Jump to content
Sign in to follow this  
DeoxyriboNucleicAcid

Cloning Bacteria; A New Experiment.

Recommended Posts

Hi Everyone.

(I do have questions, but want to give you my background info first.)

I am planning on doing an experiment to make one species of bacteria into another.

I plan to:

Take E. coli bacteria and extract all of their DNA; their entire genome.

 

Then, I will take another species of bacteria (strep) and remove ALL DNA from them.

 

I shall next take the E. coli DNA and force it into the strep.

 

I want to see if the strep cells will indeed turn into the E. coli bacteria.

None of this is finalized. The bacteria species and types may vary if it is easier to do this experiment. The DNA may be cut with enzymes, or whatever, as long as it goes into the 2nd bacteria type.

Efficiency rates are not a problem, as long as there is a small or some percent of chance of success.

 

=====

 

MY QUESTIONS:

 

What are the lab methods of extracting a full "loop" of DNA from bacteria?

Is there a way at all to extract all of the DNA in a cell (many cells will be used when I do this.)

 

If the bacterial DNA is broken into many segments when extracting (if that is the only way,) will it reassemble with sticky ends inside the host (2nd) bacteria species.

 

What are the methods of destroying/ taking out all of the DNA in a cell without killing that cell. Can it be done?

 

What are the methods of forcing DNA into bacteria cells without it.

 

Basically, this is like a clone, with no egg cell.

 

ANY IDEAS/ RECOURCES WILL HELP ALOT.

Has it been done before? Where? How can it be reproduced.

Share this post


Link to post
Share on other sites

 

What are the lab methods of extracting a full "loop" of DNA from bacteria?

Is there a way at all to extract all of the DNA in a cell (many cells will be used when I do this.)

 

What are the methods of destroying/ taking out all of the DNA in a cell without killing that cell. Can it be done?

 

What are the methods of forcing DNA into bacteria cells without it.

 

Basically' date=' this is like a clone, with no egg cell.

 

ANY IDEAS/ RECOURCES WILL HELP ALOT.

Has it been done before? Where? How can it be reproduced.[/quote']

 

i dont think that this has been done before, ever, and i dont think it is possible, although, as some would say "anything is possible". so maybe it is, but it is very hard... good luck, because i think that you will need it,

(if you do suceed, tell us about it.... we love to hear this kinda thing)

Share this post


Link to post
Share on other sites

What is your experience in microbiology? This sounds like a tough experiment to start out with. I took a micro lab once and I remember we were just happy when we could show that we grew antibiotic resistant strains.

Share this post


Link to post
Share on other sites

I have been working in the lab for a few year now, so I am fairly experienced. I believe that if I put my mind to it, I could probably pull this off (with the help of a few professors.)

Share this post


Link to post
Share on other sites

Im not too sure about this experiement. Ive had a quick look over it, and there are a few problems in performing the experiment to begin with.

 

Firstly, there are a number of kits available for genomic DNA purification, as well as the "old-school" methods which can be time-consuming and can shear the DNA. Look at QIAgen, Promega, most of the good big companies would provide good kits for extraction.

 

The transfer the DNA into the donor cell, well the donor cell generally has to be competent, and DNA transfer into bacterial cells (well in the case of plasmids anyhow) is generally achieved through either electroporation or via heat-shock. The number of positively-transformed cells would be very low, i would imagine, if it is even possible with a whole string of genomic DNA to begin with. Id also imagine that you would probably have to use a vector of some sort to deliver the DNA into the donor bacteria.

 

Ill look a little further into it for you (at work at the moment, and my brain is a little fried) :) But changing species, well.... I wouldnt place my bets on it....

Share this post


Link to post
Share on other sites

The pores produced by heat shock are usually too small for even a large plasmid, so that's pretty unlikely. Electroporesis is better for bigger things but I don't know if it'd work.

 

I imagine that if you transplanted a genome into another species the restriction enzymes would chop it up.

Share this post


Link to post
Share on other sites

Thanks for your replies inamorata and Skye,

I am thinking along the lines of electroporesis, maybe even a combo of heat shock as well. I will have to get these pores as big as possible, and quickly too, to produce suction. Ill look into those companies, that will solve my first problem of getting the full genome.

 

However, the host cell must loose it's DNA to do the experiment.

 

QUESTION:

Are there any methods of neuralising DNA inside a cell. without killing it? If the DNA is neutralized, it wont make RNA, and Rhibosomes will have nothing to do. There is a lag time between Neutralizing, and the cell haveing nothing to do, correct?

Maybe the DNA will be inserted within that time frame?

Share this post


Link to post
Share on other sites

Hmm....Inamorata and Skye have answered pretty much everything but maybe it's worth looking at other transfection methods...although I really don't know if liposome based methods work in bacteria...I only transfect eukaryote cells. What about nucleofection (look up Amaxa) and anyway, as you say, getting rid of the host DNA would be a bit problematic. What's your objective in doing this anyway? Or is that a huge secret? If you have a totally exciting (and reasonable) rationale behind this idea that's fine...but if it's just curiosity you'll never get funding!

Share this post


Link to post
Share on other sites

Create an account or sign in to comment

You need to be a member in order to leave a comment

Create an account

Sign up for a new account in our community. It's easy!

Register a new account

Sign in

Already have an account? Sign in here.

Sign In Now
Sign in to follow this  

×
×
  • Create New...

Important Information

We have placed cookies on your device to help make this website better. You can adjust your cookie settings, otherwise we'll assume you're okay to continue.