Combinatorial Chemistry and Drug Discovery

 

Lab Procedure:

 

NOTE: KEEP PETRI DISHES COVERED AT ALL TIMES! REDUCE THE RISK OF CONTAMINATION; UNCOVER THEM ONLY WHEN USING THEM.

 

1.   Using a marker, label the bottoms of two petri dishes as shown below.  The bottom is the side that contains the yellow agar.  Do not remove the top cover of the plates.  Divide each of the petri dishes into 3 sections.  Label the sections of one plate M1, M2, M3, and the sections of the second plate M4, M5, M6.  In addition, label the plates with your initials to distinguish them from your classmates.

 

 

 

 

 

 


2.   Turn the plates back over so the bottom is down and the top is up.  Remove the lid and pour ~5 mL of the bacterial culture E. coli TG1 1/100 onto the M1, M2, M3 plate.  Recover the plate and swirl it gently to ensure the liquid culture has completely covered the solid agar.

 

3.   Remove the lid of the M1, M2, M3 plate and the M4, M5, M6 plate and pour the liquid culture from the M1, M2, M3 plate onto the M4, M5, M6 plate.  Cover the M4, M5, M6 plate immediately.

 

4.   Using a transfer pipette, remove the excess liquid from the M1, M2, M3 plate by tilting the plate towards you and sucking off the liquid that collects at the bottom edge of the plate.  Cover the M1, M2, and M3 plate immediately.  Dispose of the excess liquid in the E. coli TG1 1/100 test tube.  Save the transfer pipette for use in step 6.

 

5.   Swirl the M4, M5, M6 plate gently to ensure the liquid culture has completely covered the solid agar.  Remove the lid of the M4, M5, M6 plate and pour the liquid culture into the E. coli TG1 1/100 test tube.

 

6.   Using the transfer pipette from step 4, remove the excess liquid from the M4, M5, M6 plate by tilting the plate towards you and sucking off the liquid that collects at the bottom edge of the plate.  Cover the M4, M5, M6 plate immediately.  Dispose of the excess liquid in the E. coli TG1 1/100 test tube.  Dispose of the transfer pipette in a biohazard waste bag.

 

7.   Using the unlabelled petri dish, practice-making cups in the agar with the glass Pasteur pipette.  Use the large end of the pipette to bore a hole in the agar by pushing it straight down into the agar and then lifting straight up.  Then use the small end of the pipette to remove the small cylinder of agar.  This is best done by pushing the small end of the pipette into the crevice between the small cylinder of agar and the rest of the agar and then leveraging the small cylinder out.  Dispose of the cylinder of agar in the biohazard waste bag.

 

8.   Use the glass Pasteur pipette to make cups in center of each of the "pie sections" of the M1, M2, M3 plate and the M4, M5, M6 plate.  When finished your plate should look like those below.  Dispose of the glass Pasteur pipette in a sharp glass disposal container.

 

 

 

 

 

 


9.   Label the six 1.5 mL tubes M1, M2, M3, M4, M5, and M6.

 

10.  Using the transfer pipette for each solution, add the appropriate reagents to each of the six 1.5 mL tubes according to the table below.  Be sure to add the reagents in the proper order and be sure to add the correct number of drops.  For instance, to prepare M1, add 5 drops B1 to tube M1, and then add 5 drops B2 to tube M1, and then add 5 drops B3 to tube M1, and then add 15 drops A1 to tube M1.

 

 

 

 

 

 

Tube #

Add 5 drops

Then 5 drops

Then 5 drops

Then 15 drops

M1

B1

B2

B3

A1

M2

B1

B2

B3

A2

M3

B1

B2

B3

A3

M4

A1

A2

A3

B1

M5

A1

A2

A3

B2

M6

A1

A2

A3

B3

     Record any observations you make during the addition.

11.  Cap each tube and shake for 10-15 seconds.  Again record your observations of the mixtures.

 

12.  Using a new transfer pipette for each mixture, transfer 2 drops of each mixture to the corresponding cup in the agar plates.  This takes a delicate touch.  If you are right-handed, hold the transfer pipette in your right hand.  Rest your left fist on the bench top and use your left index finger to steady the tip of the pipette.

 

13.  Incubate the two agar plates at 37 °C for 24 h.  Be careful when carrying the plates to the incubator - do not spill any of the liquid out of the cups.

 

Combinatorial Chemistry and Drug Discovery

Data Analysis:

 

  1. Using the table below, record the results of the experiment.  Place a "+" in the table for mixtures that resulted in an inhibition of growth.  Place a "-" in the table for mixtures that resulted in no inhibition of growth.

 

Mixture

Contents

Result

M1

A1, B1, B2, B3

 

M2

A2, B1, B2, B3

 

M3

A3, B1, B2, B3

 

M4

B1, A1, A2, A3

 

M5

B2, A1, A2, A3

 

M6

B3, A1, A2, A3

 

     Each mixture contains three compounds.  For instance, M1 contains the compounds A1-B1, A1- B2, and A1-B3.  Therefore, in the mixtures that show inhibition of growth (antibiotic activity) there are three compounds.  How can we tell which of the three compounds is the one with the antibiotic activity?  In fact, we have already done the experiments needed to make this determination.

 

2.   The table below enables us to determine all the compounds we made.  The outside of the table shows the starting materials for the reactions we performed:  A1, A2, A3, B1, B2, and B3.  Whenever an A molecule comes in contact with a B molecule they react to form A-B molecules.  We can see this in the table by looking at the inside:  when we trace A1 down in the first column, we see that it forms A1-B1, A1- B2, and A1-B3 molecules.  Fill in the rest of the table in the same way by tracing across and down.

 

 

A1

A2

A3

B1

A1-B1

A2-B1

A3-B1

B2

A1-B2

 

 

B3

A1-B3

 

 

 

  1. The inside of the table represents the contents of each mixture M1-M6.  For instance, the mixture M1 is represented by the first column in the table (see the shading in the left table).  Likewise, the second and third columns represent M2 and M3.  Mixture M4 is represented by the first row in the table.

(See the shading in the right table).  Similarly, M5 and M6 are represented by the second and third rows of the table.

 

 

 

 

 

 

 

 

A1-B1

A2-B1

A3-B1

 

A1-B2

 

 

 

A1-B3

 

 

 

 

 

 

A1-B1

A2-B1

A3-B1

 

A1-B2

 

 

 

A1-B3

 

 

 

You can use this table to determine which compound in our active mixtures shows antibiotic activity.  Fill in the table below as you did in part 2.  Shade in the column that corresponds to the mixture M1-M3 that shows antibiotic activity.  Now shade in the row that corresponds to the mixture M4-M6 that shows antibiotic activity.  The position in the table where the shaded row and shaded column intersect is the active compound.

 

 

 

 

 

 

 

A1-B1

A2-B1

A3-B1

 

A1-B2

 

 

 

A1-B3

 

 

 

 

 

 

 

 

 

5.   What is the advantage to working with mixtures?  How many reactions would we need to run in order to make each compound individually?  How many antibiotic activity tests would we need to run in order to screen each compound individually?  Compare these numbers with the number of reactions and antibiotic activity screens we ran in this experiment.