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Question in gateway system, LR reaction


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#1 fonie

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Posted 25 July 2015 - 11:04 AM

Hello all,

 

I asked this on another forum before, but was hoping to get some more insight in this problem.

 

 

Has anyone here every noticed something weird when doing a LR reaction, using the gateway system, in terms of getting some sort of mixture of plasmids? Like some sort of hybrid plasmid?

I get a very low efficiency after the LR reaction (only a few cells per plate). The destination vector is fine (all gateway sequences are ok), but after the LR reaction I get only a few colonies and when I sequence them I get something really strange.
I use 3 primers:
1 primer on my destination vector before the GOI (primer 1)
1 primer after my GOI on the destination vector (to cover the gene and some part of my destination vector) (primer 2).
1 primer on the GOI , going towards my destination vector (primer 3).

The first 2 primers give me what I want:
Primer 1: piece of my destination (now expression vector) , attb1 site, followed by the GOI

Primer 2: piece of my destination vector , attb2 site, followed by the GOI

The above primers, "added" together give me: destination vector - GOI - destination vector.
So far so good.

However: using primer 3 (that binds my GOI) I get : GOI followed by a little piece of the attb1 site and then the donor vector...
This makes no sense to me.

Do I have some sort of hybrid plasmid? But how is this possible? Or do I have a mixture of plasmids? But this seems also weird since its from a single colony (I hardly have any colonies on my plate anyway so its really form a single CFU).


I know that something is wrong since I only get a few colonies per LR reaction after plating, but I really do not get this hybrid/weird plasmid.

 



#2 miST32

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Posted 26 July 2015 - 12:41 PM

Hi fonie,

Interesting observations.  One of my colleagues recently got some weird results with their LR clones, too, so this may be common.  At least you aren't alone!

When you say that you see your donor vector, do you mean the GOI-bearing entry vector?  With Primer #3 do you see attL or attP site as well as your attB1 fragment and GEO?  The donor vector is regenerated in the LR reaction, but it should have different antibiotic resistance than the destination vector (like the Entry vector).  Very odd indeed.  I don't know if it's possible to pick up and re-purify the donor/entry vectors from the plate that receives your transformation.  Seems highly unlikely - this is usually only a problem with colony PCR.  That makes me think you do have a hybrid of some sort.

If I were in this position and suspected multiple plasmids, I would try a few things:

1) Transform the sequenced product into new cells and re-prep a few more colonies to see if the pattern repeats in another sequencing round. This should dilute out contaminant plasmids.

2) Use a restriction digest to screen your sample.  If possible, select a unique cut site that is in the region of your GOI covered by Primer #3.  Two products vs. a linearized plasmid might indicate a hybrid plasmid or multiple plasmids in your prep.  Also try digests with enzymes that only recognize your entry and destination vectors, respectively.

3) Start over.  Verify your entry and destination sequences, get super clean DNA and perform the LR reaction again.  The amount of DNA can be varied in the reaction according to the LR clonase manual - so I might play around with the amount of each vector.  Definitely consider the stoichiometry of your entry and destination vectors (i.e. how many molecules do you have of each per given mass of DNA).

If you have multiple plasmids, I can only surmise that you've picked one up from a contaminating source in the lab (plasmids get everywhere).  Re-transforming and carefully prepping your colonies should clean it up.  If this were the case though, I'd also suspect that your sequencing traces would show dirty base calls from primer #3, indicative of two different, equal-length termination products rooted with the same primer sequence.  This would begin approximately where the GOI sequence ends as that region should be common to any plasmid with the GOI insert.

Good luck



#3 fonie

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Posted 28 July 2015 - 01:41 AM

Hi fonie,

Interesting observations.  One of my colleagues recently got some weird results with their LR clones, too, so this may be common.  At least you aren't alone!

When you say that you see your donor vector, do you mean the GOI-bearing entry vector?  With Primer #3 do you see attL or attP site as well as your attB1 fragment and GEO?  The donor vector is regenerated in the LR reaction, but it should have different antibiotic resistance than the destination vector (like the Entry vector).  Very odd indeed.  I don't know if it's possible to pick up and re-purify the donor/entry vectors from the plate that receives your transformation.  Seems highly unlikely - this is usually only a problem with colony PCR.  That makes me think you do have a hybrid of some sort.

If I were in this position and suspected multiple plasmids, I would try a few things:

1) Transform the sequenced product into new cells and re-prep a few more colonies to see if the pattern repeats in another sequencing round. This should dilute out contaminant plasmids.

2) Use a restriction digest to screen your sample.  If possible, select a unique cut site that is in the region of your GOI covered by Primer #3.  Two products vs. a linearized plasmid might indicate a hybrid plasmid or multiple plasmids in your prep.  Also try digests with enzymes that only recognize your entry and destination vectors, respectively.

3) Start over.  Verify your entry and destination sequences, get super clean DNA and perform the LR reaction again.  The amount of DNA can be varied in the reaction according to the LR clonase manual - so I might play around with the amount of each vector.  Definitely consider the stoichiometry of your entry and destination vectors (i.e. how many molecules do you have of each per given mass of DNA).

