Team:BYU Provo/Notebook/SmallPhage/Winterexp/Period3/Exp/4.25 T7 phage viability test


Small Phage March - April Notebook: Experiments

Small Phage

4.25 T7 phage viability test

I) Purpose

Test the viability of the E coli BL21 and T7+
Perform T7+ titer to determine a workable concentration for selection test
Test phage viability in liquid culture

II) Expected Outcome

E coli BL21 overnight liquid culture should show normal growth; streaking should give multiple colonies
T7 spot test should give results similar to that seen in 4.8 and 4.10 phage viability assay
T7 titer should give results similar to that seen in 4.10 phage viability assay
Phage liquid culture should see clearage, but not in control liquid culture

III) Reagants Used

T7 + phage: dilution from 4.8; E coli BL21; Agar: ×2 prepared by Jordan in 500mL glass bottle; LB: prepared by us on 4.3; overnight bacteria culture: set up on Thursday at 11:15pm using 4.6 streak

IV) Actual Procedure

1. Streak test for E coli BL21

i) E coli overnight liquid culture was set up the night before. At 3.25 10:30 am obvious signs of bacterial growth were seen. Streak test was then performed to check bacteria viability and prepare for storage.
ii) As of 5:00pm NO contamination was seen on plate. We thus proceed to the next step.

2. Perform spot test

i) 0.5mL E coli overnight + 5mL ×1 top agar -> two plates: one for control, one for actual spot test.
ii) Divide spot test plate into 6 sections, labeled -4 through -9
iii) Spot 5 μL of phage solution onto each of the sections. Concentration of phage should match the labeled number.
iv)Both plates were incubated right side up.

3. Perform titer test

i) Four test tubes were labeled -4 through -7. To each of them, 0.5mL of E coli and 20 μL of phage solution (at concentration matching the labeled number) were added. Let this mixture sit for 20 minutes
ii) To each test tube add 5mL of ×1 top agar. Plate.
Note: -7 did not solidify after plating and was disposed before incubation.
iii) Incubate up side down (4.25 6:00pm – 4.26 9:30am for 15.5hr)

4. Liquid culture test

i) 2.mL LB + ½ colony of E coli BL21 was added to two test tubes
Note: colony size is based on estimation, this introduces a huge variable into the experiment.
ii) To one of the test tubes add an extra 20 μL of phage solution (-5); the other will serve as control
iii) Incubate for almost two days (4.25 6:00pm – 4.27 2:00pm)
iv) The supernatant was collected, transferred to two 1.5mL centrifuge tubes, and stored in the fridge.

V) Results

1. Plates

Plate Results
Plate Results
Streak test Multiple colonies; no infection; too many colonies and too small to be stored
Spot test - control Uniform lawn of E coli, no contamination seen
Spot test – experiment (-4 through -9) Obvious clearage for -4 through -6; -7 has a pin-size prick
Titer test -4 Overlapping plaques
Titer test -5 Individual plaques that overlaps in some places
Titer test -6 Individual plaques; streaking seen, possibly due to incubating before agar has completely solidified
Titer test -7 Did not solidify and was disposed before incubation
Liquid culture test As compared to the control, + phage is much more clear

2. Spot test results


This is similar to what we saw before finals. This confirms that our phage dilution series have survived. Hurray!

3. Titer test result


-5 and -6 will be a good place to start our selection test. We will also need to increase phage titer (phage concentration).

4. Liquid culture test (4.27 6:00pm)


Obvious clearage is seen in + phage test tube. E coli debris was also observed at the bottom of the + phage test tube.

VI) Conclusion

T7+ have survived for a week. We can continue to use the 4.8 dilution series for our experiments. This will save us some stock phage.
-5 is a good phage concentration to start our selection test.
The stock phage solution has a medium titer, we need to increase it to perform our mutation experiments.

VI) Proposed next step

Perform selection test with -5 phage.
Increase titer of stock phage for mutation experiments.