Link to this post | posted 17 Jan, 2018 20:24

lhughes

Steve, I have always been successful in doing this in the past, and it gave me the same problem just now when I tried it. My DNA Master shows Build 2534 (14 Sep 2017). The only other thing that I know has changed on my computer is that I recently did an update to the most recent version of Windows 10. Help! I'm going to need to be able to download some more genomes soon for some QC I'm working on. Lee

Link to this post | posted 29 Aug, 2017 17:15
lhughes	Tamarah Adair I am having this issue also. It is returning tRNA calls, for example on Amigo, but no genes. Help please. Tammy Tammy, This post from Debbie in another thread may answer your question about the gene calls: https://www.seaphages.org/forums/post/5735/ Lee

Link to this post | posted 27 Aug, 2017 21:21

lhughes

Another one from Abt2graduatex2: RNase adapter protein RapZ The genome has this function (not on the approved list) for gp55. I compared the HHPRED data for the equivalent gene in BabyGotBac (gp56, which was annotated as "ATPase" and found there is 100% probability, 90% coverage, and E-value of 0 to the RNase adapter protein (RapZ in E.coli). The function in Uniprot is given as "Modulates the synthesis of GlmS, by affecting the processing and stability of the regulatory small RNA GlmZ" Thoughts on this function? Lee

Link to this post | posted 24 Aug, 2017 00:59

lhughes

Here is a new one to consider: Sortase This one is described in the Abt2graduatex2 genome I am QCing, but for a phage already annotated it can be found in BabyGotBac_44. Description of "sortase" below. It looks like to me that these gene products have the conserved catalytic triad. They show 98% homology in HHPRED with an E-value of 10^-9 Sortase is described in CDD as: "Sortases are cysteine transpeptidases, mainly found in Gram-positive bacteria, which either anchor surface proteins to peptidoglycans of the bacterial cell wall envelope or link proteins together to form pili by working alone, or in concert with other enzymes. They do so by catalyzing a transpeptidation reaction in which the surface protein substrate is cleaved at a conserved cell wall sorting signal and covalently linked to peptidoglycan for display on the bacterial surface. Sortases are grouped into different classes based on sequence, membrane topology, genomic positioning, and cleavage site preference. The different classes are called class A to F sortases. Most Gram-positive bacteria contain more than one sortase and it is thought that the different sortases attach different surface protein classes. The typical eight-stranded beta-barrel fold is observed in all known sortases, along with the conserved catalytic triad consisting of cysteine, histidine and arginine residues. Some sortases contain an N-terminal signal peptide only and the C-terminus serves as a membrane anchor, which represents a type I membrane topology, with the N-terminal enzymatic portion projecting towards the bacterial surface and the C-terminal end residing in the cytoplasm. Other sortases adopt a type II membrane topology, with the N-terminal hydrophobic segment inside the cytoplasm and the C-terminal enzymatic portion located across the plasma membrane. The N-terminus either functions as both a signal peptide for secretion and a stop-transfer signal for membrane anchoring. Sortases are also present in some Gram-negative and Archaebacterial species, but the functions of these enzymes are unknown." So, what do you think? Sortase, or cysteine transpeptidase, or not a function we add to the list? Lee

Link to this post | posted 14 Aug, 2017 17:59
lhughes	Working now! Thanks, Chris

Link to this post | posted 14 Aug, 2017 16:22
lhughes	Chris - I found today that some phams aren't linking to reports from PECAAN, and then I checked your direct link above to your starterator website and found that the highest Pham # available is 32344, while the phams I was trying to view all had a higher number than that. The files all show a run date of 8/7/17. Is it possible that not all phams were updated or generated on that date? Lee

Link to this post | posted 10 Aug, 2017 17:14
lhughes	Here is some more information: BeardedLady_37 gives hits mostly to the archael HJC on HHPRED (this is a BD1 phage) Sushi23_89 gives one hit to an E.coli HJC and the rest are RuvC (this is a BE1 phage) (data screen shots attached) 265Kb

Link to this post | posted 08 Jul, 2017 20:08
lhughes	Thanks for the link, Steve.

Link to this post | posted 07 Jul, 2017 21:03
lhughes	Welkin Pope Yep, we can add officially. give me a phage name and gene number. Aaronocolus_57 I already put it down on the "pending" section of the working list. Will we go with written out "deoxynucleoside monophosphate kinase" or "dNMP kinase"?

Link to this post | posted 03 Jul, 2017 21:50

lhughes

Hi Joyce: At UNT we currently prepare spore suspensions by the following procedure: Spore Harvest Note: Prior to use, all solutions and apparati should be sterilized by autoclaving. 1. Pipet 6-8ml dH2O to a well-sporulating plate (2-3ml for slant cultures). 2. Scrape of growth with a sterile loop to release spores. 3. Collect solution off the top of the plate using a pipet and transfer all liquid into a sterile 15 conical tube. 4. Mix vigorously on a vortex mixer for 1-2 minutes. 5. Filter by vacuum through fiberglass wool to remove mycelial fragments. 6. Centrifuge at 1800 x g for 10 minutes in a Sorvall H1000B rotor (3000 RPM using the Sorvall centrifuge in the HHMI lab) 7. Carefully pour off the supernatant (leave about .5ml or last few drops in) 8. Add 1ml dH2O and mix well. 9. Transfer the mixture to a screw-top vial containing 0.5ml of 80% (w/v) glycerol to achieve a final concentration of 20% glycerol. 10. Store in the freezer at -20 degree celsius. Steve Caruso at UMBC has a slightly different protocol (maybe he can post his as well). On your second question, we use only spread plates for our lawns (the Streptomyces Phage Discovery Guide shouldn't say top agar anywhere - unless I missed an edit somewhere) with good success. We also work from liquid cultures for the spread plates (there are other protocols out there that use spores and top agar, but most require a spore germination step that takes a lot more time). I am assuming that you are planning to use S. griseus (each species has their own quirks). Hope this helps, Lee