Team:BYU Provo/Notebook/LargePhage/Summerexp/Period11/Dailylog


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Large Phage September - October Notebook: October 16 - October 31 Daily Log

Large Phage


Today we tried to finish off a mutagenesis procedure. We found that our new T4 WT Keltzie phage stock produced plaques down to between 10^-6 and 10^-9. I used 1 mL of this lysate to start the mutagenesis procedure, adding it to 25 mL of bacteria that had an optical density of 0.155. However, after 4 hours the bacterial cultures looked more like they had been cleared, rather than getting more turbid due to a superinfection. Adding chloroform confirmed this--if anything, the 5 mL sample we removed and added chloroform to got more turbid rather than clearing. This is the first time we've seen actual clearage in a liquid culture. All of the other times we've created superinfection conditions.

We added 0.5 mL of overnight bacterial culture to each flask to see if we can restart bacterial growth in the flask in superinfection conditions. We'll let this incubate overnight and harvest it tomorrow.




Today we titered out the mutagenesis stuff to see if it's high enough to run a CsCl gradient. We did spot tests of the T4 WT Keltzie, 1x mutagen in LB, 1x mutagen in M9+, and 2x mutagen in M9+. We did spots of 10^-3, -6, -9, -10, and -11. We also spot tested 10^-3 through -6 for the T4 WT liquid culture we used as a stock for all of these mutagenesis procedures.


Yesterday I plated out 10^-6 through 10^-8 for the T4 WT stock to calculate the pfus so we can use it in another mutagenesis and calculate it better. Last time we didn't ever get lysis, and looking at the spot test plates yesterday it appears our superinfection failed because we didn't ever get titers up to 10^-9 at all... some just made single plaques on 10^-6 or not even that far. The T4 WT stock had a titer of 1.2 x 10^10 pfu/mL, so I'll need ~100 uL to infect the liquid cultures.

Today I started another mutagenesis. I grew E. coli B in M9+ overnight, supplemented with 150 uL of adenine. This morning I measured the OD600 of this culture at 0.765. I added 4 mL of this culture to 21 mL of M9+ broth and measured the resulting optical density for each of the four flasks at ~0.15. I had a T4 WT flask with no supplements. Each of the other flasks 150 uL adenine, 250 uL uracil. One flask had 500 uL BrdU and 100 uL phage, another had 1 mL BrdU and 100 uL phage, and one had 500 uL BrdU and 200 uL phage. They were put on a shaker at 9:30 am.

At 2:40 pm, the flasks were still not very turbid. I measured the OD as: T4WT=0.369, M91x=0.395, M9 2x = 0.430, and M9 1x extra phage = 0.348. None of them produced visible lysis when I added chloroform but it was difficult to tell because I could see through the 15 mL tube before I added the chloroform. Some bacterial debris did appear at the interface when I shook the chloroform to mix it with the broth and bacteria, but I couldn't observe "visible lysis" like we saw last time.

We allowed the bacteria to incubate until 4:20pm, and measured the optical density again. T4WT=0.487, M91x=0.506, M9 2x = 0.527, and M9 1x extra phage = 0.442. This showed us that the bacteria was still actively dividing (hadn't all been lysed yet), but that the doubling time is very slow. We left the bacteria in the shaker overnight and refrigerated them the next day until we could process them.

10/28/13 Today we processed the mutagenized phage.