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Need Help: Bacterial gene deletion- sacB sucrose resistance - (Aug/25/2009 )

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fishdoc on Aug 28 2009, 11:32 PM said:

cloneboy on Aug 28 2009, 10:15 AM said:

I think we still come back to the starting point of essentiality again with respect to your above suggestions. As said above 3 outcomes by conjugation and i suppose mine follows the third one " third option is that the crossover ONLY occurs once, either upstream or downstream. In this case, the entire plasmid integrates into the genome at the site of recombination".

For your comments "If you're not getting any colonies immediately after plating your conjugation while selecting for single crossovers, then I think that you're not making any mutants, you've simply got a conjugation that is not resulting in any recombination, for whatever reason".
I just want you to recap the table which i posted before (added below again). If you see my WT grows only in cent and not in cent+amp+kan. If the WT incorporates the antibiotic cassette from the plasmid obviously it might not grow even in cent alone. No growth in cent+amp+kan for sure. If no recombination then i should see growth at least in the cent right?? I think you got the catch now.

Growth on Centrimide ||| Growth on Cent+kan+amp

Wild Type (WT) - G (Growth) ||| NG (No Growth)

WT recombine with
recomb plasmid but
gene NON ESSENTIAL - G ||| G


WT recombine with
recomb plasmid and
gene ESSENTIAL - NG ||| NG

No recombination - G ||| NG


Coming back to mutant i would like to define MUTANT into category,
1. Mutant (non-essential) -In this case of non-essential genes, cells can survive the disruption and form kan+amp resistant colonies.

2. Mutant (essential) - no growth due to lack of recovery of antibiotic-resistant colonies.

My mutant is essential and no matter single or double crossover, my mutant will be lethal after wild type copy gene is disturbed .

This can be even verified from the NAR paper for which i gave the link in the previous posts. Why didn't the group try all the above steps which you mentioned above?

Does this mean NAR reviewers failed to ask the above questions?


I can't speak to what the NAR reviewers did or didn't ask. I'm simply going on the information you're telling me. If you have a vector that carries kan+amp resistance in its backbone, but not in the gene mutation, then the presence of the plasmid should result in growth on kan+amp, right? When a single crossover occurs, as Homebrew described, you get a merodiploid: one mutant allele and one WT allele in the genome of a single cell. The presence of that native allele *should* complement the activity of the mutated one, and thus you should get growth on kan+amp. Because you're selecting for the maintenance of kan+amp, you're selecting for a single crossover, which would result in the presence of the WT non-mutated gene in the genome. With that there, it should still grow, correct? Only once you remove the kan+amp pressure, thus not selecting for the plasmid integration, the plasmid can excise from the genome, removing the kan+amp resistance, and leaving either a WT or a mutant allele (but not both). This is when you'd see lethality.

If you were trying to insert kan or amp into the middle of your gene of interest, thus causing a mutation, then I think the inability to grow in the presence of the antibiotic might indicate lethality of a mutant. However, since the resistance cassettes are encoded by the plasmid backbone, and not inserted into the gene, you should definitely get growth of colonies on kan+amp following conjugation. They will be your single crossovers that, while they do carry a mutated allele, will also carry the WT allele. And since that WT allele is still intact, you should get growth if indeed you are getting that single crossover.

For your table, are those results you expect to happen or are those what you have actually observed?

If you do your conjugation and immediately plate onto cent, you should still get growth, because there will still be WT PAO1 present in the conjugation. Not all of the cells will have picked up the plasmid or have had a recombination event. Also, some that do have that event will revert back to the WT and lose the plasmid, thus growing on cent alone.

That is, unless I'm really misunderstanding something big.


Your argument seems correct at times i am little confused. Let me tell my view on your above comments.

"If you have a vector that carries kan+amp resistance in its backbone, but not in the gene mutation, then the presence of the plasmid should result in growth on kan+amp, right?"


The tabulation was the result of my experiments and thats how i came to a conclusion. For the above comment that the presence of plasmid should result in growth on kan+amp i should say this will happen only when the gene is non essential and the antibiotic cassette has no effect on the chromosome and hence it will grow. The mere presence of plasmid will have no growth as i am using "cetrimide" agar very specific for PAO1 only.

