SEA-PHAGES | All posts created by smolloy123

Link to this post \| posted 24 Oct, 2018 15:35
smolloy123	Viknesh Sivanathan smolloy123 Hi All, We have been growing G. terrae in PYCa broth for four days with sterile glass beads in baffled flasks. They plate like buttah! I take the glass 3-mm beads we use for archiving (a bottle is super cheap), sterilize then in culture tubes so that I can easily pour them into flasks, and I do not use Tween ( I am too lazy to take time to sub-culture). After four days of growth, the cultures are smooth and produce perfect lawns! I wash beads and re-autoclave for re-use. Just don't get these re-used beads mixed up with the beads you would use for archiving as those should not be recycled beads. I meant to post this over the summer when I discovered this but forgot! Best, Sally That's a really neat trick, Sally. Do you know if it works for M. smeg too? I'll definitely give it a try and report back. Hi Vic, It works with M. smegmatis and fairly well with some of the clumpier species of Mycobacterium. The really clumpy ones sometimes I still need to grow with tween first. Cheers!

Link to this post | posted 24 Oct, 2018 15:35

Viknesh Sivanathan
smolloy123
Hi All,
We have been growing G. terrae in PYCa broth for four days with sterile glass beads in baffled flasks. They plate like buttah! I take the glass 3-mm beads we use for archiving (a bottle is super cheap), sterilize then in culture tubes so that I can easily pour them into flasks, and I do not use Tween ( I am too lazy to take time to sub-culture). After four days of growth, the cultures are smooth and produce perfect lawns! I wash beads and re-autoclave for re-use. Just don't get these re-used beads mixed up with the beads you would use for archiving as those should not be recycled beads. I meant to post this over the summer when I discovered this but forgot!
Best,
Sally

That's a really neat trick, Sally. Do you know if it works for M. smeg too? I'll definitely give it a try and report back.

Hi Vic,
It works with M. smegmatis and fairly well with some of the clumpier species of Mycobacterium. The really clumpy ones sometimes I still need to grow with tween first.
Cheers!

Posted in: Gordonia → Clumping in liquid cultures

Link to this post \| posted 21 Oct, 2018 12:40
smolloy123	Hi All, We have been growing G. terrae in PYCa broth for four days with sterile glass beads in baffled flasks. They plate like buttah! I take the glass 3-mm beads we use for archiving (a bottle is super cheap), sterilize then in culture tubes so that I can easily pour them into flasks, and I do not use Tween ( I am too lazy to take time to sub-culture). After four days of growth, the cultures are smooth and produce perfect lawns! I wash beads and re-autoclave for re-use. Just don't get these re-used beads mixed up with the beads you would use for archiving as those should not be recycled beads. I meant to post this over the summer when I discovered this but forgot! Best, Sally

Link to this post | posted 21 Oct, 2018 12:40

smolloy123

Hi All,
We have been growing G. terrae in PYCa broth for four days with sterile glass beads in baffled flasks. They plate like buttah! I take the glass 3-mm beads we use for archiving (a bottle is super cheap), sterilize then in culture tubes so that I can easily pour them into flasks, and I do not use Tween ( I am too lazy to take time to sub-culture). After four days of growth, the cultures are smooth and produce perfect lawns! I wash beads and re-autoclave for re-use. Just don't get these re-used beads mixed up with the beads you would use for archiving as those should not be recycled beads. I meant to post this over the summer when I discovered this but forgot!
Best,
Sally

Posted in: Gordonia → Clumping in liquid cultures

Link to this post \| posted 21 Oct, 2018 12:35
smolloy123	Hi All, We have eight Rhodococcus phage this year at UMaine and this is our first year using Rhodococcus in the classroom. Does anyone know how Rhodococcus phage are clustered? Are we using the same parameters for clustering as in mycobacteriophage or Gordonia phage? Thanks! Sally FYI I just posted this same post in the wrong forum…hope I am not in trouble with the Pope for this!

Posted in: Rhodococcus → Rhodococcus clustering

Link to this post \| posted 05 Sep, 2018 22:40
smolloy123	Hi Veronique, I just downloaded a new copy from this web site (https://baylor.app.box.com/v/DNAMasterMac). I had another Blob and had to re-install… Blobs!! argh Cheers Sally

Posted in: Using WINE to run DNA Master on a Mac → Link to Baylor box

Link to this post \| posted 25 Aug, 2018 21:14
smolloy123	Hi All, Is there a model cluster C genome I could use to guide function calls? I am QCing two cluster C genomes and I am not super familiar with their genomes. It doesn't help that phagesdb is acting super slow and I cannot capitalize on the "phagesdb" button on PECAAN to immediately see how everyone else called a particular gene within a pham. Could someone recommend a cluster C genome annotated by cluster C experts? And Dan, why is phagesdb so slow these days and why isn't GeneMark cooperating. I think I just broke forum rules sneaking that into this message….

