Download Curli Production in Escherichia coli (E

Name ________________________________
Date _______________
Investigating Curli Proteins in Escherichia coli (E. coli)
1. What are biofilms? Give several examples of where they are found.
2. What are curli? What organisms produce curli?
3. How do bacteria use curli?
4. Complete the following table.
Protein
Subunit
Formal
Name
Size
(kDa)
Function of Subunit
A
B
D
E
F
G
5. What is meant by each of the following?
CsgF :
csgA :
MC4100csg :
MC4100csgA :
6. What is a positive control in an experiment?
7. What is a negative control in an experiment?
8. Identify the positive control and negative control in this experiment.
9. What does Congo red dye bind to?
Day 1. Plating of Bacteria
Note: All bacterial strains in this study are Biosafety Level 1 organisms and are not biohazards.
1. Put on gloves. Sterilize workbench space by wiping area with 70% ethanol.
2. Bring agar plates (one LB and one YESCA per group) to room temperature on sterile workbench.
3. Using a Sharpie pen, draw lines on each plate bottom (why not the top?) to divide each plate
into quarters as indicated below.
4. Label each plate bottom along an edge with group names/initials and date. Also label each of
three sections with one of the three cell types (MC4100 WT, MC4100csg, MC4100csgA).
Label the fourth section as “MC4100csgA + csgB” for the interbacterial complementation.
5. Using a sterile plastic inoculating loop or a metal loop sterilized in a Bunsen burner flame, gather
cells of one cell type from a stock culture plate.
6. Streak the corresponding area of the LB and YESCA agar plates with the cells on the loop tip,
following the streak diagram given by your teacher. Discard the plastic loop in the biohazard
waste or reheat the metal loop in the Bunsen burner flame.
7. Repeat steps #6 and 7 above using a new inoculating loop/reheated metal loop for each of the
remaining two cell types.
8. For the interbacterial complementation, use a new inoculating loop to gather a large quantity of
MC4100csgA (acceptor) cells and streak a short vertical line in the fourth plate quadrant. Using
a new inoculating loop, gather a large quantity of MC4100csgB donor cells and streak a short
horizontal line across the first line to form a “plus.” Only do this once!
9. Cover and place the plates upside down (agar on top – why?) in the incubator at 26 oC.
10. Incubate for 48 hours.
Day 3. Checking Bacteria
1.
2.
3.
4.
5.
Put on gloves. Sterilize workbench space by wiping area with 70% ethanol.
Bring group agar plates from the refrigerator and incubator to the sterile workbench and observe.
Record observations on separate paper. Compare your observations with those of other groups.
Using a cell scraper, scrape cells off surface of agar. Record observations of the underlying agar.
Discard cell scrapers, cell products and plates in the biohazard waste bin. Wipe area with
70% ethanol.
Results
Day 1. Plating of Bacteria
Indicate below how your plates were labeled and streaked.
1. Why are plates placed upside down (agar side up) in the incubator or refrigerator?
Day 3. Checking Bacteria
1. Record your observations for each section of your LB-CR and YESCA-CR plates.
2. Compare these observations with at least two other groups. Were they consistent? Why or why
not?
3. What is the difference between LB and YESCA media in terms of curli production?
4. What can you infer regarding the need for protein subunits CsgA and CsgB to produce curli?
5. Explain what you see in the interbacterial complementation assay in terms of what the
donor/acceptor bacterial strains can donate/accept.
6. After scraping cells from the agar surface of both plates, record your observations for each
section.
7. From your observations, what can you infer regarding the production of curli and whether these
fibers are cell-associated or detached for each cell strain?
Propose a Next Experiment:
Using what you have learned about curli structure and using a Congo Red binding assay to study
curli formation, what experiment would you like to do next?
1. Formulate a hypothesis for this experiment.
2. Briefly outline what methods you would use. Indicate which cell strains you would use for
positive and negative controls as well as your experimental cell strain.
3. Last, specify what you would expect to see on your YESCA-CR plates if your hypothesis was
correct.