Strains of P. aeruginosa are identified by their production of pyocyanin, a blue, water-soluble, nonfluorescent, phenazine pigment in addition to their colonial morphology and the characteristic grapelike odor of aminoacetophenone. P. aeruginosa is the only species of Pseudomonas or gramnegative rod known to excrete pyocyanin. Cetrimide Agar, therefore, is a valuable culture medium in the identification of this organism.Other species of Pseudomonas do not produce pyocyanin, but fluoresce under UV light. Most non-Pseudomonas species are inhibited, and some species of Pseudomonas may also be inhibited.

The growth with only the plasmid can happen if i had used LB media.

Am i right?

"If you do your conjugation and immediately plate onto cent, you should still get growth, because there will still be WT PAO1 present in the conjugation. Not all of the cells will have picked up the plasmid or have had a recombination event"

May be i need to check again this part in case cells that have not picked up the plasmid can grow in cet alone. In that case i have only one option of checking teh mutant cells that don t grow ie. by keeping a control palte where i just culture PAO1 WT only and if i get say 50-100 colonies and then my conjugation plate in cet alone results say some 10 colonies then i can say that atleast 40-90 must be mutant cells that are essential and hence less growth. Does this look reasonable??

-cloneboy-

cloneboy on Aug 28 2009, 11:32 AM said:

Your argument seems correct at times i am little confused. Let me tell my view on your above comments.

"If you have a vector that carries kan+amp resistance in its backbone, but not in the gene mutation, then the presence of the plasmid should result in growth on kan+amp, right?"


The tabulation was the result of my experiments and thats how i came to a conclusion. For the above comment that the presence of plasmid should result in growth on kan+amp i should say this will happen only when the gene is non essential and the antibiotic cassette has no effect on the chromosome and hence it will grow. The mere presence of plasmid will have no growth as i am using "cetrimide" agar very specific for PAO1 only.

Strains of P. aeruginosa are identified by their production of pyocyanin, a blue, water-soluble, nonfluorescent, phenazine pigment in addition to their colonial morphology and the characteristic grapelike odor of aminoacetophenone. P. aeruginosa is the only species of Pseudomonas or gramnegative rod known to excrete pyocyanin. Cetrimide Agar, therefore, is a valuable culture medium in the identification of this organism.Other species of Pseudomonas do not produce pyocyanin, but fluoresce under UV light. Most non-Pseudomonas species are inhibited, and some species of Pseudomonas may also be inhibited.

The growth with only the plasmid can happen if i had used LB media.

Am i right?

"If you do your conjugation and immediately plate onto cent, you should still get growth, because there will still be WT PAO1 present in the conjugation. Not all of the cells will have picked up the plasmid or have had a recombination event"

May be i need to check again this part in case cells that have not picked up the plasmid can grow in cet alone. In that case i have only one option of checking teh mutant cells that don t grow ie. by keeping a control palte where i just culture PAO1 WT only and if i get say 50-100 colonies and then my conjugation plate in cet alone results say some 10 colonies then i can say that atleast 40-90 must be mutant cells that are essential and hence less growth. Does this look reasonable??



"For the above comment that the presence of plasmid should result in growth on kan+amp i should say this will happen only when the gene is non essential and the antibiotic cassette has no effect on the chromosome and hence it will grow."

I disagree with that. I think you're referring to the product of a double crossover event, which results in the exchange of the plasmid mutant allele with the genome WT allele, AND loss of the vector backbone. That would lead to a single mutant allele in the organism, and if lethal, the inability to grow at all, on any type of media.

However, if you're plating onto cent+kan+amp following conjugation, you're putting pressure on the recombination event to be a single crossover, in which the plasmid integrates into the genome. That does not mean the plasmid integrates and disrupts the specific allele, causing a mutation. As I and Homebrew have described, what occurs during a single crossover is the plasmid integrates, and you maintain the WT gene in addition to adding the mutant gene and the rest of the vector. By virtue of that WT gene still being present in the genome, PAO1 should still grow. The caveat to that, however, is if the gene you're mutating is the first gene of an operon. If that's the case, the insertion of the plasmid may disrupt downstream expression of any genes in the operon. And if that's the case, the loss of expression of one of those genes may be the cause for the inability to grow rather than the gene you're trying to mutate. If the gene is monocistronic (I've assumed it is), then that will not be the case.

