SEA-PHAGES | All posts created by welkin

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Link to this post \| posted 26 Feb, 2016 20:22
welkin	Hi Joy, It just means that you need to streak out the potential lysogens for single colonies. Here is a youtube video (tps://www.youtube.com/watch?v=Ay2hhujTuvg) You can use sterile wooden sticks, an innoculation loop, sterile toothpicks, etc. I like sticks for students, they are easier to use and tend not to accidentally gouge into the agar surface as much. The streaking serves two purposes: one, to grow up a single clonal colony to work from, and two, to remove any exogenous phage that might be left over from the initial mesa spot. Sometimes you can get a mix of resistant cells and true lysogens. Streaking to purify cells and then working from a purified clone is good microbiology practice.

Link to this post | posted 26 Feb, 2016 20:22

Hi Joy,
It just means that you need to streak out the potential lysogens for single colonies. Here is a youtube video (tps://www.youtube.com/watch?v=Ay2hhujTuvg)
You can use sterile wooden sticks, an innoculation loop, sterile toothpicks, etc. I like sticks for students, they are easier to use and tend not to accidentally gouge into the agar surface as much.

The streaking serves two purposes: one, to grow up a single clonal colony to work from, and two, to remove any exogenous phage that might be left over from the initial mesa spot. Sometimes you can get a mix of resistant cells and true lysogens. Streaking to purify cells and then working from a purified clone is good microbiology practice.

Posted in: Lysogeny/Immunity → Lysogeny experiment help

Link to this post \| posted 24 Feb, 2016 15:45
welkin	So question 1: is it the DNA primase? All the primases are split into two overlapping reading frames in the Cluster As. We are trying to figure out how this works biologically (frameshift? splice?) but don't know yet. If it is not the primase, please send us the sequence coordinates so we can make sure it isn't a sequencing error. Question 2: if the immunity repressor gets inactivated through mutation, you will no longer have a temperate phage (or be able to form a lysogen in the first place). If the phage integrates and THEN somehow the repressor got inactivated, the prophage would still come out. so either way, there would not be a lysogen around to test. To answer the other part of your question, if you had an A6 prophage, and infected that lysogen with an A1 phage (that has a different recognition sequence for the immunity repressor), yes, you would make plaques on the lysogen.

Link to this post | posted 24 Feb, 2016 15:45

welkin

So question 1: is it the DNA primase? All the primases are split into two overlapping reading frames in the Cluster As. We are trying to figure out how this works biologically (frameshift? splice?) but don't know yet. If it is not the primase, please send us the sequence coordinates so we can make sure it isn't a sequencing error.
Question 2: if the immunity repressor gets inactivated through mutation, you will no longer have a temperate phage (or be able to form a lysogen in the first place). If the phage integrates and THEN somehow the repressor got inactivated, the prophage would still come out. so either way, there would not be a lysogen around to test. To answer the other part of your question, if you had an A6 prophage, and infected that lysogen with an A1 phage (that has a different recognition sequence for the immunity repressor), yes, you would make plaques on the lysogen.

Posted in: Lysogeny/Immunity → One of our draft phage genomes has a 'stop' in the middle of the immunity repressor gene.

Link to this post \| posted 24 Feb, 2016 15:03
welkin	Nope, the tRNA cassettes are weird, and no you don't have to have a promoter for each of them or for each protein encoding gene interspersed within the cassette. We tend to steer clear of a tRNA and a protein occupying the same space, but there are definitely genomes where they get pretty close (take a look at the Ms). The only promoter rule is still about switching directions– presumably, switching transcriptional directions would still require two promoters even for tRNAs.

Link to this post | posted 24 Feb, 2016 15:03

welkin

Nope, the tRNA cassettes are weird, and no you don't have to have a promoter for each of them or for each protein encoding gene interspersed within the cassette. We tend to steer clear of a tRNA and a protein occupying the same space, but there are definitely genomes where they get pretty close (take a look at the Ms). The only promoter rule is still about switching directions– presumably, switching transcriptional directions would still require two promoters even for tRNAs.

Posted in: tRNAs → How close can one pack protein and tRNA's genes

Link to this post \| posted 24 Feb, 2016 15:00
welkin	It does not strictly refer to NCBI. Frankly, I would prefer the phagesdb match over a random gene in GenBank that was likely deposited by auto-annotation software with no personal review. The matches retrieved by BLAST at NCBI are also sorted according to age; identical newer hits are listed first. The point of listing the BLAST data is to demonstrate that you are examining similar genes and looking to see which starts have been used previously. Using the top hit for your notes is a convenience rather than a requirement: GenBank used to only list our phages rather than tons of auto-annotated prophages, so it didn't really matter where you got the alignment from. Noting the alignment of your gene to similar phage gene makes much more sense to me!

Link to this post | posted 24 Feb, 2016 15:00

welkin

It does not strictly refer to NCBI. Frankly, I would prefer the phagesdb match over a random gene in GenBank that was likely deposited by auto-annotation software with no personal review.

