Genomic integration of DNA

[Mega]

Hey guys,

I've learned very much of the function of DNA and plasmids here...

What I yet don't know:

What are the methods to get DNA into the genome?

Could you give me some keywords so that I can do a google research on that?



(I know there are e.g. Agrobacterium transformation and bacteriophage, but what about gene gun etc. ? )


Am I right, that you can insert DNA of interest into a short gene sequence of the host, and then put it into the host?
So it will make homologuos recombination with the strand and the insert is in the genome??


Thanks!

[Nathan McCorkle]

keywords:
"homologous recombination"
insertion
vector
homology

http://en.wikipedia.org/wiki/Homologous_recombination

On Tue, May 1, 2012 at 2:35 PM, Mega <masters...@gmail.com> wrote:
> Hey guys,
>
> I've learned very much of the function of DNA and plasmids here...
>
> What I yet don't know:
>
> What are the methods to get DNA into the genome?

It depends on your understanding of "genome"... it isn't the same as
chromosome. The genome of an organism contains plasmids, larger
elements like chromosomes, and potentially viruses and their
associated DNA or RNA molecules (these could be contamination or for
cloning).

For integrating with chromosomes, all I know is homologous
recombination, and synthetic assembly like they've shown at JCVI.
Traditional cloning (restriction enzymes (REs) and ligase) might work,
but I bet the efficiency would be pretty low... here's how JCVI got
their 1MBp synthetic chromosome into a DNA-free cell:

"Genome Transplantation in Bacteria: Changing One Species to Another"
http://www.scribd.com/doc/92047145/Science-2007-Lartigue-632-8

and a presentation on that paper:
http://openwetware.org/images/0/0e/Derek_Ju_Presentation_4-8-10.pdf

>
> Could you give me some keywords so that I can do a google research on that?
>
>
>
> (I know there are e.g. Agrobacterium transformation and bacteriophage, but
> what about gene gun etc. ? )

Electroporation is my favorite way, its very versatile... there are
phages, PEG buffers (which can cause cell fusion too)... Agrobacterium
tumerfaciens first needs to be transformed with an engineered Ti
plasmid, then can be used to transform plants, which it does by
injecting the Ti plasmid into plant cells (usually flowers are dipped
in Agro solution... sonoporation, gene gun

>
>
> Am I right, that you can insert DNA of interest into a short gene sequence
> of the host, and then put it into the host?
> So it will make homologuos recombination with the strand and the insert is
> in the genome??
>

--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics

[abass]

Check out Transcription Activator-Like Effector Nucleases (TALENs).  Here's a brief overview: http://en.wikipedia.org/wiki/Transcription_Activator-Like_Effector_Nuclease

Andy

[Ravasz]

Hi there!

I dunno if this has been said before, but getting foreign DNA into chromosomes depends mostly on what organism/cell line you have.

In mice or certain chicken cell lines targeted genomic integration is possible, but it is a bit time consuming. In humans this is very difficult, although there are certain cell lines (like 293) who readily integrate linear pieces of DNA randomly into themselves. In bacteria I only know E. coli, which loves to take up plasmids, although it will not actually integrate it into its genome, but it will carry it stably as an episome.

Maybe if you pick an organism what interests you the most, then we can come up with something more detailed.

Cheers,
Mat

[Andreas Sturm]

Thank you!!!!

E.g. I choose an apple tree. Or even the chloroplasts of it.

I cut the chromosomal (that's what I meant above, not genome...)  DNA into pieces. Do gel electrophoresis. And insert a gene of interest inbetween.

Then all I have to do is:  get the new DNA strand inside the nucleus  (or chloroplast) and it will make homologuos rec.?

[Mega]

Ah.... Read more about it. is it true that quite every plasmid can be integratet into the Chromosome when you keep it very long in the antibiotic media? Also in animal cells? The chromosome is big, so there probably will be a sequence for homologous recombination?

