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yeast gene deletion


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#1 yuer

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Posted 15 February 2009 - 07:25 AM

Dear All,

I am going to delete an essential gene from yeast genome. From books and papers, I learned that I have to make diploid cells transformed with a plasmid containing the URA3 and the wild type gene, and delete the gene by one step gene disruption from the diploid cells, then sporulate the diploid to get the desire haploid cells.

In my hand, I have wild type haploid cells. I was wondering If I could just transform a plasmid containing the URA3 and the wild type gene into the haploid cells and then do one step gene disruption in the haploid cells.

Could anybody help me? I am new in yeast genetics. I deeply appreciate your help in advance!

Have a great weekend and a happy holiday!

yuer

#2 yuer

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Posted 16 February 2009 - 05:58 PM

Dear all,

Please Pleeeeeeeeeeeeeeeeeeeeze help me!

yuer

#3 perneseblue

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Posted 17 February 2009 - 06:41 AM

after looking at the problem, the short answer is yes, you can do it. However depending on what you want to do you will need either a more complex system than the one you have envisioned, or take a little more risk.

Do you know the DNA sequence that flanks the essential gene which you are interested in?

Do you have access to site-specific recombinase? If yes, what is it? Is it Cre or better still unidirectional phage integrase phiC31 or phiBT1?

Most importantly, what do you want to do with the essential gene you are knocking out? Replaces it will an allele to see the effect while preserving the gene context.. ie its native promoter and copy number? Or will you be expressing the allele using multicopy plasmid and under a non-native promoter?

PS: If I am not mistaken, if you are going to carry the the allele of gene of the interest on a plasmid, normally the one copy of the gene of interest in the diploid cell is knock out first. Only then is the diploid cell transformed with the plasmid that carries the allele of interest
May your PCR products be long, your protocols short and your boss on holiday

#4 yuer

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Posted 17 February 2009 - 07:55 AM

after looking at the problem, the short answer is yes, you can do it. However depending on what you want to do you will need either a more complex system than the one you have envisioned, or take a little more risk.

Do you know the DNA sequence that flanks the essential gene which you are interested in?

Do you have access to site-specific recombinase? If yes, what is it? Is it Cre or better still unidirectional phage integrase phiC31 or phiBT1?

Most importantly, what do you want to do with the essential gene you are knocking out? Replaces it will an allele to see the effect while preserving the gene context.. ie its native promoter and copy number? Or will you be expressing the allele using multicopy plasmid and under a non-native promoter?

PS: If I am not mistaken, if you are going to carry the the allele of gene of the interest on a plasmid, normally the one copy of the gene of interest in the diploid cell is knock out first. Only then is the diploid cell transformed with the plasmid that carries the allele of interest



Dear Perneseblue,

Thanks a million for your comments! They are very useful!

My purpose of deleting the essential gene is to see the phenotype of the mutant gene. I will make a point mutation on the gene and transform the mutant gene which will be on a centremeric plasmid (low copy) with the native promoter and terminator of the gene. The sequence of the gene is known as well as its flanking sequences. To delete the gene, i am going to use pUG6 plasmid which has the loxP-kanMX-loxP deletion cassette.

I thought that the diploid cells were first transformed with the plasmid carrying the wild type gene of the interest and then knockout the genomic gene in the diploid cells. Thanks for your correcting! After reading your comments, I realized that if I transform the wild type gene of my interest (on a centromeric plasmid) into the haploid cells first, then transformation of the deletion cassette to knockout that genomic gene may also cause homologous recombination with the wild type gene on the centromeric plasmid so that the desired deletion can not be obtained. Am I right? That's why the diploid cells are always used to delete essential genes?

I really appreciate your help! Happy every day!

yuer

#5 perneseblue

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Posted 17 February 2009 - 08:49 AM

After reading your comments, I realized that if I transform the wild type gene of my interest (on a centromeric plasmid) into the haploid cells first, then transformation of the deletion cassette to knockout that genomic gene may also cause homologous recombination with the wild type gene on the centromeric plasmid so that the desired deletion can not be obtained. Am I right? That's why the diploid cells are always used to delete essential genes?
yuer


Yes, quite right. In terms of implementation, the diploid knockout is easier to accomplish.

however with a little more risk... ie screening a lot more colonies... you can transform the centromeric plasmid. And then target your gene by homologous recombination. While not impossible, it will be more difficult.


With some complexity...

you can target your gene (ygi) with a construct

seq homology---- att---ygi allele---ura3---att---seq homology.

This allows you to see immediately start testing the effect of your alleles..
once this construct is targeted the endogenus...ygi.. you treat it with Cre, which removes the ygi allele and the cell should die.

the control will be construct (for effect of the construct)
att--ygi--ura3---att

alternatively, with a similar setup, you can target with the (seq homology---- att---ygi---ura3---att---seq homology) construct. Transform with the centromeric plasmid... then delete the ygi--ura3 structure by Cre recombinase.


But overall, the diploid cell method is probably faster.
May your PCR products be long, your protocols short and your boss on holiday

#6 yuer

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Posted 17 February 2009 - 07:05 PM

Dear perneseblue,

Thank you so so much for your suggestion! It is very helpful!

