Re: [DIYbio] Re: Genomic integration of DNA

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

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

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.

 

On Tuesday, May 22, 2012 5:05:05 PM UTC-4, Mega wrote:

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?

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

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

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.

 

On Tuesday, May 22, 2012 5:05:05 PM UTC-4, Mega wrote:

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?

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

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

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.

 

On Tuesday, May 22, 2012 5:05:05 PM UTC-4, Mega wrote:

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?

--
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