Multiple genes cloning (antibacterial, antifungal and antivirus genes to be exact)

[PseudoGene]

Dear people,

I have this question on whether is it possible to clone these 3 genes which has the antibacterial, antifungal and antivirus activities into a vector and expresses them? I have tried to find journals related to this, but to no avail. Hope you guys can help out. Please let me know if it's possible, what are the requirements and how can it be done. It would be better if you can attach the links from where you obtain the information from. Thanks.

Cheers!

[CharonY]

Which three genes? Technically you can clone any gene into any organism. By adding the right promoter you can even express them. The main problem, however, is to get a functional protein out of it. So basically you require the right promoter, the right recipient and a bit of luck. The cloning process itself is pretty much the same (DNA is just DNA, after all). If you are interested in protocols I can recommend: "Miolecular cloning" by Sambrook and Maniatis. If you want to know whether it has been done already, you would have to indicate which genes you have in mind.

[PseudoGene]

Dear CharonY,

 

First of all, thanks for your reply. Perhaps it was my fault that I did not phrase my questions clearer. I know it is possible to clone and express multiple genes into a vector. But since these three genes has different functions and also different targets, my questions are what host to use? And what about the protein expression? Broad spectrum activity are not necessary, as long as they can act against ONE (antibacterial that targets one bacteria and so on) would be more than enough.

 

Once again, thanks alot!

[DrDNA]

Dear CharonY,

 

First of all, thanks for your reply. Perhaps it was my fault that I did not phrase my questions clearer. I know it is possible to clone and express multiple genes into a vector. But since these three genes has different functions and also different targets, my questions are what host to use? And what about the protein expression? Broad spectrum activity are not necessary, as long as they can act against ONE (antibacterial that targets one bacteria and so on) would be more than enough.

 

Once again, thanks alot!

 

I think this is what you are asking for:

Identify the genes of interest.

Identify restriction sites around the genes of interest.

Cut them out with REs

Puirfy them (probably PAGE).

Put them in a vector; take your pick:

http://www.addgene.org/pgvec1?f=v&cmd=listvecinfo

Add the vector any number of expression system:

again take you pick, but competent E coli cells are common.

 

Additional info and details can be found in any Molec Bio textbook.

I hope that this isn't homework, and if so, that I didn't just do your homework for you.

[CharonY]

But since these three genes has different functions and also different targets, my questions are what host to use? And what about the protein expression? Broad spectrum activity are not necessary, as long as they can act against ONE (antibacterial that targets one bacteria and so on) would be more than enough.

 

Well, it depends what you want to do with them and also what kind of proteins they encode. So, for instance you would chose a different system for membrane proteins than for soluble ones. Also it is a difference if you want to overexpress and purify them or if you want to have them active in the host organism, which requires a stricter control of protein expression. Also the host selection defines whether your protein gets folded and/or modified the right way etc.

 

In general, if you want to have them active in vivo, you should choose a host which is close to where the originated from. That is whether it is eukaryotic or prokaryotic, mammal or yeast, etc and either use a vector that has an appropriate promoter or integrate it into the genome. If you want to purify the protein it really depends if the protein in question requires special folding, co-factors and/or modifications.

 

Again, your question can only be answered if you specify your problem.

 

 

To DrDNA: Nice overview over a cloning experiment, however if sequences are known nowadays most would rather start with a PCR rather than purify DNA and then cut it.

And clean-up (PCR or restriction) would either be done with spin-columns or agarose gels. PAGEs are only really necessary for distinction of very similar length fragments but cloning can be a major pain. Just some nitpicking