Link to this post | posted 25 Aug, 2018 21:14

smolloy123

Hi All,
Is there a model cluster C genome I could use to guide function calls? I am QCing two cluster C genomes and I am not super familiar with their genomes. It doesn't help that phagesdb is acting super slow and I cannot capitalize on the "phagesdb" button on PECAAN to immediately see how everyone else called a particular gene within a pham.

Could someone recommend a cluster C genome annotated by cluster C experts?

And Dan, why is phagesdb so slow these days and why isn't GeneMark cooperating. I think I just broke forum rules sneaking that into this message…. smile

Posted in: Cluster C Annotation Tips → Model Cluster C genome

Link to this post \| posted 06 Aug, 2018 18:04
smolloy123	Debbie Jacobs-Sera Sally, Hi. Using web Phamerator's gene numbers, I would call gene 16 a TAC. Neither 15 or 18 are good candidates by HHpred (gp 15 is a ribosomal protein hit) and 18 has no significant matches). I believe that gp 16 is also a TAC, BUT I would not call it. I can't find a slippery sequence and the Pfam hit is too weak for me. Hi Deb, Thanks for your response. I already submitted it and luckily I did exactly what you prescribed! This was very helpful. Thanks so much!!! Cheers Sally

Link to this post | posted 06 Aug, 2018 18:04

smolloy123

Debbie Jacobs-Sera
Sally,
Hi. Using web Phamerator's gene numbers, I would call gene 16 a TAC. Neither 15 or 18 are good candidates by HHpred (gp 15 is a ribosomal protein hit) and 18 has no significant matches). I believe that gp 16 is also a TAC, BUT I would not call it. I can't find a slippery sequence and the Pfam hit is too weak for me.

Hi Deb,
Thanks for your response. I already submitted it and luckily I did exactly what you prescribed!
This was very helpful. Thanks so much!!!
Cheers
Sally

Posted in: Cluster Q Annotation Tips → tail assembly chaperones

Link to this post \| posted 03 Aug, 2018 18:25
smolloy123	smolloy123 Hi All, Does anyone savvy about the Q cluster know what the current rule is for calling tail assembly chaperones in the Qs? I see that the frameshift is not known in the Qs…but are we sure about which genes are the chaperones? I am QCing the Q genome Gancho. One gene that is called tail assembly chaperone in nearly all the Q genomes (Except Giles) is pham 30734 which is gp17 in most genomes. Upstream of this is a gene is a gene (gp16 in most genes including Giles, Evanesce and HH92)) that has HHPRED matches (none of them with prob greater than 90) to 50s ribosomal proteins. Two genomes (Evanesce and HH92) called this a tail assembly chaperone. Downstream of the gp17 that is called a chaperone is another gene, gp18, that has ribosomal HHPRED hits (around 30 - 40% prob) and a 22% prob match to tail assembly chaperone of a lysteria phage ( I looked at other tail assembly chaperone protein HHPRED matches from other genomes where we know they are chaperones…the first chaperone gene tends to hit ribosomal proteins and the second chaperone tends to hit bacteriophage gp15 family (gp17 of the Qs matches this same protein family). So do I call just gp17 as chaperone? Do I call the upstream, gp16 a chaperone along with gp17? or gp17 and gp18 ? Any suggestions? Welkin? I forgot to add that I used gene numbers for the majority of Q phage genomes but they are off by 1 for Gancho since it has a deletion in the very end of left arm

Link to this post | posted 03 Aug, 2018 18:25

smolloy123

smolloy123
Hi All,
Does anyone savvy about the Q cluster know what the current rule is for calling tail assembly chaperones in the Qs? I see that the frameshift is not known in the Qs…but are we sure about which genes are the chaperones? I am QCing the Q genome Gancho. One gene that is called tail assembly chaperone in nearly all the Q genomes (Except Giles) is pham 30734 which is gp17 in most genomes. Upstream of this is a gene is a gene (gp16 in most genes including Giles, Evanesce and HH92)) that has HHPRED matches (none of them with prob greater than 90) to 50s ribosomal proteins. Two genomes (Evanesce and HH92) called this a tail assembly chaperone. Downstream of the gp17 that is called a chaperone is another gene, gp18, that has ribosomal HHPRED hits (around 30 - 40% prob) and a 22% prob match to tail assembly chaperone of a lysteria phage ( I looked at other tail assembly chaperone protein HHPRED matches from other genomes where we know they are chaperones…the first chaperone gene tends to hit ribosomal proteins and the second chaperone tends to hit bacteriophage gp15 family (gp17 of the Qs matches this same protein family).

