electroporation

[ZEGSUS]

I want to make electroporation but I donit know from which microbе to extract DNA and to which to insert DNA.
Which combination is the most easy?

[JackBean]

E.coli -> E.coli

but I don’t see, why would you do that just to do that.

[ZEGSUS]

And is it possible bacillus bulgaricus –>e.coli?

[JackBean]

sure, you can do salmon’s DNA into acetobacter. It’s just questionable, whether will the DNA stay or will be degraded or what you want to do with it (but I guess nothing since you don’t care about the organisms 😀 ).

[ZEGSUS]

Well as you know I want to extract DNA from bulgaricus and to insert it in E.coli.The DNA will be in ethanol in the fridge.

[JackBean]

yeah, I understood that 😀 But what DNA will it be? Plasmid or gDNA?

But I guess you want to clone only small part and insert into some plasmid and express?

[ZEGSUS]

I will use and the both type because I haven’t equipment to separate them.No , I will not clone them , I will try directly to insert the DNA into the E.coli.

[JackBean]

you have tuo purify the DNA first and that’s were you differentiate between gDNA and plasmid. However, plasmid may be incompatible because of other replication origin. Inserting gDNA doesn’t make sense since bacteria cannot have two chromosomes and thus would one repel probably (even keeping the plasmid without antibiotic selection will be hard).

[ZEGSUS]

And how to insert gDNA into the chromosome?

[JackBean]

you could use BAC (bacterial artificial chromosome), which has high capacity, but still I’m not sure, whether will it fit whole genome of your bacteria.
You want to create gDNA library?

[ZEGSUS]

And how to use BAC?

[JackBean]

I have never worked with BAC, but I guess cut with RE, insert your DNA and transform. Although I don’t know whether electroporation will work for such a big piece.

[ZEGSUS]

And what about the plasmids?

[JackBean]

these should be fine, but I don’t know whether they have compatible the replication origins 😉

[ZEGSUS]

So if I want to insert gDNA first I need to cut it and after that to insert it?And how to transform it?

[JackBean]

I don’t know how big is the genome, but probably yes, cut, ligate, and transform. I’m not sure what is the size limit for electroporation.

[ZEGSUS]

The size ot the lactobacteria is 2.3 Mbp.The limit is <10Kbp.

[JackBean]

here http://en.wikipedia.org/wiki/Bacterial_ … chromosome they put the common size of insert into BAC is up to 350 kbp. Thus the number of clones you had to test would be N = [ln(1-P)]/[ln(1-[350/2300])] where P is the probability you will find your gene of interest or whethever are you looking for (99.5%) and 350/2300 is the ratio of insert and whole genome size. As I calculated it the N is ~4, what is odd, since the ration genome/insert is 6.5 😆 🙄

I guess best to contact someone who is experienced e.g. someone from some sequencing company/group.

What is your aim again?

[ZEGSUS]

To insert DNA from the bulgaricus in E.coli.

[ZEGSUS]

quote JackBean:

here http://en.wikipedia.org/wiki/Bacterial_ … chromosome they put the common size of insert into BAC is up to 350 kbp. Thus the number of clones you had to test would be N = [ln(1-P)]/[ln(1-[350/2300])] where P is the probability you will find your gene of interest or whethever are you looking for (99.5%) and 350/2300 is the ratio of insert and whole genome size. As I calculated it the N is ~4, what is odd, since the ration genome/insert is 6.5 😆 🙄

I guess best to contact someone who is experienced e.g. someone from some sequencing company/group.

What is your aim again?

So you mean to cut the gDNA and to attach it to F-plasmids and after that to insert the plasmids into E.coli?

[JackBean]

yes, that’s how BACs work

[ZEGSUS]

And how to get F-plasmids?

[JackBean]

sorry, I have really no idea. But I guess the material providers like Invitrogen or whatever should probably have system for making gDNA library.

[ZEGSUS]

http://aem.asm.org/cgi/reprint/60/11/4192.pdf
Well I guess that is possible to insert "naked" DNA into bacteria.

[JackBean]

what do you mean by naked?

