Wild Type Yeast?

[methionine]

Hi all,
I have a question about wild-type yeast -- just-- What are they? Does that just mean that the wild type yeast are the same kind you would find naturally (as opposed to being a laboratory strain?)
thanks,
-M

[MelissaB]

The short answer: yes.

More information can be found here: http://en.wikipedia.org/wiki/Wild_type.

[carolinethorn]

Hi M,

It depends on the context somewhat - as you can see the wikipedia entry has 2 slightly different definitions.
Geneticists tend to use "wild type" to describe a control strain/organism that has not been genetically modified as compared to one that is transgenic or is a gene knock-out. So if your context is a paper where the yeast has been transfected with a plasmid of some kind then wild type is the control yeast.

-Caroline

[methionine]

Hi,
Thanks for your responses. I understand the difference now -- so, wild type organisms are more.. hardy.
Just one more general question concerning controls and variables-- In my experiment, I used the wild type plasmids. Now I realize that ideally, I should have used a mutant plasmid. However, when I asked about this, I was told that using a mutant plasmid would just add another variable in my experiment. I was confused, because I had thought that... if you’re keeping the yeast type constant, wouldn’t it just be like a control all the same?

Thank you!
-M

[carolinethorn]

Hi M,

Please can you refresh our memories with what tests and controls you used in the end. I remember you had a plasmid with a gene that when expressed aided growth on copper medium and then were going to have different intron lengths.

-Caroline

[methionine]

Hi Caroline,

Sure-- here is a summary of my research so far: I wanted to test the effect of intron length on gene expression levels, so I artificially increased the length of the ACT1 intron within a plasmid. The ACT1 intron was engineered into the CUP1 gene, so in a sense.. those other researchers made CUP1 intron-containing. CUP1 allows yeast to grow on otherwise lethal concentrations of copper.. and the way you would test that is by seeing whether or not/how well the yeast grow on varying concentrations of copper.
My question before was if whether using a wild type plasmid as opposed to a mutant plasmid would make a difference. The thing is, the mutant plasmid would be more sensitive to copper at all levels, and that the wild type plasmid would make the yeast less sensitive overall, even if the CUP1 gene was expressed at lower levels.
When I asked about why we did not use the mutant plasmid instead (I mean, ideally, you'd want the assay to be as sensitive as possible), I was told that using a mutant plasmid would just add another variable in my experiment. I was confused because I thought that if you kept the plasmid type constant within all the yeast, it would still be controlled all the same.
Could somebody tell me why using a mutant plasmid would be introducing another variable in my experiment? I've already used the wild type and don't have enough time to redo it with a mutant plasmid, even if it was more ideal... I just really want to know or at least get a better concept of controlling experiments and variables, etc. Thank you for your help!

-M

[carolinethorn]

Hi M,

First off, this is a pretty complex experiment so it's quite possible for some of us "experts" to get the wrong end of the stick with our line of questioning. Especially as this is tricky to discuss over the message board. If you have talked over your approach with your mentor and they feel it is solid then you are the expert on your project and should have the confidence that your choices were good and sensible. With that confidence you can then relax and not worry about rushing back to the lab to do more experiments but treat peoples questions as interesting "thought experiments" or figuring out what if... or what you would do next year.

I am assuming when you say mutant plasmid that you mean the CUP1 has been mutated to effect the growth on copper. To be honest i would consider these a whole new set of experiments rather than just another variable. Your series of plasmids with different length ACT1 are hypothesised to effect the efficiency of tranlsation of CUP1 and indirectly effect the ability to grow on copper due to variable amounts of CUP1 protein being produced, whereas using a mutant CUP1 plasmids would more likely effect the protein directly and the protein to copper binding and have its effect on growth via a different mechanism. I guess, if i did not see much difference between the original ACT1 -CUP1 plasmid and the different length ACT1-CUP1 plasmids I might be interested to see what would happen in the context of the CUP1 mutants to see if the different length introns can actually increase stability above what is seen with the original plasmid - so that they might "rescue" growth defects caused by mutants in CUP1.

I guess talking about mutant plasmids you could aslo mean if you were to introduce mutations into the ACT1 intron. This would be closer to your original hypothesis (which i would assume is that intron length effects the stability of CUP1 products and thus effects growth on copper)- the hypothesis would need to be more broad, that alterations in the intron effect translational stability of the CUP1 product and thus effect growth on copper. I guess this would then be considered another variable you coud look at in your experiment. It might be interesting if you could predict which sequence motifs in the intron might be important for stability of mRNA and mutate those but that would be a lot of work. Also not something you could quickly do.

Hope my ramblings are not too confusing. As I said its a complex problem and i may not have grasped all the subtleties. The main things about controls is to think about making sure you can explain the extreme situations ie. lots of growth and no growth. If you have no growth with the yeast that had your original ACT1 CUP1 plasmid, positive control, you know soemthing went wrong with the procdure. Likewise if you had yeast with no plasmid, negative control, and they grew on the copper than something else went wrong. Hopefully the negative had no colonies, the positive had lots of colonies and your different length ACT1 plasmids were somewhere in between.

Best of luck,
Caroline

[methionine]

Hi,
Thanks, I really appreciate your helpful response, Caroline.
I understand-- I had already talked it over with my mentor, and that's just what she said about using the wild type strain vs. a mutant strain. I actually discussed my project with several professors, so that's why I had different opinions.. I just wanted to get a fuller sense of what they were all saying.

.. My hypothesis didn't have to do with the stability of the CUP1 products-- it had to do with the splicing efficiency and thus the CUP1 gene expression levels. I get to find my results in the next two weeks, so I'll decide on further experiments after that..

Oh yes-- and my negative control was going to be yeast without the CUP1 gene. I'm so happy that I finally understand positive and negative controls now!

thank you,
-M