9 replies to this topic

[joy123]

Hi! I want to do gene knockout in a G(+) bacteria.

I am new in this field, and would like to start with reading.

I didn't find any article about gene KO in this bacteria. Would you please recommend some books/ chapters that give systemic introductions about gene knockout in bacteria?

Thanks very much!

[pito]

joy123, on 06 April 2012 - 12:01 PM, said:

Hi! I want to do gene knockout in a G(+) bacteria.

I am new in this field, and would like to start with reading.

I didn't find any article about gene KO in this bacteria. Would you please recommend some books/ chapters that give systemic introductions about gene knockout in bacteria?

Thanks very much!

Did you do a search on gene knock out in general? (rather then looking for a specific gene knock out or in a specific bacterium).

Because normally you should be able to find some literature...

[joy123]

Thanks for your reply!

I didn't find any book or article introducing this technique in general.

But I did found a lot of articles about specific gene knock in specific bacteria. They are so different, making me confused about how  to set up a gene KO system in my bacteria. This is why I ask if people could give me suggestions on some general introduction about this technique.

[pito]

joy123, on 08 April 2012 - 09:51 AM, said:

Thanks for your reply!

I didn't find any book or article introducing this technique in general.

But I did found a lot of articles about specific gene knock in specific bacteria. They are so different, making me confused about how  to set up a gene KO system in my bacteria. This is why I ask if people could give me suggestions on some general introduction about this technique.

But the techniques they used.. you can look those up..

I find your question rather "strange".

If you do a general search on google (for example for) gene knock out, you should be able to find some basic techniques? For example: homologous recombination.. did you allready check that?

[joy123]

Yes, I do find some articles, and introduction about genetic information exchange.

I am reading them. And I need to set up a method for my bacteria. There are so may variables to consider. I probably need to try the conditions one by one.

I think at present, what is hard for me is to find a plasmid that is suitable for my bacteria. I have tried several, none of them was successfully transformed to the bacteria. I am concerning if the competent cells I made was good enough. But I don't know how to test the competent cells, as I don't know which vector for sure will survive in my bacteria (so I cannot find a vector as positive control to confirm my competent cell was good).

[pito]

joy123, on 09 April 2012 - 12:26 PM, said:

Yes, I do find some articles, and introduction about genetic information exchange.

I am reading them. And I need to set up a method for my bacteria. There are so may variables to consider. I probably need to try the conditions one by one.

I think at present, what is hard for me is to find a plasmid that is suitable for my bacteria. I have tried several, none of them was successfully transformed to the bacteria. I am concerning if the competent cells I made was good enough. But I don't know how to test the competent cells, as I don't know which vector for sure will survive in my bacteria (so I cannot find a vector as positive control to confirm my competent cell was good).

Well thats the problem when working with gram + bacteria and especially if you are working with a bacterium that has not been studied before in another publication.
All you can do is search the literature, check what kind of vectors are being used and see if you can use them for your bacterium.
I am not sure what you mean about the positive control. You use for example an antibiotic resistence as a control. But of course, you can not know for sure it will work if nothing is known about it in literature.
ANd I hope you allready checked that your bacterium does or does not contain a plasmid from itself..

Edited by pito, 09 April 2012 - 01:45 PM.

[joy123]

I think I should search articles that use plasmids in the bacteria that are close to mine. At present, I found people have done gene KO in some bacteria in the same "Order" with mine. I don't know if that is close enough.

I have several questions regarding: "ANd I hope you allready checked that your bacterium does or does not contain a plasmid from itself."

I read an article about a bacteria in the same "Genus" (but not in same "Species") to mine. No plasmid was found in this bacteria. And I searched on PATRIC database that the "

Number of Plasmid only genomes=0

". I want to ask: 1. is there a way to check plasmid myself? And is it necessary? 2. If there is a plasmid in it, how should I use it? Should I synthesize and modify it?

Thanks a lot!

[joy123]

And by "positive control", I mean if there is a plasmid suitable for a broad range of bacteria, including G+ and G-?

[pito]

joy123, on 10 April 2012 - 06:31 AM, said:

I think I should search articles that use plasmids in the bacteria that are close to mine. At present, I found people have done gene KO in some bacteria in the same "Order" with mine. I don't know if that is close enough.

I have several questions regarding: "ANd I hope you allready checked that your bacterium does or does not contain a plasmid from itself."

I read an article about a bacteria in the same "Genus" (but not in same "Species") to mine. No plasmid was found in this bacteria. And I searched on PATRIC database that the "

Number of Plasmid only genomes=0

". I want to ask: 1. is there a way to check plasmid myself? And is it necessary? 2. If there is a plasmid in it, how should I use it? Should I synthesize and modify it?


Thanks a lot!

Didnt the article you did read specify how they tried to extract the plasmid? ANyway, thats what you can do: try to exctract the plasmid.. if it isnt there, you wont see it on gel.
You also do a general DNA extraction and see what comes up.
And if there is a plasmid.. then you can use that one to transform the bacterium...

And about the positive control: there are indeed some plasmids that can be used in many gram+ bacteria, but its best you check the literature.
It sounds that you are going to try something without propper knowledge.
I would insist on you reading and learning ... Thats all you have to do now. There is no point trying to do practical work if you are not familiar with it.

Check for papers concerning your bacterium
check for paper that deal with transformation of gram + bacteria (see what plasmids are used etc)
Check some basic student books on transformation/plasmids etc...

[joy123]

OK! Yes, I think reading before start is important. Thanks for your suggestions!