[ZEGSUS]

gDNA not into f-Plasmid

[JackBean]

quote ZEGSUS:

http://aem.asm.org/cgi/reprint/60/11/4192.pdf
Well I guess that is possible to insert “naked” DNA into bacteria.

yeah, but I guess it depends on the species. Agrobacterium is pathogenic bacteria and some of its relatives (I don’t know whether Agrobacterium by itself) can posses linear plasmids. Such bacteria will of course accept "naked" DNA (but still the problem with replication), but it’s questionable how will E.coli do.

[ZEGSUS]

quote JackBean:

quote ZEGSUS:

http://aem.asm.org/cgi/reprint/60/11/4192.pdf
Well I guess that is possible to insert “naked” DNA into bacteria.

yeah, but I guess it depends on the species. Agrobacterium is pathogenic bacteria and some of its relatives (I don’t know whether Agrobacterium by itself) can posses linear plasmids. Such bacteria will of course accept “naked” DNA (but still the problem with replication), but it’s questionable how will E.coli do.

You mean that the linear DNA cant be save for the future generations?

[JackBean]

I really don’t know, whether E. coli is capable of that. Maybe other species would be more sutable for that 😉

[canalon]

E. coli will not readily take up naked DNA. Other species can, but not E. coli.
If you are looking for a specific gene from Acetobacter bulgaricus your best bet is indeed to try a BAC. There are many kits commercially available (Google "BAC cloning kit" and your country of residence to find some supplier, or ask your friendly local sales rep) to do just that.
And no, not all bacteria will accept DNA by electroporation. But in your case that should not be a problem.

[ZEGSUS]

In my country the gene engineering is forbidden so there is no suppliers here.And do you have a easy protocol how to insert gDNA into E.coli?

[ZEGSUS]

I want to report a little success about my project in electroporation.This is my first prototype of electroporator.I have succeeded to make it with less than 100$.What do you thing?

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[JackBean]

seems just like a bunch of wires to me 😀 (I don’t have much big screen here). Just curious, where are you from, that gene engineering is forbidden? You mean even manipulation with bacteria, right?

[ZEGSUS]

http://en.wikipedia.org/wiki/Bulgaria
Yes even manipulation with bacteria. 🙁 The "eco organisation" provides a lot of pressure on the government. 🙁 And the government ban GMO. 👿

[JackBean]

I see. So even Academy is not allowed to work with GM? And I though that EU has strict rules 😆 🙁

[ZEGSUS]

Yes.But remember -here is Bulgaria.

[JackBean]

oh man, you’re part of EU, right? 🙄 😳 My apologies 🙁

[canalon]

I have to say that it is hard to see much from the picture, but if you have managed to generate nice square shocks with the possibility to control the intensity and duration, kudos to you.

[ZEGSUS]

Yes , I have.It was difficult but after many attempts I succeeded.

[ZEGSUS]

And what about plants?How to insert DNA from plant to plant without plasmids?

[JackBean]

for that you would need golden (or from other heavy metal) microparticles with use high pressure to bombard the plant tissue.
http://www.nepadbiosafety.net/for-regul … ombardment

[ZEGSUS]

I know that but is there a chance to insert a genomic DNA with only electricty?

[JackBean]

I highly doubt so, because the plant cells have the cell wall, which is pretty good barrier for such transformation. Better way is to use Agrobacterium, which infects plants.
You want to insert full gDNA again?

[ZEGSUS]

Yes.Ok I will use Agrobacterium.Is it possible to transfer gDNA form plant to Agrobacterium?

[JackBean]

I though that since you have banned gene manipulation in Bulgaria, you won’t have access to Agrobacterium neither. That’s why I didn’t mention it.
However, you cannot insert full gDNA using Agrobacterium, since plant genomes consist of several chromosomes and the Agrobacterium transfers only "small" portion from its Ti-plasmid. Nowadays there are available kits for Agrobacterium, which work with two plasmids, thus the inserted DNA may be longer, but it still won’t be full chromosome.
(and I really don’t see any reason, why to do that)

[ZEGSUS]

Well.Lets just say that I have Agrobacterium from somewhere.And thanks about information.

[JackBean]

😆 you’re welcome 🙂

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