Link to this post | posted 05 Sep, 2018 13:12

debbie

Jeremy, I think the simplest way to think about that is that "old" bacteria would be a selection bias. Because of the large number of phages that plaque on "old" bacteria, I don't think that is what would stop you from finding phages. But, it will stop you from finding phages we haven't ever found before (because we may have been using "old" bacteria). We have an anecdotal report from Northwestern College that they found a Mycobacterium phage singleton because they selected for tiny plaques and they were using "fresh" smeg. It is an N of 1, but still interesting. Good microbiology says to not use old cultures. So it will never hurt to grow them weekly. The most likely causes of not finding phages from enriched samples are: The smeg is clumpy. Use a Tween starter culture, so you start with individual cells. Keep shaking the culture for 3 days (you can use it as soon as you have a dense enough culture, but then put it back on the shaker. Do not let it sit still on your bench until you are sure cells have reached stationary phase. Otherwise a biofilm will form and clump your culture. Enrichment time is not long enough (or too long). How many days are you enriching? The number of phages found increases with the right timing. Are you enriching at 37? Plate at the same temperature as your enrichments. When (and how often) do you check your plates for plaques? Sometimes, the plaques are so turbid, they can overgrow to look like an uninfected bacterial lawn. Other times, a plaque - especially from the original sample - takes a few days longer to 'appear'. Good luck, debbie

Link to this post | posted 28 Aug, 2018 20:56
debbie	Kayla, I think everything looks as I would expect. Do you think it is all correct now? debbie

Link to this post | posted 27 Aug, 2018 18:56
debbie	Kayla, Would you do dilutions of D29 and not just a single spot? You streak from P1FF streak from stock #2 looks like smeg. When you see 4 day colonies, are they dry and wrinkled? (as in, not smooth and glistening?)

Link to this post | posted 23 Aug, 2018 19:59
debbie	Be careful! The auto-annotation predicts forward strand proteins in the middle of the genome. The proteins in the reverse strand are better supported.

Link to this post | posted 23 Aug, 2018 14:38
debbie	At this time (8-23-18 ), we felt it prudent to call the DNA binding proteins around the integrases "helix-turn-helix DNA binding proteins", even thought 2 of them are hitting Lambda's CI. (Genome used in this evaluation are Nandita and Ryan). Edited 23 Aug, 2018 14:39

Link to this post | posted 23 Aug, 2018 14:35
debbie	Cluster F genomes have 2 tyrosine integrases.

Link to this post | posted 23 Aug, 2018 14:33
debbie	Corgi gp19 and 20 HHPred hit the same domain of the crystal structure of Mycobacteriophage Pukovinik (gp37) excise. Call this a helix-turn-helix DNA binding protein because that is the domain that it hits.

Link to this post | posted 23 Aug, 2018 14:29
debbie	The 4 genes between capsid and tape measure (Corgi gp 8-11) include (in this order) head-to-tail adaptor, head-to-tail stopper, major tail, hypothetical protein. the hypothetical protein in these genomes hits HK97 gp10. Bob Duda of the Hendrix lab (who extensively has studied the structural genes of HK97) agrees the function of this gene, though structural, is unknown.

Link to this post | posted 23 Aug, 2018 14:26
debbie	No tail assembly chaperones have been found….yet.

Link to this post | posted 23 Aug, 2018 14:24
debbie	The cpapsid is fused to the capsid maturation protease (gp7 in Corgi).