So do I call just gp17 as chaperone?
Do I call the upstream, gp16 a chaperone along with gp17?
or gp17 and gp18 ?

Any suggestions? Welkin?

I forgot to add that I used gene numbers for the majority of Q phage genomes but they are off by 1 for Gancho since it has a deletion in the very end of left arm

Posted in: Cluster Q Annotation Tips → tail assembly chaperones

Link to this post \| posted 03 Aug, 2018 18:23
smolloy123	Hi All, Does anyone savvy about the Q cluster know what the current rule is for calling tail assembly chaperones in the Qs? I see that the frameshift is not known in the Qs…but are we sure about which genes are the chaperones? I am QCing the Q genome Gancho. One gene that is called tail assembly chaperone in nearly all the Q genomes (Except Giles) is pham 30734 which is gp17 in most genomes. Upstream of this is a gene is a gene (gp16 in most genes including Giles, Evanesce and HH92)) that has HHPRED matches (none of them with prob greater than 90) to 50s ribosomal proteins. Two genomes (Evanesce and HH92) called this a tail assembly chaperone. Downstream of the gp17 that is called a chaperone is another gene, gp18, that has ribosomal HHPRED hits (around 30 - 40% prob) and a 22% prob match to tail assembly chaperone of a lysteria phage ( I looked at other tail assembly chaperone protein HHPRED matches from other genomes where we know they are chaperones…the first chaperone gene tends to hit ribosomal proteins and the second chaperone tends to hit bacteriophage gp15 family (gp17 of the Qs matches this same protein family). So do I call just gp17 as chaperone? Do I call the upstream, gp16 a chaperone along with gp17? or gp17 and gp18 ? Any suggestions? Welkin?

Link to this post | posted 03 Aug, 2018 18:23

smolloy123

Hi All,
Does anyone savvy about the Q cluster know what the current rule is for calling tail assembly chaperones in the Qs? I see that the frameshift is not known in the Qs…but are we sure about which genes are the chaperones? I am QCing the Q genome Gancho. One gene that is called tail assembly chaperone in nearly all the Q genomes (Except Giles) is pham 30734 which is gp17 in most genomes. Upstream of this is a gene is a gene (gp16 in most genes including Giles, Evanesce and HH92)) that has HHPRED matches (none of them with prob greater than 90) to 50s ribosomal proteins. Two genomes (Evanesce and HH92) called this a tail assembly chaperone. Downstream of the gp17 that is called a chaperone is another gene, gp18, that has ribosomal HHPRED hits (around 30 - 40% prob) and a 22% prob match to tail assembly chaperone of a lysteria phage ( I looked at other tail assembly chaperone protein HHPRED matches from other genomes where we know they are chaperones…the first chaperone gene tends to hit ribosomal proteins and the second chaperone tends to hit bacteriophage gp15 family (gp17 of the Qs matches this same protein family).

So do I call just gp17 as chaperone?
Do I call the upstream, gp16 a chaperone along with gp17?
or gp17 and gp18 ?

Any suggestions? Welkin?
smile

Posted in: Cluster Q Annotation Tips → tail assembly chaperones

Link to this post \| posted 17 Jul, 2018 18:30
smolloy123	I fixed it by deleting all the projects in GenBank submission window. There were two or three projects that were causing the error. It wouldn't let me delete the files directly but somehow as I deleted the others it eventually allowed me to delete the problem files. I still don't know what was going on but managed to export the project. smolloy123 Hi Veronique, Thanks for getting back to me. Given that I can't even open up Submit To GenBank without getting this error, I think that I am still stuck…I think it is weird that it is happening on both versions of my DNA Master (lap top and desktop versions) Cheers Sally Veronique Delesalle Hi Sally I had the same issue two years ago. Debbie had to go to Jeffrey Lawrence for that one! Happily I still have his email response which I paste below: "That is a warning/error form the database engine. A BLOB is a "Binary Large Object"; it is a field in the database used to store large fields of information (like a BLAST alignment for a hit). That error arises when one tries to open the same record more than once; the BLOB is already open elsewhere. This warning could arise if the same sequence is loaded form the database more than once, or if the same gene is examined in more than one way that would open a BLOB field. I would need far more details to determine the actual cause, but it may not be an error. It is only an error if it persists after it is clear that the object should have been closed. One possible source is that the record for the gene may not have been save prior to opening the BLAST tab. Try to post the record first." I can't remember what I did (it was two years ago) but the problem did resolve itself…