If you have multiple plasmids, I can only surmise that you've picked one up from a contaminating source in the lab (plasmids get everywhere).  Re-transforming and carefully prepping your colonies should clean it up.  If this were the case though, I'd also suspect that your sequencing traces would show dirty base calls from primer #3, indicative of two different, equal-length termination products rooted with the same primer sequence.  This would begin approximately where the GOI sequence ends as that region should be common to any plasmid with the GOI insert.

Good luck

 

Yes, by the donor vector I did mean the entry vector with my gene of interest

 

With primer 3 I see my gene of interest (where the primer binds) followed by a small part of the Attb1 site, followed by the entry vector (I guess a piece of the attL site).

 

So it looks like an incomplete LR reaction somehow.

 

But its weird I also (with primers 1 and 2) I get the correct expression clone.

 

And yes: it has a different antibiotic selection, so its weird I do seem to find it.

I also find it, in general, weird I would have 2 plasmids in 1 E.coli? Or maybe 1 hybrid plasmid that contains the gene twice? But this seems weird, I should see this in a gel, that my vector is too large for what it should be!

 

 

1) I did this and it seemed to have done the trick for 2 plasmids!

 

2) Working on this one.

 

3) well... its pretty expensive... I did it 4 times already by now and I changed pretty much everything I could change...

 

 

 

About the sequencing: you are right, when the donor/entry vector pops up in the sequencing results, it seems to be that there are 2 plasmids present... 

After resequencing some plasmids that I transformed again, it gave me the correct sequence (the expression vector) and it seemed ok in terms of sequencing (nice peaks).

Biut this only worked for 3 plasmids out of 10 or something.



#4 miST32

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Posted 28 July 2015 - 07:56 AM

Hi fonie,

That's great news!  Glad to hear you got the correct sequence isolated through your efforts.

It does seem odd that you had multiple plasmids in the first place, but your results suggest this to be the case.  

Although somewhat rare, it's quite possible that you had a single CFU but multiple plasmids in that initial cell - particularly if you used a rapid plating strategy where plasmid incompatibility can be (briefly) overcome.  This article discusses the issue and might be helpful:  http://bitesizebio.c...smid-retention/  

This suggests that the outgrowth step might be particularly important here, such that the bacteria have a chance to grow/divide and dilute out the multiple plasmids into individual "progeny" cells before being plated on selective LB.

As far as why so many clones (7 of 10?) had an apparent multiple plasmid problem, I have to wonder if this is due to the recombination kinetics.  Speculatively, I wonder if the recombination proceeds slowly enough that intermediate products are co-transformed at a higher rate if the reaction is terminated to early.  I'm not familiar enough with the technology to really know.

Are you using a particularly large destination vector?  Maybe doing a 16+ hour LR reaction and reducing the dominant molar species of plasmid (probably the entry vector) will yield more correct, individual clones?

Anyway - hope your luck keeps up.  Cheers



#5 Davegatc

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Posted 29 July 2015 - 08:45 AM

 -deleted-


Edited by Davegatc, 29 July 2015 - 08:51 AM.


#6 fonie

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Posted 29 July 2015 - 09:03 AM

I use simple TOP10 cells...

And yes, its a very simple protocol for the transformation and plating is done with beads, quickly after the recovery that only takes places for 40-50 minutes (after the heat shock).

 

Well, no, pretty much all of the plasmids gave me the problem, but after retransforming I managed to get 3 good ones.

But it took a lot of time and effort.

 

It should not be the case, if the recombination is not completed the plasmid should not survive since its another selection marker.

 

 

Yes ! Its a large destination vector (10kb) and I already do an overnight LR reaction, so yes, often 16 hours or longer.

 

Hi fonie,

That's great news!  Glad to hear you got the correct sequence isolated through your efforts.

It does seem odd that you had multiple plasmids in the first place, but your results suggest this to be the case.  

Although somewhat rare, it's quite possible that you had a single CFU but multiple plasmids in that initial cell - particularly if you used a rapid plating strategy where plasmid incompatibility can be (briefly) overcome.  This article discusses the issue and might be helpful:  http://bitesizebio.c...smid-retention/  

This suggests that the outgrowth step might be particularly important here, such that the bacteria have a chance to grow/divide and dilute out the multiple plasmids into individual "progeny" cells before being plated on selective LB.

As far as why so many clones (7 of 10?) had an apparent multiple plasmid problem, I have to wonder if this is due to the recombination kinetics.  Speculatively, I wonder if the recombination proceeds slowly enough that intermediate products are co-transformed at a higher rate if the reaction is terminated to early.  I'm not familiar enough with the technology to really know.

Are you using a particularly large destination vector?  Maybe doing a 16+ hour LR reaction and reducing the dominant molar species of plasmid (probably the entry vector) will yield more correct, individual clones?

Anyway - hope your luck keeps up.  Cheers

 






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