Link to this post | posted 24 Apr, 2024 18:47
dane.bowder	Ah- There it is- I was not looking AFTER the lysins for transmembrane domains I was thinking they'd be upstream for some reason- makes total sense now. Those will all end up membrane proteins. Thanks for clarification Debbie!

Link to this post | posted 24 Apr, 2024 15:42

dane.bowder

Hello! WE are in the process of calling functions for the CS2 phage, BigShaq. My students have found a gene that we are struggling with whether to call holin or not- gene 32. The genes in this pham are often called holins in other phages, but not particularly often in CS2s and I am wondering why? There are 4 transmembrane domains found by DeepTMHMM- argues in favor of the holin call It is just upstream of the two lysin A genes, also in favor. We have hits in HHPRED to holin in two chunks that are back to back (both are hitting different parts of PF16081.9), each has great probability, but low E values, I think because there are two hits separated the e value is low. Argues in favor of the holin call- kind of. I don't see any other transmembrane proteins in the region- also argues in favor. So, I would argue that we should call it a holin, but I am wondering if I am missing something that is preventing other phages from calling this holin (other CS2s are also not calling it a membrane protein) Any guidance? Edited 24 Apr, 2024 15:43

Link to this post | posted 28 Sep, 2023 17:08

dane.bowder

Hi Debbie! The patches were from colonies generated from a streak from a mesa that I patch tested. Not totally sure what the plan will be with these- right now I am just exploring! I think some of these lysogeny experiments would be good for our senior research students, so just kind of testing the waters. For now, I will keep working with all four and will test for homoimmunity and phage release in culture. Thanks for the advice! Dane

Link to this post | posted 28 Sep, 2023 16:24

dane.bowder

Hi all- I have been working on generating some lysogens for the Gordonia phage BearBQ. My patch test worked well, and I got good clearing around 8/9 streaks. I have now streaked out the patches to start purification of the bacteria. My question is a practical one- how many of these should I pursue? I streaked out 4 of 8, but do I need to keep all of these going? I guess I am just wondering what the success rate is at this point and how many "backups" I might need to generate a stable lysogen for immunity experiments. Let me know! Dane

Link to this post | posted 31 May, 2023 22:18
dane.bowder	Interesting Debbie- I see what I did wrong. We weren't running UniProt as part of our HHPRED runs, we were using SCOPE instead. I still don't see it in PECAAN. We ultimately did call it an NKF but noted it on our cover sheet. Thanks for the response!

Link to this post | posted 18 May, 2023 16:37

dane.bowder

Hi all- I am running into the same problem with BearBQ gene 4 (stop coordinate is 4232). I am seeing the same mix of MuF-like minor capsid protein (I know to ignore this from the approved functions) and capsid maturation protease in other phages, but I am just not seeing the evidence for the latter. Our only HHPRED hits with E values worth anything are both MuF minor capsids. Seems like maybe this should be an NKF in spite of all of the other phages that have a function called?

Link to this post | posted 12 May, 2023 20:41
dane.bowder	Hi! I have a follow up on this thread. We are looking at BearBQ_21. We are seeing hits in HHPRED for both an M15 peptidase domain and the L-ala-D-glu domains. Is there a preferable choice here? M15 is ranking a bit higher in this particular HHPRED search, but every other phage with this gene calls the L-ala-D-glu. Any feedback?

Link to this post | posted 19 Apr, 2022 18:51

dane.bowder

Hi all, we are annotating ViaConlectus and we found an interesting gene at gp16. We have found that this gene has a MuF-like domain and most other phages call it that- we saw on the boards and the function list that this is no longer an allowed function, so we won't go with MuF in this case. However, we also have strong HHPRED hits to a minor capsid protein. Of note is that this gene clearly has a Hedgehog intein domain as well. We are considering calling it "minor capsid protein with intein domain", does this sound like a reasonable call?