Link to this post | posted 17 Jul, 2018 18:30

smolloy123

I fixed it by deleting all the projects in GenBank submission window. There were two or three projects that were causing the error. It wouldn't let me delete the files directly but somehow as I deleted the others it eventually allowed me to delete the problem files. I still don't know what was going on but managed to export the project.

smolloy123
Hi Veronique,
Thanks for getting back to me. Given that I can't even open up Submit To GenBank without getting this error, I think that I am still stuck…I think it is weird that it is happening on both versions of my DNA Master (lap top and desktop versions)
Cheers
Sally

Veronique Delesalle
Hi Sally

I had the same issue two years ago. Debbie had to go to Jeffrey Lawrence for that one! Happily I still have his email response which I paste below:

"That is a warning/error form the database engine. A BLOB is a "Binary
Large Object"; it is a field in the database used to store large fields
of information (like a BLAST alignment for a hit). That error arises
when one tries to open the same record more than once; the BLOB is
already open elsewhere. This warning could arise if the same sequence is
loaded form the database more than once, or if the same gene is examined
in more than one way that would open a BLOB field. I would need far more
details to determine the actual cause, but it may not be an error. It is
only an error if it persists after it is clear that the object should
have been closed.

One possible source is that the record for the gene may not have been
save prior to opening the BLAST tab. Try to post the record first."

I can't remember what I did (it was two years ago) but the problem did resolve itself…

Posted in: DNA Master → Running DNA Master on a Mac using Wine

Link to this post \| posted 17 Jul, 2018 18:00
smolloy123	Hi Veronique, Thanks for getting back to me. Given that I can't even open up Submit To GenBank without getting this error, I think that I am still stuck…I think it is weird that it is happening on both versions of my DNA Master (lap top and desktop versions) Cheers Sally Veronique Delesalle Hi Sally I had the same issue two years ago. Debbie had to go to Jeffrey Lawrence for that one! Happily I still have his email response which I paste below: "That is a warning/error form the database engine. A BLOB is a "Binary Large Object"; it is a field in the database used to store large fields of information (like a BLAST alignment for a hit). That error arises when one tries to open the same record more than once; the BLOB is already open elsewhere. This warning could arise if the same sequence is loaded form the database more than once, or if the same gene is examined in more than one way that would open a BLOB field. I would need far more details to determine the actual cause, but it may not be an error. It is only an error if it persists after it is clear that the object should have been closed. One possible source is that the record for the gene may not have been save prior to opening the BLAST tab. Try to post the record first." I can't remember what I did (it was two years ago) but the problem did resolve itself…

Link to this post | posted 17 Jul, 2018 18:00

smolloy123

Hi Veronique,
Thanks for getting back to me. Given that I can't even open up Submit To GenBank without getting this error, I think that I am still stuck…I think it is weird that it is happening on both versions of my DNA Master (lap top and desktop versions)
Cheers
Sally

Veronique Delesalle
Hi Sally

I had the same issue two years ago. Debbie had to go to Jeffrey Lawrence for that one! Happily I still have his email response which I paste below:

"That is a warning/error form the database engine. A BLOB is a "Binary
Large Object"; it is a field in the database used to store large fields
of information (like a BLAST alignment for a hit). That error arises
when one tries to open the same record more than once; the BLOB is
already open elsewhere. This warning could arise if the same sequence is
loaded form the database more than once, or if the same gene is examined
in more than one way that would open a BLOB field. I would need far more
details to determine the actual cause, but it may not be an error. It is
only an error if it persists after it is clear that the object should
have been closed.

One possible source is that the record for the gene may not have been
save prior to opening the BLAST tab. Try to post the record first."

I can't remember what I did (it was two years ago) but the problem did resolve itself…

Posted in: DNA Master → Running DNA Master on a Mac using Wine

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