I realize that the E. coli donor cannot grow on cent. If I insinuated anywhere above that it could, my apologies as I misspoke. PAO1 carrying an integrated plasmid which has kan+amp resistance, however, should grow still grow. The plasmid is not freely replicating on its own, obviously, because pir is not present, but kan+amp can still express if the plasmid is integrated into the genome, and that is why I think you should still see growth on cent+kan+amp following conjugation.


"May be i need to check again this part in case cells that have not picked up the plasmid can grow in cet alone. In that case i have only one option of checking teh mutant cells that don t grow ie. by keeping a control palte where i just culture PAO1 WT only and if i get say 50-100 colonies and then my conjugation plate in cet alone results say some 10 colonies then i can say that atleast 40-90 must be mutant cells that are essential and hence less growth. Does this look reasonable??"


I don't think that would work, but I'm not certain I understand your plan. You would need to conjugate your control WT cells the same way you did your experimental ones (with a "non-lethal" mutation or something), not just culturing PAO1 onto cent media.



What it boils down to is the way recombination works, a single-crossover integration of a plasmid leaves a WT gene intact, as well as adding the mutant gene. Only a double crossover would result in one or the other being present, and plating on cent+kan+amp does not does not select for double crossovers, only singles.

-fishdoc-

fishdoc on Aug 29 2009, 12:58 AM said:

cloneboy on Aug 28 2009, 11:32 AM said:

Your argument seems correct at times i am little confused. Let me tell my view on your above comments.

"If you have a vector that carries kan+amp resistance in its backbone, but not in the gene mutation, then the presence of the plasmid should result in growth on kan+amp, right?"


The tabulation was the result of my experiments and thats how i came to a conclusion. For the above comment that the presence of plasmid should result in growth on kan+amp i should say this will happen only when the gene is non essential and the antibiotic cassette has no effect on the chromosome and hence it will grow. The mere presence of plasmid will have no growth as i am using "cetrimide" agar very specific for PAO1 only.

Strains of P. aeruginosa are identified by their production of pyocyanin, a blue, water-soluble, nonfluorescent, phenazine pigment in addition to their colonial morphology and the characteristic grapelike odor of aminoacetophenone. P. aeruginosa is the only species of Pseudomonas or gramnegative rod known to excrete pyocyanin. Cetrimide Agar, therefore, is a valuable culture medium in the identification of this organism.Other species of Pseudomonas do not produce pyocyanin, but fluoresce under UV light. Most non-Pseudomonas species are inhibited, and some species of Pseudomonas may also be inhibited.

The growth with only the plasmid can happen if i had used LB media.

Am i right?

"If you do your conjugation and immediately plate onto cent, you should still get growth, because there will still be WT PAO1 present in the conjugation. Not all of the cells will have picked up the plasmid or have had a recombination event"

May be i need to check again this part in case cells that have not picked up the plasmid can grow in cet alone. In that case i have only one option of checking teh mutant cells that don t grow ie. by keeping a control palte where i just culture PAO1 WT only and if i get say 50-100 colonies and then my conjugation plate in cet alone results say some 10 colonies then i can say that atleast 40-90 must be mutant cells that are essential and hence less growth. Does this look reasonable??



"For the above comment that the presence of plasmid should result in growth on kan+amp i should say this will happen only when the gene is non essential and the antibiotic cassette has no effect on the chromosome and hence it will grow."

I disagree with that. I think you're referring to the product of a double crossover event, which results in the exchange of the plasmid mutant allele with the genome WT allele, AND loss of the vector backbone. That would lead to a single mutant allele in the organism, and if lethal, the inability to grow at all, on any type of media.

However, if you're plating onto cent+kan+amp following conjugation, you're putting pressure on the recombination event to be a single crossover, in which the plasmid integrates into the genome. That does not mean the plasmid integrates and disrupts the specific allele, causing a mutation. As I and Homebrew have described, what occurs during a single crossover is the plasmid integrates, and you maintain the WT gene in addition to adding the mutant gene and the rest of the vector. By virtue of that WT gene still being present in the genome, PAO1 should still grow. The caveat to that, however, is if the gene you're mutating is the first gene of an operon. If that's the case, the insertion of the plasmid may disrupt downstream expression of any genes in the operon. And if that's the case, the loss of expression of one of those genes may be the cause for the inability to grow rather than the gene you're trying to mutate. If the gene is monocistronic (I've assumed it is), then that will not be the case.

