PM 703 Pharmaceutical Biotechnology Practical Course Revision Fall 2015 Part I. Fermentation Products: Biomass and Glucose Consumption Measurement 06-Dec-15PM703 Pharmaceutical Biotechnology Fall 20152 Biomass and glucose concentrations for an experiment involving yeast growth Time Growth (OD 600 ) Glucose Concentration (mg/dl) Dec-15PM703 Pharmaceutical Biotechnology Fall 20153 Graph-plot showing the increase in biomass and decrease in glucose levels over 120 h 06-Dec-15PM703 Pharmaceutical Biotechnology Fall 20154 Determine the bioburden and glucose levels at 60 h Bioburden = 0.85 at 60 h Glucose Concentration = 100 mg/dl at 60 h 06-Dec-15PM703 Pharmaceutical Biotechnology Fall 20155 Part II. Determination of Gas (CO 2 ) Production by Yeast Fermentation 06-Dec-15PM703 Pharmaceutical Biotechnology Fall 20156 Procedure Add 10 ml Distilled Water Add 10 ml 1% Glucose Solution cT 06-Dec-15PM703 Pharmaceutical Biotechnology Fall 20157 Procedure (Contd) Add 5 ml Yeast Extract cT 06-Dec-15PM703 Pharmaceutical Biotechnology Fall 20158 Procedure (Contd) cT Place in Water Bath at 40C and remove after 15 min. 06-Dec-15PM703 Pharmaceutical Biotechnology Fall 20159 Procedure (Contd) cT Falcon Tube (TEST) showing bubbles while (CONTROL) doesnt show any, thats due to CO 2 production from fermentation of glucose in the TEST. 06-Dec-15PM703 Pharmaceutical Biotechnology Fall Part II.Determination of Amylase Activity in Starch Degrading Soil Isolates 06-Dec-15PM703 Pharmaceutical Biotechnology Fall Initial Test Tube BTest Tube A Add 1 ml of Phospate Buffer Saline (PBS), pH 7 to Positive Control test tube and 1 ml of Supernatant A to Test Tube A and 1 ml of Supernatant B to Test Tube B Procedure 06-Dec-15PM703 Pharmaceutical Biotechnology Fall Initial Test Tube BTest Tube A Add 0.5 ml of Starch Solution to each test tube Incubate at 37C or 50C for 5 min. Procedure 06-Dec-15PM703 Pharmaceutical Biotechnology Fall Positive Control Test Tube BTest Tube A Stop the reaction by adding 1 ml of 0.1 M HCl to each test tube Add 1 ml of I 2 /KI solution to each test tube Measure blue color at 620 nm Blank is done containing all the ingredients except starch and supernatant (instead add 1.5 ml PBS) Procedure 06-Dec-15PM703 Pharmaceutical Biotechnology Fall Calculations and Interpretation of Results Using the equation mentioned above, determine the activity of each supernatant. The supernatant with a greater value has a greater enzyme activity. 06-Dec-15PM703 Pharmaceutical Biotechnology Fall PM703 - Clinical Pharmaceutical Biotechnology Fall Weak Amylase ActivityModerate Amylase Activity High Amylase Activity Little or no hydrolysisPartial hydrolysisComplete hydrolysis Degree of hydrolysis of starch Dark blueGreenish blueyellow Color appears after + Iodine solution Needs dilution *Take 1ml and add to 9 ml water.read abs **Abs is then divided by the dilution factor (0.1) **Mainly in case of tube (I) readable Spectrophoto meter device Dil. Part III. Important Spots 06-Dec-15PM703 Pharmaceutical Biotechnology Fall Biosurfactant Activity: Emulsification Index (E 24 ) Questions What is the name of the test? Emulsification Index What is the result of the test? = 2/4 X 100 = 50 % What is the use of the test? Qualitative determination of biosurfactant activity 2 cm 4 cm 06-Dec-15PM703 Pharmaceutical Biotechnology Fall Biosurfactant Activity: Cetyl Trimethyl Ammonium Bromide (CTAB) Plates Questions What is the name of the test? CTAB Which of the three SAA has the most activity? Sort according to the greatest zone of inhibition What is the use of the test? Semi-quantitative determination of biosurfactant activity of anionic biosurfactants 06-Dec-15PM703 Pharmaceutical Biotechnology Fall Amylase Activity Questions What is the name of the test? Determination of amylase activity in soil degrading isolates Does the bacteria have amylase activity? Negative result Positive result 06-Dec-15PM703 Pharmaceutical Biotechnology Fall Screening for Lactic Acid Bacteria in Dairy Products Questions What is the name of Spot A? Lactobacillus spp. growing on MRS Agar Medium What is the name of Spot B? Lactic Acid Bacteria stained using Crystal Violet (Simple Staining) A B 06-Dec-15PM703 Pharmaceutical Biotechnology Fall Screening for some Protease- containing Drugs Questions What is the name of the test? Determination of protease activity in different preparations in the market Which of the preparations shows the greatest protease activity? Sort according to the greatest zone of inhibition 06-Dec-15PM703 Pharmaceutical Biotechnology Fall Part IV. Bioinformatics The Genetic Code 06-Dec-15PM703 Pharmaceutical Biotechnology Fall Do You Speak MolBio? What is the reverse, the complement, the reverse complement, and the message of: AAGGCCTTGCTTCG What is the amino acid encoded by: GUC Translate: AGC ATG ATT CTG GAA TAG CTA G Reverse translate these peptides: PHARMACY, HAPPY Write your name using the genetic code (ignore non- applicable letters) 06-Dec-15PM703 Pharmaceutical Biotechnology Fall Questions Transcription Convert DNA to RNA sequence Translation Convert RNA sequence to protein 06-Dec-15PM703 Pharmaceutical Biotechnology Fall Good luck from the PM703 Course teaching team 06-Dec-15PM703 Pharmaceutical Biotechnology Fall