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Multiple genes cloning

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Multiple genes cloning

Postby PseudoGene » Wed Jan 14, 2009 3:19 pm

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!
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Postby mith » Thu Jan 15, 2009 7:03 am

kills the bacteria if you put it into a bacteria lol.
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Postby GreenDog » Thu Jan 15, 2009 9:33 am

1. Yes it is possible.
2. As for sources, it's not written someplace people just know how to do this and they teach each other, you can also look at the sites of various biotech companies which sell stuff for molecular biology.
3. The antifungal and antiviral proteins may be transfected to bacteria, the antibacterial to yeast.
You should prepare the appropriate expression vectors, pay attention to the size, promoter, selection marker. Treat the cells accordingly and transfect.
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Re: Multiple genes cloning

Postby PseudoGene » Thu Jan 15, 2009 1:41 pm

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.

To mith, I know about that. That is why one of the main question would be which host to use?

To Greendog, I am sorry but what do you mean by transfecting antiviral and antifungal proteins to bacteria and antibacterial to yeast? Are you implying that 2 hosts should be use here? Actually, my question is would it be possible to express these genes into one single host since they have different activities.

Thanks alot though. Cheers!
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Postby mith » Fri Jan 16, 2009 5:28 am

could try a mammalian cell
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Postby GreenDog » Sun Jan 18, 2009 6:55 am

I thought you just needed to get the proteins doesn't matter how and you could use two hosts. If you want all of them together on the same plasmid and in the same host it's a different question entirely.
If you can choose the protein, pick an antibacterial gene which works on gram positive cells, and transfect to gram negative.
However this will be a large plasmid…
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Postby Cat » Tue Jan 20, 2009 2:17 am

You can construct a vector with all three genes in tandem antibiotic resistance gene (ex. NPTII) under control of the constitutive promoter while the other two under the control of the inducible plant promoters (there are many different ones) and transform plant cells. Then you will regenerate plats and grow them on the antibiotic containing media to select for transformants. Then you spay them with the inducer (some chemical or hormone) to get the expression of the other 2 genes.
Theoretically, you can put all three genes under the control of the constitutive plant promoters, however, I have no idea whether or not they will interfere with the normal cell function (I know that antibiotic resistance genes will not).

Do you need to isolate those proteins???
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Re: Multiple genes cloning

Postby PseudoGene » Sun Feb 01, 2009 10:50 am

Dear people,
Thanks a lot for the various opinions.

Cat: Nope. Just to express it. Basically, its just to protect the organism of interest for bacteria, fungi and virus.
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