[shamrock]

Not being a plant biologist I can't speak directly about integration into the apple genome but I can give you some general input about genomic integration. First of all you will some sort of selectable marker in your input DNA-this is usually a drug resistance gene, or a gene necessary for biosynthesis of an essential nutrient. You will probably have to search through the literature to find what types of markers are being used in apples. Then your going to have to identify a target site that you wish to integrate into so that you can design your input DNA to have homology at the ends to the target site. So a basic DNA construct for integration would look like this: DNA sequence homologous to the target site followed by your gene of interest followed by a selectable marker (both of these will need appropriate promoters) followed by DNA sequence homologous to the target site. Some constructs also have sequences flanking the selectable marker so that it can be excised at a later date.

 

Some organisms can be very effeciently transformed and integrate DNA into the chromosome (i.e. yeast- Saccharomyces), others such as E. coli will degrade incoming linear DNA so you need to coexpress a recombinase. Not sure what is required for apples-I again would search the literature.

 

I would caution you to think twice about fooling around with agriculturally important species such as apples or other food crops for a couple of reasons; first of all there is quite a bit of controversy and regulation (depending on where you live) concerning GMO crops. If you start messing around in your basement you might attract a lot of attention that you probably don't want and may have some government agency come shut down your home lab for violating some obscure regulation. Secondly I think you have to think seriously about containment and making sure that what ever you create doesn't escape. It's unlikely, but possible, that what you create could spread through apple populations with really unknown consequences. Before proceeding you should do a very through risk assesment and consult with plant biologists from the apple community.

 

Some may shrug at my caution but I think that each of us owes it to the larger DIY-Bio community to be extremly circumspect about what we are doing in our labs. Theres plenty of suspision and apprehension about DIY Bio and all it would really take is some inadverdent release or accident to raise the barriers to these kinds of activities.

 

My two cents!  

[Claudio Capitao]

Hi

Unfortunatlly homologous recombination is a very difficult process to achieve. Here you can read some articles about it:

http://www.unige.ch/sciences/biologie/plantsciences/grpaszkowski/Paszkowski_lab/Publications_files/Current%20Opinion%20in%20Plant%20Biology%202003%20Hanin.pdf

http://www.pnas.org/content/96/13/7398.full.pdf

http://www.botanik2.uni-karlsruhe.de/download/pmb02r.pdf

The most common method for plant transformation usually consists in the use of Agrobacterium tunisienses. It is a very simple method to transform Arabidopsis using flower dipping. However for other plants (like crops) this method has to be combined with plant cell cultures transformation or tissue transformation followed by regeneration in both cases to obtain the transformant. Other methods are: gene gun, electroporation or Transcription Activator-Like Effector Nucleases (TALENs). All methods are based in random insertion of the DNA sequence, only the last one allow to target it to a specific genome region.

I agree with shamrock, you have to be very careful with this, because GMOs have hard laws in them, especially in Europe. I don’t know but I think that you should get first a license to work with GMOs out of research facilities. The other possibility is to do the work with cooperation with a University lab. I am only saying this because the main reason taking me so much time to start to do something related with DIYBio is related with the laws in my country and all the licenses that need to be obtained.

I hope that this could help

[Nathan McCorkle]

On May 2, 2012 10:47 AM, "Andreas Sturm" <masters...@gmail.com> wrote:
>
> Thank you!!!!
>
> E.g. I choose an apple tree. Or even the chloroplasts of it.
>

Apples aren't good enough already???

[Andreas Sturm]

Hey, you are right.

Apples are not good. (Anyway it was just an example.) Better, to use willows or maples those have no food purpose thus they are less risky to work with.

[John Griessen]

On 05/17/2012 03:35 AM, Andreas Sturm wrote:
> Better, to use willows or maples those have no food purpose thus they are less risky to work with.

They still have plenty of purpose in the world, so the same caution applies.

[Mega]

Because of the flower dip method, I assume plant pollen (male gametophytes) can be transformed too by Agrobacterium??

[Mega]

[Cory Tobin]

> Because of the flower dip method, I assume plant pollen (male gametophytes) can be transformed too by Agrobacterium??

