Beta-lactamase Gene Cloning

[cowboy]

Hi everyone...I'm new to this forum. If you think my questions are really dumb, please don't laugh at me...because I'm not that smart...

 

I came across this problem about beta-lactamase cloning. Say, we're given an isolate of bacteria which has beta-lactamase gene and we want to clone beta-lactamase from this bacteria with the help of e.coli.

 

what are the procedures involved? Is this correct?-->

- treat the bacteria with restriction enzymes to cut the b-lac gene (but I don't know which enzyme is appropriate - shall I use restr-enzyme like HindIII, BamH1?)

- choose a cloning vector. I'm confused at this part too, which plasmid to use? If I am not mistaken, E.coli plasmids contain amp-resistance gene as well like beta-lactamase, so how can i separate the transformed e.coli and the normal e.coli without inserts?

-ligate the fragments with cloning vector

-transform into e.coli

-select e.coli with insert (how to select? can't use phenotypic selection since e.coli without insert has amp-resistance as well? or use blue-white screening perhaps?)

 

Note: please assume that we don't know where the gene for beta-lactamase is..

 

 

I'm just so confused at so many things....I hope you all are patient enough to clear my confusion. Thanks a lot.

[rakuenso]

make sure you run SDS-PAGE or some other kind of electrophoresis after the procedure to make sure it has been the target gene of interest has been inserted in the right place

[Bluenoise]

make sure you run SDS-PAGE or some other kind of electrophoresis after the procedure to make sure it has been the target gene of interest has been inserted in the right place

 

Well from that answer it's obvious you don't know what SDS-PAGE is.

 

 

Anyways in response to the original post it all depends on what you know about the beta lactamase gene. If you know the sequence or at least the boardering sequences you can purifiy the genomic dna of the organism you're trying to isolate from and then use pcr to amplify out the gene. If all you know is protein sequence then you may need to generate degenerate primers to get the gene out. If you don't even know that. Well then you have quite a bit of work to find the gene or protein responsible. And I'm not going into that.

But lets say you do have the primers well then you can use PCR to get your gene amplified out of the organism genomic dna. Ligate this into a vector for cloning. If you're smart you would have used pcr primers to add restriction enzyme cutting sites to your gene of interest that match those in your vector. Then transform your E. coli cells with it using electroporation or maybe CaCl2 competant cells. A good standard vector, one like pBS/SK with an antibiotic resistance selectable marker will help ensure you only get colonies with that are transformed with the plasmid, as well as a second screenable marker for colonies that actualy take up a plasmid that has the gene inserted like blue/white screening.

[rakuenso]

Why can't SDS-PAGE be used?

 

You would have a marker that tells you the relative position of the Beta-lactamase gene, and if it is successfully integrated into the genome such that it's expressed, you should be able to detect beta-lactamase via SDS-PAGE after lysing the cell and purifying it with centrifugation?

[Bluenoise]

Well at first I thought you wanted to use sds to for dna electrophoresis. Since even thought sds is used for proteins. Dna electrophoresis of the purified and digested plasmid would be a way to make sure your insert is there. So that's why I said that. I see what you ment.

 

You want to express the protien then look for it? That's alot of work, and a waste of time/money. And even then you wouldn't be sure you're looking at the right protein necessarily with all the other stuff present. Also for cloning purposes you don't want to be expressing the gene. Metabolic load and all.

 

Just use blue/white screening and then mimiprep, digest then run that on a gel to show it's the right fragment. If you're really paranoid send the fragment for sequencing. But proteins expression is a pretty painstaking way to do it.

 

You're not integrating it into the genome you just using E. Coli as a host for cloning. Plus you don't need to integrate it into the genome to express it anyways a plasmid will do. Also your not going to get much purification with centrifugation, you'll just get some rough cuts.

[rakuenso]

The problem with DNA electrophoresis is that you can never be sure if the gene is actually going to be expressed. I'm not sure if plasmids also follow similar promoter/operon/repressor commands similar to the that of normal "DNA" and if plasmid DNA will always be expressed (not my field =) )

 

I guess you are right with the excess effort involved though.

[cowboy]

First of all, thanks guys for the answers.

 

Well from that answer it's obvious you don't know what SDS-PAGE is.

