Team:BYU Provo/Notebook/SmallPhage/Springexp/Period3/Exp/6.12 Mutagen Concentration Test - Perfected Protocol


Small Phage May - June Notebook: Experiments

Small Phage

6.12 Mutagen Concentration Test - Second Protocol

I) Purpose

To test the perfected protocol for applying 5-bromodeoxyuridine and inducing mutation.

II) Expected Outcome

- A decrease in phage viability with increasing mutagen concentration.
- A few random mutations that produces smaller phage and consequently larger plaques during selection with x8 agar.

III) Reagents Used

5-bromodeoxyuridine stock in freezer labeled T7 (10mg/mL); 40mL of M9+ prepared by the large phage group; BL21 overnight; 5.3 phage stock; x8 top agar; uracil solution (2.5mg/mL); adenine solution (5mg/mL).

IV) Procedure

1) Applying the mutagen (6.12)

- From 5.13 Determining E coli concentration with spectrophotometer, we know that the concentration of E coli BL21 in a liquid overnight culture is about 3.12E8 cell/mL. But we need to resuspend it in M9+ medium.
- To make the M9+ suspension medium supplemented with the appropriate amount of uracil and adenine, 160uL of adenine solution and 320uL of uracil was add 40mL of M9+.
- Label 4 centrifuge tubes (15mL) 0, 100, 200, and 500. To each of these test tubes, add 10 mL of E coli overnight. Cells were then pelleted via centrifuge at 4000rpm for 10min at 7 Celsius. Supernatant was taken out using pipets and discarded. E coli was then resuspended in 10mL M9+ supplemented with uracil and adenine.
- To the four centrifuge tubes, add 0, 100, 200, and 500 uL of 5-bromodeoxyuridine. The volume of the mutagen should correspond to the label on the test tube. Because 5-bromodeoxyuridine stock is a 10mg/mL aqueous solution, the final mutagen concentration in the five test tubes will be 0, 100, 200, and 500 ug/mL.
- Let the bacterial suspension incubate with mutagen for approximately 1h 45min.
- 30uL of 5.3 phage stock was added to each centrifuge tube. From 5.15 Titer Test on 5.3 T7 new Phage Stock, we know that 5.3 phage stock has 7E8 particle/20uL. Thus, to each of the test tubes, we are adding approximately 1.2E9 particle.
- Incubate phage on shaker at 37C for approximately 30min.
- Liquid culture from the four test tubes is then centrifuged at 4000 rpm for 10 minutes to pellet cell debris. Supernatant containing phage particles is removed and 1mL of chloroform is added destroy any remaining E coli cells.
- The stock solutions are now stored in 4 Celsius.

2) Titer to determine phage concentration (6.14)

- For each mutagenesis, specifically 0, 100, 200, and 500ug/mL, dilution series were performed to generate 0 through -6 dilutions (a total of 28).
- For each dilution, a test tube was assigned to it. To each test tube, 0.75mL of BL21 liquid culture overnight was added and then transfected with 30uL of phage dilution. After 20 minutes of incubation, 7mL of x8 agar was added to each test tube and the content was then plated

3) Selection Test 1 (6.17)

- Titering result revealed an adequate plaque number at -2 dilution for each mutagen concentration. We thus plate 50 plates using 500ug -2 dilution and x8 agar. As control, 5 plates were plated using 0ug -2 dilution and x8 agar.

4) Large Plaque Confirmation 1 (6.19)

- We selected 12 of the largest plaques that we could find from our Selection Test 1
- For each plaque, we scrapped off of the plaque with a pipet tip and then dipped it in 100ul of LB in an eppendorf tube.
- To 12 test tubes each, we added 750ul of BL21 overnight.
- We then added 30ul of phage from each eppendorf tube into one of the 12 test tubes and let it incubate at room temperature for 20 minutes.
- Next, we added 7ml of x8 top agar to each test tube and plated it on 12 LB plates.
- Placed in 37 C incubator for about 24 hours.

5) Large Plaque Confirmation 2 (6.24)

- Using the 70uL (100uL - the 30uL used) stock from 6.19, we performed dilution series 1:100 to generate -2 and -4 stock.
- 0, -2, and -4 stock from each plaque was plated using 0.75mL of BL21 overnight, 30uL of phage, and 7mL of x8 top agar. Similar procedures as those in 6.19 were used.
- The plates were incubated in 37 C incubator for approximately 24 hours.

V) Results

1) Applying the mutagen

No obvious signs of clearage was seen after 30 minutes of incubation with phage.

2) Titer to determine phage concentration

There was a little variation in the number of plaques as the mutagen concentration increased, but there was no clear sign of phage death due to the mutagen. Each dilution showed plaques up to -3.

3) Selection Test 1

Each plate had roughly 25 to 50 plaques on it with natural variation in plaque sizes. From all these plaques, we selected 12 that appeared to be bigger than normal.

4) Large Plaque Confirmation 1

All the plates seemed to have been completely or almost cleared by the phage, leaving no visible plaque sizes. Also, the plates were contaminated.

5) Large Plaque Confirmation 2

No abnormally large plaque detected. Plaques are actually smaller than the ones we originally picked from. This could most likely be due to differences in agar concentration.

VI) Conclusion

We decided that mutagenesis did not occur properly again because there was no evidence of phage death with increasing mutagen concentration and the large plaques that we isolated did not retain their size once they were replicated. Poor mutagenesis probably occurred because we only incubated the phage for 30 minutes. In order to attempt to fix this, we will redo mutagenesis, but this time we will incubate the phage until clearage.