Combinatorial Chemistry Lab A - Reagent and Media Prep

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Objective

Students will successfully perform mathematical calculations required to prepare the media, reagents, and compounds needed for the research and development of a novel drug therapy based on techniques used in the field of Combinatorial Chemistry.

Big Idea

Mathematics is the foundation of any type of scientific research!

Introduction

10 minutes

Combinatorial Chemistry is a technique used to synthesize a library of compounds and screen for a desired property. Instead of screening one compound at a time, the compounds are screened more efficiently in mixtures which maximize the use of resources while increasing the possibility of identifying an active compound.  This is a key goal of the pharmaceutical industry and thus a common practice in drug discovery.

This lab protocol was adapted from the original work of Scott Wolkenberg and Andrew Su of The Scripps Research Institute (TSRI) in La Jolla, California. The experiment was originally published in the June 2001 issue of the Journal of Chemical Education (Wolkenberg, Scott E.; Su, Andrew I. J Chem.Educ. 2001 78 784) and implemented in the San Diego area from May 2002 thru 2005.  Through collaboration between the Southern California Biotechnology Center at Miramar College, Junipero Serra High School Biotechnology and Dr. Floyd Romesberg, an expert in the area of antibiotic resistance and professor of chemistry at TSRI, the protocol was revised for implementation in a high school setting.

Revision by: Jasmine Erfe Miramar College/Lab Technician, Ericka Senegar-Mitchell Science in the City/Director; Junipero Serra High School/Science Educator, Sandra Slivka Southern CA Biotechnology Center @Miramar College/Director

Objective(s):

  • Students will successfully perform mathematical calculations required to prepare the media, reagents, and compounds needed for the research and development of a novel drug therapy based on techniques used in the field of Combinatorial Chemistry.(Lab A)
  • Students will identify a drug that kills bacteria by producing libraries of compounds based on the A-B model. (Lab B)
  • Students will test the mixtures for antibiotic activity and then isolate the individual compound(s) which possess antibiotic properties. (Lab C)
  • Students will screen the mixtures by utilizing techniques used to conduct Kirby-Bauer and Ouchterlony tests. (Lab C)

 

NOTE: Instructor's are encouraged to complete the pre-lab activities found in the lesson entitled, "Combinatorial Chemistry - Designing Novel Drug Therapies to Combat Antibiotic Resistance" before completing this laboratory investigation series.

Materials

10 minutes

For the Instructor:

PREPARING FROZEN LOG PHASE E. COLI (For use in Combinatorial Chemistry Experiment)

An E. coli culture will be grown into log phase using a spectrophotometer and then frozen with %15 glycerol in 1ml aliquots. Depending on the number of samples taken from the culture for spectrophotometer readings, this protocol will supply approximately 18-26 1-ml aliquots of culture, each sufficient for 1 Combinatorial Chemistry experiment. Each student group will be provided 1.0 mL of frozen log phase E. coli in 15% glycerol sufficient for 3 plates (~300ul/plate). Store and thaw E. coli on ice until ready to use. 

MATERIALS

Frozen E. coli stock in 30% glycerol, non-competent (LMG used in protocol)

35ml sterile LB broth, room temperature

1 - culture tube + cap, autoclaved

1 - 50 ml Erlenmeyer flask + cap, autoclaved

~20 – 1ml cryogenic tubes

1 inoculating loop

3-5 test tubes

Kimwipes

1000µl micropipette + tips

Spectrophotometer

 

For the Student:

MEDIA AND REAGENT PREP CALCULATIONS (Combinatorial Chemistry Experiment)

Students should complete the compound prep worksheets Part 1 and 2 in lab groups of 2-3 students.

MATERIALS

Combi Chem Student Protocol

Combi Chem and Drug Discovery Student Compound Prep WS Part 1

Combi Chem and Drug Discovery Student Compound Prep WS Part 2

Calculator

Methods

45 minutes

For the Instructor:

Preparing overnight culture of E. coli

1. Transfer 10ml of sterile LB broth in a culture tube.
2. To take a sample of E.coli, take the inoculating loop and dip into the frozen E. coli stock, making sure that some of the stock is on the loop.
3. Dip the inoculating loop into the culture tube containing the 10ml of LB broth. Stir the broth using the loop to ensure that the E. coli is thoroughly mixed in with the broth.
4. Cap the tube and incubate the E. coli culture overnight at 37°C with agitation.

Growing E. Coli into Log Phase

1. Transfer 25ml of sterile LB broth in a 50ml Erlenmeyer flask.
2. Take 1ml of the prepared overnight E. coli culture and transfer into the Erlenmeyer flask containing 25ml of LB broth.
3. Cap the Erlenmeyer flask and incubate at 37°C with agitation.
4. Meanwhile, turn on the spectrophotometer and set to O.D. 550.
5. After 30 min, take a 3ml sample of the E. coli culture and transfer into a test tube. Wipe and handle the test tube with Kimwipes and place into the spectrophotometer.
6. Note the reading. The culture will reach log phase once the O.D.550 reaches 0.3-0.4.
7. If the reading has not reached log phase, continue steps 5 and 6 until the culture has reached log phase, using a new test tube for each reading.
8. Once the culture has reached log phase, note the total volume of culture that remains in the flask. Based on this amount, add 15% of glycerol into the flask and stir.
9. Using a 1000µl micropipette, immediately transfer 1ml aliquots of the log phase culture into the cryogenic tubes and freeze at -80°C.

For the Student:

1. Students should review the Combi Chem Student Protocol as well as the Molarity Concentration Equation (Part I) and the % Mass/Volume Concentration Equation (Part II).

2. Instructors will provide information such as total volume wanted of each type of media, reagent, or compound being prepared.

3. Complete calculations using the tables provided.

Results

15 minutes

Final Calculations for the Preparation of Solutions

ID

Compound Name

Molecular Weight (MW)

Mass to Add (mg)

Volume of diH2O (mL)

A1

2-nitrobenzaldehyde

151.12

54

12

A2

5-nitro-2-furaldehyde

141.09

51

12

A3

3-nitrobenzaldehyde

151.12

54

12

B1

4-bromophenylhydrazine hydrochloride

223.52

80

12

B2

4-cyanophenylhydrazine hydrochloride

169.62

61

12

B3

Aminoguanidine bicarbonate

136.11

49

12

 

Students should plan to complete calculations that would enable each group to prepare 12 mL of each .03M solution. It is important the concentrations of the solutions be correct. Before using, tightly cap each tube and shake vigorously for approximately 10 seconds.

Discussion

30 minutes

Instructors may want to take advantage of the natural pause in the lab and discuss the lab write-up component of the investigation. Group Lab Report A and Lab Report B are examples of lab reports completed by students. After discussing the application of the scientific method in this manner groups should be given an opportunity to begin composing the initial sections of their group lab report (i.e. focus question, hypothesis, materials, and procedure).