 

 

Anyways in response to the original post it all depends on what you know about the beta lactamase gene. If you know the sequence or at least the boardering sequences you can purifiy the genomic dna of the organism you're trying to isolate from and then use pcr to amplify out the gene. If all you know is protein sequence then you may need to generate degenerate primers to get the gene out. If you don't even know that. Well then you have quite a bit of work to find the gene or protein responsible. And I'm not going into that.

But lets say you do have the primers well then you can use PCR to get your gene amplified out of the organism genomic dna. Ligate this into a vector for cloning. If you're smart you would have used pcr primers to add restriction enzyme cutting sites to your gene of interest that match those in your vector. Then transform your E. coli cells with it using electroporation or maybe CaCl2 competant cells. A good standard vector' date=' one like pBS/SK with an antibiotic resistance selectable marker will help ensure you only get colonies with that are transformed with the plasmid, as well as a second screenable marker for colonies that actualy take up a plasmid that has the gene inserted like blue/white screening.[/quote']

 

 

It is assumed that we don't know anything about the gene, so PCR is completely ruled out.

 

I kinda understand something now. We can't use blue/white screening to check whether the right gene has been inserted or not (because we can only screen out the vector without insert but not the vector without beta-lactamase gene), right? Hmm....so is it correct to just use a plasmid which has no ampicillin resistant (i dont know what plasmid though), then screen out the vector by phenotypic selection (grow colonies on agar plate containing ampicillin - vector with insert has amp-resistance so it will grow), is that right?

 

Then, next thing to do, like you've mentioned....transform into e.coli using electroporation or competent e.coli cells. Then shall I just use the same amp-resistance to screen out e.coli cells without the plasmid of interest? Or does e.coli plasmids has amp-r as well, so I can't use this method? Confused again...

 

[Bluenoise]

Well if you don't know anything about the gene than you're going to have to find it first. So maybe look for a mutant that's defective in the genes function then I guess try to isolate that gene, or protein by comparison to a wild type. If you find the protein, you can use degenerate primers to fish it out. But this is a whole other question in itself.

 

I'm sorry you're going to have to rephrase that second part again. I'm not sure what you're trying to say.

 

A plasmid with amp resistane works to makes sure that only colonies with the plasmid grow if you plate them onto medium containing ampicillin.

 

A plasmid that allows for blue/white screening allows you to screen the colonies that have grow for just those who's plasmid has an insert in it. Now if you set up your ligation in such a manner as there can really only be 1 possible insert well then you'll have a good idea what it will be.

[ecoli]

I'm doing something similiar in my research right now, asa matter of fact.

 

I'm using the pGem vector because it has the right sites for my desired inserts. The first step is to find which vector is best for your inserts and find restriction ezymes that will cut in the desired places.

[cowboy]

Hmmm....I'm still quite confused at some parts. Pardon me as I've just learned this dna cloning only about several days ago.

 

If it's not too difficult, could you guys write down your step by step guide and I'll ask questions from there? Thanks a lot.

[Bluenoise]

That would be too much work. I don't have a problem answering questions, but doing someones homework for them when I should be doing my own isn't wise.

[cowboy]

That would be too much work. I don't have a problem answering questions, but doing someones homework for them when I should be doing my own isn't wise.

 

 

I'm not asking you to write an essay / or answer in full .... Maybe a rough step-by-step guide like what I've done on my first post will do.

 

I don't understand the part on why we need to know the gene of beta-lactamase. Is it not possible to just clone it into e.coli without knowing the gene? We know that it is amp-resistance, so if e.coli is resistance to amp, the e.coli must have the insert, right?

 

and which screening to use? amp-r or blue/white and which is better? if I could insert the gene into a plasmid which has no amp-resistance and has lacZ operon, I could use both screening method right? Since the plasmid with insert will be amp-resistance and disrupted lacZ (so will appear white in blue/white screening).

[cowboy]

After I read some reference books again and again, I think I finally understood the screening process. Anyway, here's my rough plan on how to clone the beta lactamase gene into e.coli. Please see where I've done wrong and also anything else that needed to be added in? Will there be any problems if I actually follow these steps in the lab? Thanks a lot....