The matches retrieved by BLAST at NCBI are also sorted according to age; identical newer hits are listed first. The point of listing the BLAST data is to demonstrate that you are examining similar genes and looking to see which starts have been used previously. Using the top hit for your notes is a convenience rather than a requirement: GenBank used to only list our phages rather than tons of auto-annotated prophages, so it didn't really matter where you got the alignment from. Noting the alignment of your gene to similar phage gene makes much more sense to me!

Posted in: Notes and Final Files → BLAST notes: PhagesDB or GenBank?

Link to this post \| posted 16 Feb, 2016 18:46
welkin	It does not mean accession number. The conserved domain database is part of NCBI and is searched by default when you BLAST a protein against GenBank through your browser rather than through DNA Master. If you have a CDD match, you will see a separate page with your CDD results, like in your attached figure. For this result, you would write "CDD, phage capsid". Phage capsid is the domain ID. You can also see CDD matches in Phamerator (the yellow boxes that appear on your genome map in your genes that you can browse over).

Link to this post | posted 16 Feb, 2016 18:46

welkin

It does not mean accession number. The conserved domain database is part of NCBI and is searched by default when you BLAST a protein against GenBank through your browser rather than through DNA Master. If you have a CDD match, you will see a separate page with your CDD results, like in your attached figure. For this result, you would write "CDD, phage capsid".
Phage capsid is the domain ID.
You can also see CDD matches in Phamerator (the yellow boxes that appear on your genome map in your genes that you can browse over).

Posted in: Notes and Final Files → Conserved Domain ID in notes

Link to this post \| posted 16 Feb, 2016 18:34
welkin	GregFrederick@letu.edu We want to make certain we are completing the information in DNA Master correctly. At our recent training we were given the two choices for the "LO:" description. LO: Longest Reasonable ORF -or- Not Longest Reasonable ORF (explain in notes below) I think we have some confusion over this language and a worksheet we 'borrowed' from another school. That worksheet actually has the student record the length of the longest open reading frame. Am I correct that the DNA Master "Notes" section does not require this information? Correct. you do not need to include the length of the open reading frame. When would one choose an ORF that is not the longest reasonable ORF? If there was a long overlap, that would make it unreasonable? (We are finding some 90+bp overlaps that have been used in PhageDB.) If the SD sequence was 3' of the ATG that would make it unreasonable? What else might cause one to select an ORF other than the "Longest Reasonable ORF"? the most common example of "not the longest reasonable ORF" is that the start chosen leaves a gap between the gene you are working on and the upstream gene that could be made smaller by choosing a alternate start. So I am not looking so much for reasonable vs not reasonable, as longest vs not longest. People were taking "longest" as the most important criteria, and saying that genes were not the longest ORF because there was one distant upstream start codon that could cause a 90% overlap with the upstream gene. That is not a "reasonable" start to consider. If something has a 90bp overlap, I definitely want to know about it. Can you give me some scenarios that might lead one to select the "longest 'unreasonable' ORF" or a "shorter-than-longest reasonable ORF"? Sure. when the comparative genomics, like through STarterator or BLAST, shows that the start that gives you the longest ORF in your phage gene in your genome isn't present in closely related genes, and one that gives you a shorter gene product is. Solid comparative data trumps everything. I hope that helps!

Link to this post | posted 16 Feb, 2016 18:34

welkin

GregFrederick@letu.edu
We want to make certain we are completing the information in DNA Master correctly. At our recent training we were given the two choices for the "LO:" description.

LO: Longest Reasonable ORF -or-
Not Longest Reasonable ORF (explain in notes below)

I think we have some confusion over this language and a worksheet we 'borrowed' from another school. That worksheet actually has the student record the length of the longest open reading frame. Am I correct that the DNA Master "Notes" section does not require this information?

Correct. you do not need to include the length of the open reading frame.

When would one choose an ORF that is not the longest reasonable ORF? If there was a long overlap, that would make it unreasonable? (We are finding some 90+bp overlaps that have been used in PhageDB.) If the SD sequence was 3' of the ATG that would make it unreasonable? What else might cause one to select an ORF other than the "Longest Reasonable ORF"?

the most common example of "not the longest reasonable ORF" is that the start chosen leaves a gap between the gene you are working on and the upstream gene that could be made smaller by choosing a alternate start. So I am not looking so much for reasonable vs not reasonable, as longest vs not longest. People were taking "longest" as the most important criteria, and saying that genes were not the longest ORF because there was one distant upstream start codon that could cause a 90% overlap with the upstream gene. That is not a "reasonable" start to consider.

If something has a 90bp overlap, I definitely want to know about it.

Can you give me some scenarios that might lead one to select the "longest 'unreasonable' ORF" or a "shorter-than-longest reasonable ORF"?

Sure. when the comparative genomics, like through STarterator or BLAST, shows that the start that gives you the longest ORF in your phage gene in your genome isn't present in closely related genes, and one that gives you a shorter gene product is. Solid comparative data trumps everything.

I hope that helps!

Posted in: DNA Master → LO: Designation Question

Link to this post \| posted 06 Oct, 2015 15:14
welkin	Yes. Your IT people can install DNA Master and give it admin rights for all users regardless of their privileges. It will run fine. Best, Welkin

Posted in: DNA Master → Running with Administrator Privileges

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