Faster, Better rAmylase Project

Ellyn DaughertySMBCP, San Mateo CAwww.BiotechEd.comwww.SMBiotech.com

www.emcp.com/biotechwww.sargentwelch.com/biotech

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 4j/8b/8g 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 goal of The rAmylase Project is to

• 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

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 products2)Produce sugar from starch3)Processing of cellulostic biofuel

Assay to Confirm Amylase Presence

SDS-PAGE Gels (Lab 5f )

On denaturing gels, can learn:

• # of chains in the protein

• Size (kD) chain and approximate #AA

• Size (kD) protein and approximate #AA

• A band on a gel is like a fingerprint of the polypeptide or protein

Modified Lab 5f protocol

Improvements:

• Add BME to loading dye

• NuSep gels run at 250-300 V (30-40 min)

• Use new NuSep Fast PAGE gels with UV indicator (instant viewing)

• Use new 15 min (fast) NuSep Stain

Lab 5f mini-protocol for workshop

Procedure: Wear gloves and googles to set up the gel.1. Prepare two 10-well, 10% NuSep SDS-PAGE gels by rinsing off the preservative and placing each in the correct orientation (wells exposed to the middle of the gel box) in the gel holder cassette of a vertical gel box. 2. Fill the inner chamber of the gel box with Laemmli (SDS-PAGE) running buffer. If there is a well comb, remove it. 3. Use a micropipet and tip to gently flush each well with running buffer (3X 35 µL). 4. Obtain a sample of 5-mg/mL amylase in Sample Prep Loading Dye Buffer/BME that has been heated for 3 min at 90°C. 5. Load wells 1-4 and 6-9 with 30 µL of amylase sample each.  6. Load 10 µL of protein standard markers in to wells 5 and 10 of each gel. 7. Hook up the power supply and run the gel at 200-300 volts for about 30-40 minutes, until the loading dye reaches the bottom of the gel about 20 min. 8. Stop the gel run and gently remove the gels from the cassette. Place them in a tray and rinse with dH 2O. 9. Place the gel on a UV light box for 2 min to visualize the banding pattern. 10. Place the gel back in to a staining tray and add NuBlu® Express Stain to just cover the gel. Cover the tray with foil and stain for 15-30 min. Observe the banding pattern of amylase.

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

Confirm the Protein’s ActivityProcedure * Caution: Treat saliva as a biohazard. Wear gloves and dispose of properly. * Note: Change tips with each dispensing. 1. Collect approximately 500 µL of saliva into a 1.7 mL tube.  2. Obtain a clean, 24-well plate. Study the labels heading the columns and rows. 3. Place 1 mL of 2% starch solution (Alfa Aesar® modified soluble starch solution in dH2O) in the wells of columns 1, 2, 3, and 4 rows A, B, and C, a total of 12 wells. 4. To Column 1, add 300 μL of human salivary amylase solution to each of the starch-filled wells. Mix well. 5. To Column 2, add 300 μL of 5 mg/mL bacterial amylase (in 100 mM KH2PO4, 6 mM NaCl, pH 6.9) to each of the starch-filled wells. Mix well. 6. To Column 3, add 300 μL of 1 mg/mL bacterial amylase (in 100 mM KH2PO4, 6 mM NaCl, pH 6.9) to each of the starch-filled wells. Mix well. 7. To Column 4, add 300 μL of the 100 mM KH2PO4, 6 mM NaCl, pH 6.9 buffer to each of the starch-filled wells. Mix well. 8. Allow the samples to incubate at room temperature for 15 min.  9. While waiting, identify the positive and negative controls in this experiment. Make and record predictions as to what may occur in each well, including the expected color changes. 10. Add 20 μL of iodine to each well. Mix each for 2 seconds with the pipet tip.  111. Place the 24-well tray on a piece of white paper, out of direct light. Iodine decolorizes in light.  12. After 15 min, measure the amount of starch breakdown in each well by recording the degree of lightening of the iodine solution from black (0) to a light golden-brown color (5).

Amylase Activity Assay

Fast Restriction Digestions (HF)and Fast Agarose Gels (LB)NEB High Fidelity (HF) Restriction Enzymeshttp://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

Faster Better Restriction DigestionProcedure1.Obtain 1 of the reaction tubes. These already contain 4 µL of 0.2 µg/µL pAmylase, H20, and 10x reaction buffer.2. Depending on the tube label, add one of the restriction enzymes listed in the reaction matrix below:

3. Finger flick the tube to ensure it is well mixed. Wrist-flick the sample to the bottom of the tube.4. Label the tube and make sure it is securely closed.5. Place samples in 37°C heat block for 5-15 min.6. Locate a six-well, 0.8% agarose in 1X LB buffer, covered with about 2 mm of 1X LB buffer. 7. Add 4 µL of 5x LB loading dye into the digested samples.8. Load all 24 µL of sample and 7 µL of standards into a well in the gel. Load as follows:

9. Put lid on gel box and plug into power supply. Run the gel at as close as possible to 300 V. Record the current >>>_____mA.10. Run the gel until the Orange G loading dye is 1 cm from the end of the gel (about 15 min).11. Put on gloves and remove gel from the gel box and place in weigh boat. 12. Submerge gel in 0.5 µg/mL EtBr.13. Allow the gel to stain for 20 min and visualize on a UV light box. 

New, Improved rAmylase Project

Biotechnology Curricular Support

www.emcp.com/biotechCheryl Monson

CMonson@emcp.comfor books/IRC help

www.BiotechEd.comfor PPTS, Syllabi, Acitivities,

Hints, Links, etcEllyn@BiotechEd.com

www.SargentWelch.com/biotechJana Penders

for materials/equipment support Jana_Penders@vwreducation.com