Capturing expression in Canto (with implications for website allele /genotype)

[ValWood]

1. The ability to specify the ~plasmid~ promoter (mostly of these are stored in names, background and comments)

~2. If overexpression is selected, the ability to specify the "fold" overexpression (I.e 2X, 10X, 100X) We probably need to discuss the details. We need this to a) be able to distinguish different expression levels (and hence phenotypes) for overexpression alleles (and knockdowns) b) to clean up backgrounds and comments c) To fix some allele names which do not follow standard naming conventions d) also ectopic expression~

example Check cut12-s11 https://www.pombase.org/genotype/cut12.s11-G71V-amino_acid_mutation-expression-not_assayed https://www.pombase.org/genotype/cut12.s11-G71V-amino_acid_mutation-expression-wild_type_product_level

[ValWood]

Currently we have:

When people choose WT or not assayed they don't need to add a promoter (fine).

When we say "exogenous promoter" does this "introduced into a cell or organism from an external source" mean just that it is plasmid expressed, or that it is 'external DNA' . I guess with the current labelling I would not know which 'type ' to use for the S. cerevisiae ADH promoter)

• should be precise relabel "gene" to "promoter swap (fission yeast)" ?
• Plasmid expressed (which would cover everything expressed on a plasmid)

@manulera I'm not sure what the best axis of classification are here. Are we more bothered about plasmid vs genome integrated ?

This would only work if "exogenous promoters (i.e promoters from other species)" are always introduced on plasmids and not integrated (in which case "plasmid expressed + exogenous integrated).

Does this make sense?

[manulera]

should be precise relabel "gene" to "promoter swap (fission yeast)" ?

I agree with this

Plasmid expressed (which would cover everything expressed on a plasmid)

I don't think that when we started talking about this I was thinking of making a difference between genome-integrated constructs vs. plasmids. I can see that this is important because cells may have more than one copy of the plasmid, but this is parallel to the type of promoter used (see below).

This would only work if "exogenous promoters (i.e promoters from other species)" are always introduced on plasmids and not integrated (in which case "plasmid expressed + exogenous integrated).

That's not the case, you could drive the expression of a gene under the same exogenous promoter from a plasmid or from the genome.

Sorry not to bring a solution here... I personally don't think that the distinction plasmid / genome is so important, especially for the ectopic expression case (the altered context of expression is what matters, which is in principle evident from the promoter).

For overexpression/knockdown, the level of overexpression is given by the plasmid number & primer, but what ultimately matters is that the protein is overexpressed, not so much how.

The only case that remains is how we capture the fact that sometimes people will overexpress an allele of a gene from a plasmid, in a strain where the wild-type allele is still there. In that case, I guess we can just force the users to add the wild-type allele to the genotype (e.g. ase1+ ase1-S3A(Overexpression)).

[ValWood]

So are you saying we need not worry about the plasmid at all, we expect people to specify P3nmt1 as promoter swap nmt1? and only use exogenous for foreign promoters? this would simplify and get around all the different names used.....

In that case, I guess we can just force the users to add the wild-type allele to the genotype (e.g. ase1+ ase1-S3A(Overexpression)).

this is what I have done in the past (usually in the background)

[manulera]

Yes, I think that is a good compromise.

Also I am remembering now that we discussed at some point about overexpression of non-wild-type alleles. In the example above ase1+ ase1-S3A, if ase1-S3A is expressed from a nmt1 promoter from a plasmid, the control should be a strain (over)expressing wild-type ase1 (ase1+) from the same plasmid. Because we are comparing those two conditions, in principle the phenotype comes from the difference between ase1+ and ase1-S3A, and not from the overexpression, so I would not annotate it as overexpression now, but I am sure I have done this in the past.

[ValWood]

Hi Kim, Sorry for the dealy on this one. We can change the text to "promoter swap (fission yeast)" and make this live

[kimrutherford]

Sorry for the dealy on this one. We can change the text to "promoter swap (fission yeast)" and make this live

I've merged the code into the branch. It took a while because there were a lot of conflicts to sort out. I'll deploy the change when oliver1 is back.

[kimrutherford]

"promoter swap (fission yeast)"

Would "promoter swap (Schizosaccharomyces pombe)" or "promoter swap (S. pombe)" be OK? We don't have "fission yeast" in the config or in the Canto database (this has to work for non-pombe Canto instances too).

[kimrutherford]

I've merged the code into the branch

I meant, I've merged the promoter branch into the main branch.

[kimrutherford]

Would "promoter swap (Schizosaccharomyces pombe)" or "promoter swap (S. pombe)" be OK?

[ValWood]

Would "promoter swap (Schizosaccharomyces pombe)" or "promoter swap (S. pombe)" be OK?

yep

[kimrutherford]

OK, great. I've implemented that ready for when oliver1 is back.

[ValWood]

@PCarme and I tried to use the new promoter feature but we couldn't see it?

[kimrutherford]

Ah, sorry. I never got around to updating the main Canto with the change. We will need 10-20 minutes of down time to do the upgrade.

[ValWood]

OK, lets wait until the weekend (maybe your Monday morning), because I sent tonnes of sessions out today. We are already getting a few back.... don't want to disrupt their flow!

[kimrutherford]

That makes sense. I'll update when it's quiet. First thing Monday morning NZ is a good time.

[kimrutherford]

I never got around to updating the main Canto with the change. We will need 10-20 minutes of down time to do the upgrade.

That's done now.

We should discuss how the promoters should look in the genotype list.

[ValWood]

I'm going to do this session now and we can use as an example

mad1/~10%,SPBC3D6.04c:allele-28,nmt81 promoter,{ed9652a888c24c94} mad1/~30%,SPBC3D6.04c:allele-29,bub3 promoter,{ed9652a888c24c94} mad1/~300%,SPBC3D6.04c:allele-31,ark1 promoter,{ed9652a888c24c94} mad1/~500%,SPBC3D6.04c:allele-30,nmt1 promoter,{ed9652a888c24c94} mad2/~10%,SPBC20F10.06:allele-14,mad3 promoter,{ed9652a888c24c94} mad2/~65%,SPBC20F10.06:allele-11,hphNT1 cassette in promoter,{ed9652a888c24c94} mad3/~30%,SPCC1795.01c:allele-10,hphNT1 cassette in promoter,{ed9652a888c24c94}

[ValWood]

I've done this one, it looks like this in Canto genotype list:

which is fine?

[kimrutherford]

Yep, that's how it looks for now. I'll check Chado in the morning.

[ValWood]

Yep, promoter should be in a separate column to be precise.

I also used the genotype comment fie;ld to recorded the % overexpression