 

Purification of DNA from the isolate

- isolate of bacteria is grown in liquid culture medium

- isolate is harvested by centrifugation, bacteria will form pellet at the bottom. Medium is poured off.

- Break open the cystoplasmic membrane and cell wall by using chemicals or enzyme. Cell lysis. Eg of enzyme: lysozyme, ethylenediamine tetraacetate (EDTA) or both.

- Centrifuge to remove digested fractions of cell wall in pellet. Supernatant contains DNA, RNA and protein (called cell extract)

- Purify DNA from the cell extract. To remove protein, add organic solvents like phenol and chloroform, protein will be precipitated. Centrifuge to remove the aqueous layer which contains DNA and RNA. To remove RNA, ribonuclease is added – an enzyme which break down RNA into its subunits.

- DNA can then be precipitated by adding ethanol. Solution of DNA is added with ethanol and then centrifuged. DNA precipitate will be pellet, and can be redissolved in water.

 

Problems

- removal of protein. If there are a lot of protein, treat with proteases which break down proteins into monomers, which is easier to be extracted by phenol

 

 

Cutting, joining and inserting into plasmid vectors

- Since we do not have the knowledge of where beta-lactamase gene is located, DNA treated with a restriction enzyme will produce many fragments, one of which will contain the beta-lactamase gene. For screening purposes, the plasmid used must not have amp-resistance. Therefore, any plasmid with the insert of beta-lactamase gene will have the amp-resistance. Clone gene will be used as the selective marker.

- Restriction digestion - So both DNA and plasmid will be treated with the same restriction enzyme to produce the same sticky ends. Sticky ends is preferred, because with the overhangs, complimentary strands can come together more easily and can be ligated.

- Plasmid has to be treated with alkaline phophatase to prevent it to recircularised. 5’ phosphate removed from vector, so cannot be ligated together. Can still accept phosphorylated insert.

- Make a gene bank by gel electrophoresis. Cut DNA is run in the gel along with a marker and the original uncut DNA. Fragments will be separated according to sizes, cut off gel from each band, and elute DNA from the gel. Proceed to ligation for each band of fragments.

- Fragments ligated into vector. Ensure that insert is phosphorylated at one of the 5’ overhang.

- PROBLEMS: recircularisation of vector, as solved above.

 

Transform into E.Coli

- via electroporation or treatment with chemical. Add iced CaCl2 etc

 

Selection/Screening

- Phenotypic selection, first screening – antibiotic to select the cloning vector + XGal , blue white screening, white is the vector of interest.

- Second screening – antibiotic to select cloning vector + antibiotic ampicillin to select beta-lactamase gene.

- If none is found, repeat for DNA with different fragment size from the genebank.

[zyncod]

That's probably (read: definitely) not going to work. You would have to insert into the plasmid an entire B-lactamase gene, in frame, right next to the promoter. The chances of picking a restriction enzyme that would do that are almost infinitesimal. The traditional way of doing this is to first run a Southern using E coli B-lactamase as a probe (or B-lactamase from a bacterium similar to yours). If there's enough homology for the probe to bind, you then create a plasmid library in E coli with your bacterial genomic DNA and then do a screen of the library with the probe. If you have a large enough library, you'll pick up the entire B-lactamase gene in one or more of the colonies.

[cowboy]

Hmm..let say I dont have e.coli b-lactamase nor any other b-lactamase from any other bacteria. If that's the case, I wont be able to use the probing method, is that right? I need to know a small sequence of the gene in order to run a southern blotting.

 

How about this way:

- create a gene library by cutting the genomic dna into fragments with restriction enzymes (partial digestion)

- run the dna fragments in gel electrophoresis, compare with a known marker

- select different fragments and proceed to ligate to plasmid vectors

- then everything else same as my post above.

 

Will that work?? My understanding of gene library might be wrong...please do correct me.

[zyncod]

First of all, if you run a genomic digest on a gel, it will just be a smear - there won't be separate bands. The only two ways I can think of to do this without taking years of time are to probe for the gene or purify the B-lactamase protein and sequence the amino acids.

[cowboy]

First of all, if you run a genomic digest on a gel, it will just be a smear - there won't be separate bands. The only two ways I can think of to do this without taking years of time are to probe for the gene or purify the B-lactamase protein and sequence the amino acids.