About the construct "seq homology---- att---ygi allele---ura3---att---seq homology", I've never read any paper about it. Could you please send me a link for a paper discussing about this construct?

Another question:

I have to make double deletion with two essential genes. I have one haploid strain with one essential gene1 deletion (by HIS3) covered by a LEU2 marker CEN plasmid expressing a point mutation of that gene1. The haploid strain is viable. I am going to use this strain to delete the other essential gene2 first (with your very useful suggestion, probably i will make diploid cells since it is easier). Then, I am going to transform a TRP1 marker CEN plasmid expressing the point mutation of the gene2 into the desired strain with double gene deletion. My purpose is to test if the double mutant is viable.

My question is whether I can delete the essential gene2 with LYS2 marker since this is the only nutritional marker I can use. I don't know if this can be used as a deletion marker since it is so large (4795bp). In one paper, the HIS3 was used again to delete the gene2. The genotype of haploid cells is gene1::HIS3 gene2::HIS3. I really don't understand how they could use the same marker to delete two gene in the same strain. Could you tell me whether I can use HIS3 marker to delete the gene2?

I deeply appreciate your big help!

best wishes,

yuer

Edited by yuer, 17 February 2009 - 07:06 PM.


#7 perneseblue

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Posted 18 February 2009 - 03:47 AM

"seq homology---- loxP---ygi allele---ura3---loxP---seq homology"

I made up this construct, so it doesn't appear in any paper.


My question is whether I can delete the essential gene2 with LYS2 marker since this is the only nutritional marker I can use. I don't know if this can be used as a deletion marker since it is so large (4795bp). In one paper, the HIS3 was used again to delete the gene2. The genotype of haploid cells is gene1::HIS3 gene2::HIS3. I really don't understand how they could use the same marker to delete two gene in the same strain. Could you tell me whether I can use HIS3 marker to delete the gene2?


Thank you for the information. Even with a google search, I am unable to discover how the HIS3 marker could be used twice in the same strain. HIS3 alleles do not appear to give a distinguishable phenotype in histidine limiting conditions. (Unlike the ade1 and ade2 alleles, where the cell turns red. And thus complementation can be used) Would it be possible to have a look at the paper?

Without any more additional information, I would say no. It is not possible to delete gene2 with HIS3 marker, when that marker is already in use. Could they have used a system to remove HIS3 marker after deleting gene1?

As for LYS2 marker, you can use it, even if it is on the large side. But don't you have the ura3 marker and the kan marker? As the Kan marker is in loxP-kan-loxP construct. You can delete that marker after use by Cre. Alternatively you could engineer a loxP-ura3-loxP marker. And then use Cre and counter against it. With ura3 counterselection, you can even use that to make a construct that can delete itself using the cell's natural high frequency of homologous recombination
May your PCR products be long, your protocols short and your boss on holiday

#8 yuer

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Posted 18 February 2009 - 07:40 PM

Thank you for the information. Even with a google search, I am unable to discover how the HIS3 marker could be used twice in the same strain. HIS3 alleles do not appear to give a distinguishable phenotype in histidine limiting conditions. (Unlike the ade1 and ade2 alleles, where the cell turns red. And thus complementation can be used) Would it be possible to have a look at the paper?

Without any more additional information, I would say no. It is not possible to delete gene2 with HIS3 marker, when that marker is already in use. Could they have used a system to remove HIS3 marker after deleting gene1?

As for LYS2 marker, you can use it, even if it is on the large side. But don't you have the ura3 marker and the kan marker? As the Kan marker is in loxP-kan-loxP construct. You can delete that marker after use by Cre. Alternatively you could engineer a loxP-ura3-loxP marker. And then use Cre and counter against it. With ura3 counterselection, you can even use that to make a construct that can delete itself using the cell's natural high frequency of homologous recombination


Dear Perneseblue,

Thank you so so much for your suggestions and your patience!

The paper about the double mutant using the HIS3 marker is attached. Please just take a look at the strain SY147 the table I on page 1411. I am not working on these two genes, but i feel weird after seeing the genotype of the strain.

I can't use URA3 as a marker because I will this marker for plasmid shuffling for the essential gene. I asked my boss if I can use KanMX as marker, she said that i should use nutritional markers since there are a lot these makers. Actually, based on the genotype of the strain I have, I can only use LYS2 as the deletion nutritional marker.
Do you know if there is any drawbacks of the KanMX marker?

It is very nice of you and thank you again!

yuer

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#9 perneseblue

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Posted 19 February 2009 - 03:03 AM

kan marker uses G418 as a selection agent. Compared to nutritional markers, G418 is far more expensive. Thus the use of the kan marker is avoided to save cost.

do you have lacZ gene around? Could use that as a marker.

Hmm...I was unable to follow the trail of papers that would describe the strain. So I am still at a lost.

One last thought, is gene1 and gene2 close together on the same chromosome? If they may not segregate independently at a high frequency and that would cause trouble.

Will more markers be required after creating this double knock out haploid strain? You might want to do flank the marker used for the gene knockout with loxP sote so you can delete them with cre.
May your PCR products be long, your protocols short and your boss on holiday




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