Link to this post | posted 08 Dec, 2023 00:12
debbie	The transposase interrupts the N terminus of the immunity repressor (gp 43), and does not appear to be expressed (preliminary testing in the Hatfull lab). Therefore no immunity repressor gene has been called. Some details are in the attached document. 12Kb

Link to this post | posted 08 Dec, 2023 00:06
debbie	The immunity repressor in Feyre (gp 43) is interrupted by the insertion of the transposase. Preliminary testing shows that Feyre does not make a lysogen (and the immunity repressor is not working).

Link to this post | posted 01 Dec, 2023 04:59

debbie

More papers about cluster K phages: Mycobacteriophage ZoeJ: A broad host-range close relative of mycobacteriophage TM4 https://pubmed.ncbi.nlm.nih.gov/30948168/ A peptidoglycan hydrolase motif within the mycobacteriophage TM4 tape measure protein promotes efficient infection of stationary phase cells https://pubmed.ncbi.nlm.nih.gov/17083467/ Mycobacteriophage TM4: genome structure and gene expression https://pubmed.ncbi.nlm.nih.gov/10645443/ Genome Sequences of Mycobacteriophages Amgine, Amohnition, Bella96, Cain, DarthP, Hammy, Krueger, LastHope, Peanam, PhelpsODU, Phrank, SirPhilip, Slimphazie, and Unicorn https://pubmed.ncbi.nlm.nih.gov/29217790/ Genome Sequences of Mycobacteriophages Findley, Hurricane, and TBond007. https://pubmed.ncbi.nlm.nih.gov/29122862/ Engineered bacteriophages for treatment of a patient with a disseminated drug-resistant Mycobacterium abscessus https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6557439/ Edited 01 Dec, 2023 05:05

Link to this post | posted 05 Oct, 2023 17:48
debbie	Hi all, I chatted with Graham about this and here's what i have gleaned from the conversation. Temperature and pH can affect how well the calcium goes into the solution. Maybe try a different source of peptone or yeast extract to see if it improves. best, debbie

Link to this post | posted 05 Oct, 2023 15:16
debbie	Eric, Yep. Calcium does precipitate out of solution at high temps. You can omit Ca in the autoclaved recipe and then add it back as you use the agar. You can also play with the concentrations. What you can't do is omit calcium. Many phages need calcium for adsorption, infection and stability. Make sense? debbie

Link to this post | posted 28 Sep, 2023 17:02

debbie

Hi Dane, In general, we keep one. But until the 'one' has been verified, meaning that the lysogen infection pattern 1) shows homoimmunity to the phage and 2)the phage is detected in the supernatant of the lysogen, more than 1 is advisable. The answer is also dependent on how the different streaks were obtained - from single colonies or out of a 'mesa', because having 4 different clones would be advisable depending on what you want to do. So what questions are you going to investigate with your newly made lysogens? Best, debbie

Link to this post | posted 21 Aug, 2023 16:35

debbie

I agree with your student. I would not call it. From my collegue, Christian Gauthier, "It looks to have an extremely poor D-loop and a weird anticodon loop. Acceptor stem and pseudouridine stem/loop both look OK. If I were a gambling man, I’d speculate that there used to be a tRNA there, but it has undergone some mutational decay and is almost certainly defective. I wouldn’t be inclined to annotate it." Best, debbie

Link to this post | posted 08 Aug, 2023 13:24

debbie

The immunity repressor (pham 99175) from cluster A is present in other cluster genomes along with their own cluster specific immunity repressor– specifically C1 (LittleE) , K (, and F1(DLane), CA (Phrankenstein), J(Courthouse), and K (SamScheppers). Graham described this “immunity theft” in Pope et al 2011 in PLoS One https://pubmed.ncbi.nlm.nih.gov/21298013/ From Rick Pollenz: Some of the cluster F1 (and the others Debbie mentioned) have TWO “immunity repressors”, one that is grouped to those mostly from cluster A (Pham 99175) and a second one that is specific to the cluster phage. These rogue one is typically found in a set of 2-3 reverse genes upstream of the integrase. The 2nd repressor is found downstream of the integrase in a 2nd set of reverse genes and groups to a different PHAM. Both sets of reverse genes are on during lysogeny and this is nicely illustrated in the RNA seq figure 4 for phage Fruitloop in the gp52 Wag paper (https://onlinelibrary.wiley.com/doi/10.1111/mmi.13946) where gp37 is the “rogue” hetroimmunity repressor and gp44 is the putative immunity repressor.

Link to this post | posted 08 Aug, 2023 13:22

debbie

The immunity repressor (pham 99175) from cluster A is present in other cluster genomes along with their own cluster specific immunity repressor– specifically C1 (LittleE) , K (, and F1(DLane), CA (Phrankenstein), J(Courthouse), and K (SamScheppers). Graham described this “immunity theft” in Pope et al 2011 in PLoS One https://pubmed.ncbi.nlm.nih.gov/21298013/ From Rick Pollenz: Some of the cluster F1 (and the others Debbie mentioned) have TWO “immunity repressors”, one that is grouped to those mostly from cluster A (Pham 99175) and a second one that is specific to the cluster phage. These rogue one is typically found in a set of 2-3 reverse genes upstream of the integrase. The 2nd repressor is found downstream of the integrase in a 2nd set of reverse genes and groups to a different PHAM. Both sets of reverse genes are on during lysogeny and this is nicely illustrated in the RNA seq figure 4 for phage Fruitloop in the gp52 Wag paper (https://onlinelibrary.wiley.com/doi/10.1111/mmi.13946) where gp37 is the “rogue” hetroimmunity repressor and gp44 is the putative immunity repressor.

Link to this post | posted 03 Aug, 2023 20:20
debbie	Hi Steve, Will you record? Thanks, debbie