I realize that the E. coli donor cannot grow on cent. If I insinuated anywhere above that it could, my apologies as I misspoke. PAO1 carrying an integrated plasmid which has kan+amp resistance, however, should grow still grow. The plasmid is not freely replicating on its own, obviously, because pir is not present, but kan+amp can still express if the plasmid is integrated into the genome, and that is why I think you should still see growth on cent+kan+amp following conjugation.


"May be i need to check again this part in case cells that have not picked up the plasmid can grow in cet alone. In that case i have only one option of checking teh mutant cells that don t grow ie. by keeping a control palte where i just culture PAO1 WT only and if i get say 50-100 colonies and then my conjugation plate in cet alone results say some 10 colonies then i can say that atleast 40-90 must be mutant cells that are essential and hence less growth. Does this look reasonable??"


I don't think that would work, but I'm not certain I understand your plan. You would need to conjugate your control WT cells the same way you did your experimental ones (with a "non-lethal" mutation or something), not just culturing PAO1 onto cent media.



What it boils down to is the way recombination works, a single-crossover integration of a plasmid leaves a WT gene intact, as well as adding the mutant gene. Only a double crossover would result in one or the other being present, and plating on cent+kan+amp does not does not select for double crossovers, only singles.


I read many papers related to gene disruption and finding the gene essentiality. Hence i designed my experiments according to that. From your comments above i would could understand that you try to explain two different concepts 1. Plasmid insertion mutagenesis 2. Allelic replacement.

I refer to another paper by Pucci 2006 on the above concepts.

http://www.ncbi.nlm.nih.gov/pubmed/16412986

If you go through this paper he has mentioned the following in section 1.2 Fig 2:
Fig. 2 (A) Plasmid insertion mutagenesis. An internal fragment of the target gene is cloned onto a plasmid containing a selectable marker. The suicide plasmid vector (incapable of replication in the host cell) is introduced into the bacterium under selection for the antibiotic resistance marker. Bacterial colonies result from homologous recombination of the entire plasmid into the chromosomal gene followed by a single-crossover, insertion-duplication event for the target gene where two incomplete and inactive gene copies are generated. If the target gene is essential, this will be a lethal event and cells will not be viable.

(:lol: Allelic replacement. An antibiotic resistance gene is cloned between flanking sequences of the target gene resulting in replacement of the target with the resistance marker. When introduced into the bacterial cell under antibiotic selection, a double-crossover event results in a replacement of the chromosomal gene copy with the resistance gene. If the target gene is essential, this will be a lethal event cells will not be viable.

If you see he explains that by plasmid insertion by a single crossover will result in WT as well as mutant copy of the gene (both partial) and if the gene is essential then this would result in no growth. He went on to cite the NAR paper as reference.

Am i being misguided by the papers??

-cloneboy-

cloneboy on Aug 29 2009, 09:07 AM said:

I refer to another paper by Pucci 2006 on the above concepts.

http://www.ncbi.nlm.nih.gov/pubmed/16412986

If you go through this paper he has mentioned the following in section 1.2 Fig 2:
Fig. 2 (A) Plasmid insertion mutagenesis. An internal fragment of the target gene is cloned onto a plasmid containing a selectable marker. The suicide plasmid vector (incapable of replication in the host cell) is introduced into the bacterium under selection for the antibiotic resistance marker. Bacterial colonies result from homologous recombination of the entire plasmid into the chromosomal gene followed by a single-crossover, insertion-duplication event for the target gene where two incomplete and inactive gene copies are generated. If the target gene is essential, this will be a lethal event and cells will not be viable.

(:lol: Allelic replacement. An antibiotic resistance gene is cloned between flanking sequences of the target gene resulting in replacement of the target with the resistance marker. When introduced into the bacterial cell under antibiotic selection, a double-crossover event results in a replacement of the chromosomal gene copy with the resistance gene. If the target gene is essential, this will be a lethal event cells will not be viable.

If you see he explains that by plasmid insertion by a single crossover will result in WT as well as mutant copy of the gene (both partial) and if the gene is essential then this would result in no growth. He went on to cite the NAR paper as reference.

Am i being misguided by the papers??


