Plasmids as a vector

[Louis Weber]

Hey Guys

First of all sorry for my bad English. I`m a beginner and need some help about plasmid.

So you can use plasmids for cloning (to copy a DNA-Part many time in a Bacteria), but also as a vector for gene transfection or transformation is that right?

So for Example, can I design a Vector and put it with for example with electroporation in the cell, and the Cell would, if I had de right Promoter, transcript the DNA and produce the Protein that I had on the Vector?

If that’s right, I had two questions…

1 How can I bring the Vector that is in the cell plasma because of de Electroporation into the Genome of the Cell?

2 I have seen the website http://www.addgene.org/ there it gives many plasmid. Are this also vectors? And in there Plasmid it has Promoters, Parts for restriction enzyme and ORFs. But is in the ORFs already some codes for Proteins or must I add this? And can I find there a vector for plants?

Sorry for my  questions but I am a beginner and found not very much information on the internet.

Louis

[Jeswin]

I don't know too much about this, but I think you should be looking
into expression vectors. I'm not sure how you would incorporate the
plasmid DNA into the genome, however, you can use plasmid expression
vectors to produce proteins of interest.

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[Dakota Hamill]

Off the top of my head, I don't know of any integrative plasmids for E. Coli.  But a quick google had this on the top, http://www.jbioleng.org/content/7/1/12  which explored chromosomal integration of BioBrick parts.  In general, most of the mechanisns seem to revolve around techniques that viruses have used to "take over" a host cell.

I've switched to focusing mainly on Streptomyces and Actinomyces now, and there is a phic31 integrase vector that exploits a viral mechanism for integration of your plasmid DNA Into the chromosome of the bacteria.  

I am guessing there are also integrative plasmids in E. Coli, but I don't know what size limitations they have, nor do I know the names of the plasmids.  Some literature searches should probably give you an answer though.

Do you have a specific protein in mind?

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[Jeswin]

On Sun, Feb 15, 2015 at 2:54 PM, Dakota Hamill <dko...@gmail.com> wrote:
> Off the top of my head, I don't know of any integrative plasmids for E.
> Coli. But a quick google had this on the top,
> http://www.jbioleng.org/content/7/1/12 which explored chromosomal
> integration of BioBrick parts. In general, most of the mechanisns seem to

These allow DNA to be added to the genome? I had wondered if Louis was
talking about this, but I think he is looking for a protein expression
vector. Yeah...not knowing terminology can really hurt. At one point
in my lab experience, I thought heat blocks needed water in them, so I
referred to them as water blocks...so colleagues gave me that funny
look until I described it.

[Koeng]

A lot of times vector and plasmid are used as interchangeable terms (although they do refer to different things) because almost all vectors are plasmids. There are a few different vectors, however, like viral vectors. Even those are usually based on plasmids. Almost all plasmids are vectors. A vector usually refers to something that transmits something from one organism to another, like a vector for disease. A plasmid on the other hand is a piece of DNA that replicates independently inside of a cell. Scientists use plasmids to transmit cloned information from one organism to another, so that's why they're almost always called vectors. (Just how I think of the terminology)

Most scientists use E coli, which the plasmid for cloning is the 'vector' for gene transformation. Since you can just use a straight up expression plasmid as an expression vector, nothing really needs to be done. However some organisms don't have plasmids, and that's where you want to use integration. (talking expression vectors here)

Integration of parts into the genome is sometimes more difficult, such as integration into E coli. Lambda red (in plasmid pKD46) works well for that organism. That's probably what the biobrick people were using. Other organisms, like S cerevisiae, are even better at integration and will readily integrate any DNA you have (given homologous sites). pGREEN is a plant vector I know of that I think integrates into the genome, but I'm not sure. Might want to investigate further on that one.

ORF stands for "open reading frame", also known as a protein. Most often you want to add in your own ORF or protein.

-Koeng

[Mega [Andreas Stuermer]]

The chromosomal DNA contains all the essential information (all enzymes how to degrade sugar, proteins and fat and build up new - and when to do this) and is pretty big, like the hard drive of your computer. A plasmid is very small, often contianing only a handful of genes that may be beneficial (antibiotic resistance), Plasmids are easily mobilizeable between species and in this comparison are USB drives. 

[Mega [Andreas Stuermer]]

http://www.sigmaaldrich.com/content/dam/sigma-aldrich/articles/biology/plasmid-map.jpg 

Here is a map of a plasmid. Actually, the KanR also has a promoter, but usually people are lazy and don't draw promoters.

All of these genes should also have terminators btw. 

For the palsmid to replicate in E coli you only need the pUC ori. To be able to kill bacteria that haven't taken up the plasmid DNA, you need the antibiotic resistance (KanR), The other stuff is payload. And the payload also needs promters and terminators to be expressed. Else the bacterium has the blueprint, but never produces it. 

I suggest having a look at this file 

http://www.snapgene.com/resources/plasmid_files/image_consortium_plasmids/pUC19/ 

[Ravasz]

The other replies probably give a lot fo info for you already, but here are my two cents:

1 How can I bring the Vector that is in the cell plasma because of de
Electroporation into the Genome of the Cell?

Are you talking about the E. coli bacterium? If so, then you don't need to integrate the plasmid into the bacterial genome. The genes you cloned in will express fine from a separate plasmid after electroporation. This is the standard procedure to express foreign genes in E. coli. Although there is technology available to integrate plasmids into the E. coli genome (as someone mentioned above), you don't need it. If just prepare a plasmid with the right gene on it and put it into the bacteria, then it will express fine as it is.

 
2 I have seen the website http://www.addgene.org/ there it gives many
plasmid. Are this also vectors? And in there Plasmid it has Promoters,
Parts for restriction enzyme and ORFs. But is in the ORFs already some
codes for Proteins or must I add this? And can I find there a vector for
plants?

The first question answered was perfectly answered above.
For the second, if you look at an expression plasmid, then it should have the proper expression cassette (promoter - multiple cloning site - end of gene) to work. If it does, then you need to cut the plasmid at the multiple cloning site, insert your protein code of interest, and your expression plasmid is then ready.
Transfecting plants is a lot harder than transfecting bacteria. Some plants can be transfected via the agrobacterium system, others using plant viruses, and for most plants, we don't really know yet how to transfect them. Either way, its a lot more challenging than E. coli. So I wouldn't attempt transfecting plants without a solid knowledge of cloning in E. coli.
Also, don't forget that cloning is under strict regulation in most places, so unless you have the proper clearances, you cannot perform such experiments.