run on agarose gel and visualized on UV imager.
•Band pattern can confirm size and sequence of plasmid.
Fast Restriction Digestions
(HF)and Fast Agarose Gels (LB)
NEB High Fidelity (HF)
Restriction Enzymes
http://www.neb.com
These engineered enzymes have
the same specificity as their
established counterpart with the
benefit of reduced star activity and:
•Fast digestion times (5-15 min)
•Work in same buffer (Buffer 4)
BamHI-HF™
HindIII-HF™
Lithium Borate(LB) Buffers
http://www.fasterbettermedia.com
Lithium borate conducts electricity
better than TRIS so you can turn up
the voltage and current on agarose
gels :
• Fast gel run times (8-15 min)
• Same protocol as TAE gels just
substitute 1X LB buffer for
1X TAE
• Purchase as 10X or 20X
concentrate
Lab 5f (Modified) Amylase on vertical SDS-PAGE
Basic Procedure:
• Prepare diluted samples of known amylase concentration with
SPLD/BME.
• Load and run on NuSep gels at 200-250V ! for 30 min.
• View immediately on UV imaging system and/or stain with
Coomassie blue (long or short method).
Visualizing Protein Samples on Gels
NuSep gel – UV photo:
• Rinse in dH2O, then to viewing
• 2 min UV exposure
• Amylase #1-4, Rennin #5-8
NuSep gel – NuBlue stain photo:
• 15- 30 min stain, no de-stain
• May reuse
• Rennin #3-6, Amylase #7-10
Using Chromatography to Study and Separate Molecules
Paper chromatography. Molecules separate as they move up the paper.
The distance that the molecules travel depends on their size and solubility in the
solvent.
Thin-layer chromatography.
Molecules separate as they move
through the silica gel.
Thin-layer chromatography is used to
separate small molecules, such as
amino acids.
Column Chromatograaphy
Open or Gravity Column
Ion-Exchange Chromatography
Ion Exchange Resin.
Resins are manufactured
with ions attached. The ions
present a certain degree of
positive or negative charge,
depending on the buffer pH.
Column
Chromatography in
Biomanufacturing
Fast-Performance Liquid
Chromatography (FPLC)
Pumps push the buffer or
sample through tubing, into
and through the column.
As fractions come off the
column, they are run
through a
spectrophotometer that
determines the protein
concentration of the sample.
Column Chromatography in Biomanufacturing
Fast-Performance Liquid Chromatography (FPLC)
High-Performance Liquid Chromatography (HPLC)
Greatly improved ability to separate, purify, identify, and qualify samples.
Get more help…
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