I am confused by the legend of Figure 2a -- could you post a pdf of the paper, or at least an image of that figure?

-HomeBrew-

HomeBrew on Aug 29 2009, 09:41 PM said:

cloneboy on Aug 29 2009, 09:07 AM said:

I refer to another paper by Pucci 2006 on the above concepts.

http://www.ncbi.nlm.nih.gov/pubmed/16412986

If you go through this paper he has mentioned the following in section 1.2 Fig 2:
Fig. 2 (A) Plasmid insertion mutagenesis. An internal fragment of the target gene is cloned onto a plasmid containing a selectable marker. The suicide plasmid vector (incapable of replication in the host cell) is introduced into the bacterium under selection for the antibiotic resistance marker. Bacterial colonies result from homologous recombination of the entire plasmid into the chromosomal gene followed by a single-crossover, insertion-duplication event for the target gene where two incomplete and inactive gene copies are generated. If the target gene is essential, this will be a lethal event and cells will not be viable.

(:lol: Allelic replacement. An antibiotic resistance gene is cloned between flanking sequences of the target gene resulting in replacement of the target with the resistance marker. When introduced into the bacterial cell under antibiotic selection, a double-crossover event results in a replacement of the chromosomal gene copy with the resistance gene. If the target gene is essential, this will be a lethal event cells will not be viable.

If you see he explains that by plasmid insertion by a single crossover will result in WT as well as mutant copy of the gene (both partial) and if the gene is essential then this would result in no growth. He went on to cite the NAR paper as reference.

Am i being misguided by the papers??


I am confused by the legend of Figure 2a -- could you post a pdf of the paper, or at least an image of that figure?

attached the paper.
Attached File

-cloneboy-

cloneboy on Aug 29 2009, 08:07 AM said:

I read many papers related to gene disruption and finding the gene essentiality. Hence i designed my experiments according to that. From your comments above i would could understand that you try to explain two different concepts 1. Plasmid insertion mutagenesis 2. Allelic replacement.

I refer to another paper by Pucci 2006 on the above concepts.

http://www.ncbi.nlm.nih.gov/pubmed/16412986

If you go through this paper he has mentioned the following in section 1.2 Fig 2:
Fig. 2 (A) Plasmid insertion mutagenesis. An internal fragment of the target gene is cloned onto a plasmid containing a selectable marker. The suicide plasmid vector (incapable of replication in the host cell) is introduced into the bacterium under selection for the antibiotic resistance marker. Bacterial colonies result from homologous recombination of the entire plasmid into the chromosomal gene followed by a single-crossover, insertion-duplication event for the target gene where two incomplete and inactive gene copies are generated. If the target gene is essential, this will be a lethal event and cells will not be viable.

(:lol: Allelic replacement. An antibiotic resistance gene is cloned between flanking sequences of the target gene resulting in replacement of the target with the resistance marker. When introduced into the bacterial cell under antibiotic selection, a double-crossover event results in a replacement of the chromosomal gene copy with the resistance gene. If the target gene is essential, this will be a lethal event cells will not be viable.

If you see he explains that by plasmid insertion by a single crossover will result in WT as well as mutant copy of the gene (both partial) and if the gene is essential then this would result in no growth. He went on to cite the NAR paper as reference.

Am i being misguided by the papers??



OK, I see what's going on. I did not understand how you were constructing the suicide vector (with just a gene fragment rather than another construct such as a deletion or a insertion). In that single crossover event, because you only have a fragment of the target gene (not a complete 5' or 3' end), homologous recombination (single event) results in one copy that doesn't have a 5' end and one that doesn't have a 3' end.

By that method, I agree you would inactivate the gene (both copies).


With that said, I think there are much better ways of of going about such a study (just my opinion), but since you've already done a majority of the work, you can't go back now and change it.

-fishdoc-

fishdoc on Aug 29 2009, 11:47 PM said:

cloneboy on Aug 29 2009, 08:07 AM said:

I read many papers related to gene disruption and finding the gene essentiality. Hence i designed my experiments according to that. From your comments above i would could understand that you try to explain two different concepts 1. Plasmid insertion mutagenesis 2. Allelic replacement.

