Team:BYU Provo/Notebook/SmallPhage/Summerexp/8.16 Modeling phage plaque size

Small Phage

March-April

May-June

July-August

September-October

• Utilizing knowledge gained from 8.2 Modeling Phage Plaque Size - Preliminary Experiments, we will model phage plaque size against phage particle size and genome size.

II) Expected Outcome

• A inverse relationship between phage plaque size and phage particle size/genome size.

III) Reagents Used

• Accurate x2 top agar made using a volumetric flask.
• LB
• E coli B.
• Stocks of phage: T1, T2, T3, T4, and T7.

IV) Procedure

1) Plating (8.16)

• To see a statistical significance in plaque sizes between different phages, many plaques are needed. Thus 10 plates were used for T1, T2, T3, and T7. Because T4 form much smaller plaques, only 3 plates were used.

• Experimental procedures are as follow: 0.5mL of E coli B (OD 0.5, adjusted after initial measurement of 0.558) was infected with 20uL of respective phage dilution samples for 10 minutes. 5mL x1 top agar was then added to each test tube, and the content was plated. The plates were incubated at 37 Celsius for 24 hours.

2) Propagation and spot test of T1 (8.12-8.20)

• Propagated T1 by infection 1mL E coli B and 4mL of LB with 10uL of stock phage. Complete clearage was observed in two days before purification via centrifuge (3000rpm for 5min) and chloroform (100uL of chloroform to 1mL of lysis). This stock of T1 is labeled T1P.

• Dilution series -2 through -12 was performed for T1P. 5uL of each dilution sample was spotted onto plates overlaid with 0.5mL of E coli B and 5mL of x1 agar.

3) Spot test for T2 from stock (8.20)

• Dilution series -2 through -7 was performed for T2 from stock. 5uL of each dilution sample was spotted onto plates overlaid with 0.5mL of E coli B and 5mL of x1 agar.

4) Preliminary Titer (9.8)

• Based on results from spot test for T1P and T2, titer was performed for T1P at -6, -7, and -8; and T2 at -2, -3, and -4. Specifically, 0.5mL of E coli B liquid culture overnight was infected with 20uL of respective phage sample. After 15 minutes of incubation, 5mL of x1 top agar (not precise) was added to each test tube, and the content was plated. The plates were incubated at 37 Celsius for 20 hours.

5) Repeated plating for T1 and T2 (9.9)

• Preliminary titer revealed that T1P at -7 and T2 at -4 have adequate concentrations for plating. Plating procedures were the same as those recorded on 8.16. The only difference is the initial OD 600 reading of E coli B liquid culture overnight and subsequent dilutions: the average between two OD 600 readings was 0.513, but dilution was performed right after to adjust it to 0.5.

V) Results

1) Plating

• T1 plates had obvious contamination. Results need to be repeated.

• T2 still formed larger than expected plaques.

2) T1 and T2 spot test

• T1 showed plaques up to -8 after propagation, while -2 showed plaques up to -4.

VI) Conclusion