Attack of the Superbugs Lab Introduction Mrs. Stewart Medical Interventions

Superbugs What is a “superbug”? Bacteria that have become stronger and less responsive to antibiotic treatment

How can bacteria share resistance? Genetic Transfer Method Description Conjugation The one-way transfer of DNA (plasmid) between bacteria in close cellular contact Transformation The genetic modification of a bacterium by incorporation of free DNA from the surrounding environment (usually caused by another ruptured bacterial cell) Transduction The transfer of genetic material from one bacterium to another by a genetic vector (bacteriophage virus)

This lab uses 2 types of Bacteria: E. coli I contains a gene found on the chromosomal DNA coding for streptomycin resistance E. coli II contains a gene found on the plasmid DNA coding for ampicillin resistance.

4 types growth plates LB Agar LB + Str LB = Luria Broth - growth medium for bacterial cells *Str = Streptomycin LB /Str /Amp LB + Amp *Str/Amp = Streptomycin & Ampicillin *Amp = Ampicillin

Lab Day 1: Confirmation Plates Obtain 1 of each type of growth plate 4 plates total Streak E. coli I on 4 plates Streak E. coli II on 4 plates Incubate overnight

How to label the plates I II I II Group Name Group Name

Evaluate Today’s lab (step #1) is called “preparing the confirmation plates”. What are we confirming? These plates will confirm that our E. coli strains are resistant to the antibiotic we said they were

Lab Day 2: Observe confirmation plate results Prepare “mix” plate You will literally be mixing the two types of bacteria together onto one plate Incubate overnight

LB agar LB + Str LB + Amp LB + Str + Amp Predicted Results Observed Results E. coli I E. coli II LB agar LB + Str LB + Amp LB + Str + Amp + = Growth -- = No Growth

Prediction Predict the results from your confirmation plates that you will expect to record

Predict What will happen when the E. coli I strain is mixed with the E. coli II strain?

Evaluate Look at the genes each strain of E. coli carries. If conjugation occurs, specifically describe which strain will transfer what DNA.

Lab Day 3: Use new “mixed” bacterial cultures and spread onto the 3 types of antibiotic plates Incubate overnight

Predict What would you expect to see on these growth plates? Why?

Lab Day 4 Observe and record your results Remember: Pictures of lab results will be required in your lab reports *Growth plates will be discarded after today. Any results not recorded will be lost.

Conclusion Questions Why did the E. coli I strain grow on both the LB agar plate and the LB agar plate with streptomycin but not grow on the LB agar plate with ampicillin? Explain what the results indicate about the new strain of bacteria produced when both strains of E. coli were mixed together. Based on the results of the experiment as well as what you learned about the mechanism of gene transfer between bacterial cells, was the streptomycin resistant gene transferred from Strain I to Strain II, or was the ampicillin resistant gene transferred from Strain II to Strain I? How do you know?

Student Lab Schedule Day: Time Needed: Activity: How to Store: Lab Day 1 ~ 30 minutes Students prepare confirmation plates. Invert the plates, label, and incubate at 37˚C for 24 hours. Lab Day 2 Students observe confirmation plates and prepare “Mix Plate.” Lab Day 3 Students streak cultures from “Mix Plate” onto antibiotic plates. Lab Day 4 ~ 10 minutes Students observe & record results Clean-up all materials.