I refer to another paper by Pucci 2006 on the above concepts.

http://www.ncbi.nlm.nih.gov/pubmed/16412986

If you go through this paper he has mentioned the following in section 1.2 Fig 2:
Fig. 2 (A) Plasmid insertion mutagenesis. An internal fragment of the target gene is cloned onto a plasmid containing a selectable marker. The suicide plasmid vector (incapable of replication in the host cell) is introduced into the bacterium under selection for the antibiotic resistance marker. Bacterial colonies result from homologous recombination of the entire plasmid into the chromosomal gene followed by a single-crossover, insertion-duplication event for the target gene where two incomplete and inactive gene copies are generated. If the target gene is essential, this will be a lethal event and cells will not be viable.

(:lol: Allelic replacement. An antibiotic resistance gene is cloned between flanking sequences of the target gene resulting in replacement of the target with the resistance marker. When introduced into the bacterial cell under antibiotic selection, a double-crossover event results in a replacement of the chromosomal gene copy with the resistance gene. If the target gene is essential, this will be a lethal event cells will not be viable.

If you see he explains that by plasmid insertion by a single crossover will result in WT as well as mutant copy of the gene (both partial) and if the gene is essential then this would result in no growth. He went on to cite the NAR paper as reference.

Am i being misguided by the papers??



OK, I see what's going on. I did not understand how you were constructing the suicide vector (with just a gene fragment rather than another construct such as a deletion or a insertion). In that single crossover event, because you only have a fragment of the target gene (not a complete 5' or 3' end), homologous recombination (single event) results in one copy that doesn't have a 5' end and one that doesn't have a 3' end.

By that method, I agree you would inactivate the gene (both copies).


With that said, I think there are much better ways of of going about such a study (just my opinion), but since you've already done a majority of the work, you can't go back now and change it.


Hope i made you understand the methodology i followed. Can i conclude from your above comment that i was right in following the knockout procedure and hence my control results (table) are in right direction?

-cloneboy-

cloneboy on Aug 26 2009, 02:06 AM said:

I am trying to knockout a gene which is essential for growth of the pathogen Pseudomonas aeruginosa PAO1 (has no sacB gene in it). I check for the gene knockout in "centrimide+kan+amp" plate with PAO1 using the gene inserted suicide vector pFS100 and get no growth (showing the gene is essential) whereas the control plate without antibiotic markers show growth.


Yes, the confusion was coming from our assumption that you had cloned the whole gene into pFS100, and not just a fragment of the gene...
Attached File

-HomeBrew-

cloneboy on Aug 29 2009, 10:58 AM said:

Hope i made you understand the methodology i followed. Can i conclude from your above comment that i was right in following the knockout procedure and hence my control results (table) are in right direction?



Yes, I now understand the methodology.

However, I don't know that I completely buy the conclusions of the results (that it's a lethal knockout). I realize there is literature out there that rationalizes that conclusion, and maybe it indeed is the correct conclusion, I don't know. But as I said, I think there are better methods available, that can provide better quantitative and qualitative data. I think there are too many variables involved to assume that because there is no growth that the mutation is lethal. You have a sort of control in that you have a "nonessential" gene that results in growth, but there could be gene-to-gene variability in recombination. The one that you get growth with may recombine easier due to differences in sequence, differences in surrounding DNA that lead to changes in any secondary structure of the DNA, or anything of that sort. Furthermore, depending on what is around the gene your mutating, insertion of a large plasmid could disrupt transcriptional regulatory regions of surrounding genes.

These things are possibilities, but in no way am I saying they are likely. However, I think they are questions that could be raised, depending on who may review the work.

The one thing that may be able to squash those questions is having that complementation plasmid in place to show that the bacteria survives the recombination event in which 2 incomplete alleles are in the genome as long as it is provided in trans from a plasmid, thus showing there are no effects of the plasmid integration on surrounding DNA and the plasmid is indeed integrating.

I just really have a hard time seeing "no growth" as a final result and concluding what is happening without being able to actually confirm it, particularly when there are other methods available that can give more information. Those sorts of experiments are discussed/described earlier in the thread.

-fishdoc-

I agree. Were I reviewing your paper, I'd ask for experiments demonstrating restoration of viability by complementation in trans before accepting the conclusion that the gene is essential, and experiments demonstrating that the plasmid integrated into the chromosome in such a way as to disrupt the gene you're saying it did -- essentially, these are the .the same two pieces of data I suggested you needed in this post.

-HomeBrew-
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