In Arabidopsis only the ovules are transformed by Agro [1] but in
other species there have been reports of pollen being transformed [2],
although it was done with in-vitro culture of haploid pollen which was
then converted to diploid via colchicine treatment (dihaploid
induction).

[1] http://www.jstor.org/stable/10.2307/4279321
[2] http://www.sciencedirect.com/science/article/pii/016894529390083C


-cory

[Mega]

I found pBluescript.

It is a mixture of usual plasmid + phage DNA.

When circular it can be multiplied just like plasmids.
But when linear, it will infect cells.


My question:
A helper phage is needed. Does that mean, you have to insert the phagemid into a virus and this infects E.Coli?

Or do you transform E.Coli and  then add the helper phages which will integrate your phagmid into the chromosome?

[shamrock]

Hi Mega,

 

The Bluescript phagemid was designed so that one could easily generate single stranded DNA for use in the old sanger dideoxy sequencing method. It has all of the bells and whistles of a plasmid (ori, selectable marker, MCS, lacZ color screening) and a filamentous phage origin of replication. The idea was that you could maintain the plasmid in the host cell and then when you wanted to sequence the insert you had cloned in you add the helper phage. The helper phage triggers replication from the bluescript phage origin producing lots of single stranded DNA that is packaged and extruded from the cell (filamentous phage generally don't lyse the cell). You then collect the phage particles, do a quick protein extraction and DNA precipitation and viola you have gobs of single stranded DNA that you can then use for sequencing or mutagenesis. The phagemid DNA doesn't integrate.

 

If you want to do integration in E. coli then you need to have access to whats called the lambda red system - this is a plasmid that has the recombinase genes from phage lambda controled by an inducible promoter (ara, lac, or). You transform the lambda red plasmid into the E. coli cell and then transform in a linear piece of DNA (PCR product). The linear DNA needs to have homology (35-40 bases) to your target site and a selectable marker. Once you have transformed in your linear DNA you select transformants based on the selectable marker that you are using then take those cells and grow on non selectable conditions-you should lose the lambda red plasmid but the integrant should remain-you can do PCR to verify that your integration was at the correct place.

 

There are other ways of integrating into E. coli genome that use transposons or lambda phage but the system described above is easier and faster.

 

If you want to play with integrating into the genome try yeast-Saccharomyces. The transformation procedures are pretty easy and integration takes place very efficiently without other recombinases or other things. You do still need to design your integrating DNA so that it has homology at the ends to the target site, or you can transform with a nonreplicating plasmid (yIP) that has your favorite gene cloned into it-that results in a different kind of integrant that incorporates the entire plasmid.

[Andreas Sturm]

Thanks. That was very interesting! Have to google "lambda red system" ;)

2012/5/22 shamrock <thmsb...@gmail.com>

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[shamrock]

Here's a paper that describes the system.

2012/5/22 shamrock <thmsb...@gmail.com>

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2012/5/22 shamrock <thmsb...@gmail.com>

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[Andreas Sturm]

About the Red-System (Red - lambda phage):

It works just for E.Coli and specifically only in E.Coli K12 ??

So it can't be used to engineer Cyanobacteria, B.Subrilis, etc. ??


Are there ready-to use systems (you just buy and use) for those organisms??


Thx,

[Slangtry]

Phage transduction is a pretty tedious and slow process.  I like the flp-frt system for homologous recombination.  I've only used it in E. coli K12, but there are many papers about the use of this method across a wide range of species.  The methodology is fairly simple and straight forward and plasmids are available from an open-source depository.  It can get tricky when integrating into a site that is resistant to recombination, but that usually for a good reason

http://www.igmm.cnrs.fr/IMG/pdf/flpin_pog44_man-2.pdf

http://www.pnas.org/content/93/12/6191.short

[Andreas Sturm]

But, as I understood, only E.Coli K12 has the Insertion Sequence needed in its chromosome???
Am I wrong???

2012/6/1 Slangtry <sarah....@gmail.com>

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