 

Yeah, you are right. I will get only a smear because there are so many different fragments. So it is really not possible to cut a portion of the smear, elute the DNA and ligate it with the plasmids, portion by portion? Is this what we call creating the gene library? Since bacteria genome is small, it wont take very long, will it?

 

Can you explain more about the two ways you mentioned? I'm not sure about the steps. How do we create a probe for the gene? How do we sequence the amino acid of b-lactamase protein?

[zyncod]

The probe for the gene requires that you know something about the B-lactamase sequence, which you don't, apparently. The other method is to use chromatography techniques to purify out the B-lactamase protein (you could use X-gal hydrolysis as a test for the presence of the protein). Once you have a relatively pure sample of the B-lactamase protein, there are a number of ways to get the amino acid sequence (mass spec is the most common now). With the amino acid sequence, you can make degenerate probes for the gene.

[cowboy]

The probe for the gene requires that you know something about the B-lactamase sequence, which you don't, apparently. The other method is to use chromatography techniques to purify out the B-lactamase protein (you could use X-gal hydrolysis as a test for the presence of the protein). Once you have a relatively pure sample of the B-lactamase protein, there are a number of ways to get the amino acid sequence (mass spec is the most common now). With the amino acid sequence, you can make degenerate probes for the gene.

 

Hmm...that is like an entire whole new experiment on its own... hmm.. But thanks a lot for your help.

 

How about this question of mine? Is it really not possible or is it really stupid to do so?

So it is really not possible to cut a portion of the smear, elute the DNA and ligate it with the plasmids, portion by portion? Is this what we call creating the gene library? Since bacteria genome is small, it wont take very long, will it?

[Aubiegirl]

Are you actually going to have to perform this procedure, or are you just asked to describe how you would hypothetically do it?

[cowboy]

Are you actually going to have to perform this procedure, or are you just asked to describe how you would hypothetically do it?

 

Does it make any difference? I want a method that is hypothetically correct and can be performed in the lab as well.. ?

[Aubiegirl]

I was just curious, I couldn't tell from your responses. The only difference it makes depends on your access to resources, lab time, etc, the duration of time you had to complete the experiment, and what equipment/supplies you'd be able to obtain to do the procedure.

[zyncod]

So it is really not possible to cut a portion of the smear, elute the DNA and ligate it with the plasmids, portion by portion? Is this what we call creating the gene library? Since bacteria genome is small, it wont take very long, will it?

 

That is possible, but what you get won't be that different than a traditional plasmid library where you just ligate everything into E coli plasmids at once. And once you do that, you still come up against the problem of how you find the B-lactamase gene in that library. Unless you chance to insert the gene exactly correctly into the plasmid, you won't get B-lactamase expression. So you either need at least a small sequence of the B-lactamase gene or you need to purify the protein.

[Bluenoise]

Hey sorry I didn't respond earlier. BTW the big point your missing is that beta-lactamase codes for ampicillin resistance. Beta-lactamase codes for an enzyme which breaks down the latam ring found in many penicillin type anti-biotics. I think not pointing this out is what is causing the difficulty in getting you a good answer. I had known this previously but it took a little while to come back to me. Doh!

 

So if you digest the genomic dna from your host than shot gun it into a few clones for expression all you need to do is grow the clones on amp containing medium and only those with your gene will survive. Much simpler eh?

 

You may have to try a few different digest methods till you find one that doesn't disrupt your gene of interest. Sonication and blunt end ligation would probably be a good way to go about it.

 

Yeah so you don't really need to do any special selection or screening, functional complementation by the beta-lactamase gene will be enough. So to make that clear blue/white or antibiotic resistance on your plasmid is a complete waste of time. You just need a plasmid capable of expressing the gene. Plus you don't need to know anything about the gene before hand except for it's function (which you do).

 

I hope that helps. I'd write you up a proceedure, but I'm pretty swamped for time as is sorry.

[ecoli]

You may have to try a few different digest methods till you find one that doesn't disrupt your gene of interest. Sonication and blunt end ligation would probably be a good way to go about it.

 

I'm using blunt-end ligation to good effect here. I, too, am doing Beta-Lactamase gene cloning as well. If you're having problems with your assay, PM me. I'll give you my notes on the subject.