Bacterocins as an alternative selection method

[Koeng]

Hello everyone!

Recently there was talk of synthesizing Kanamycin production operon for use in diy biology. I wanted to know if anyone had looked into E coli bacterocins as an alternative. They would be cheaper to synthesize, and even better the strain to get them from could possibly come from a genetic stock center. Probably the best part is they are almost guaranteed  to work in E coli. Just an interesting research option

-Koeng

[Cathal Garvey]

:) Give me a week, and I'll have a fun project to share with you!

[Keoni Gandall]

I'll wait till then then!

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[Matt Lawes]

OK I'm confused. Synthesizing kanamycin? Really? Why?

Or kanamycin resistance genes?

>matt

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[Koeng]

Synthesize kanamycin synthesis and resistance genes and put them on a plasmids, therefore the plasmid selects for itself

[Matt Lawes]

OK....

Still lost on why. Is kanamycin such an issue, or is DIY about being totally off the grid? I get KanR but synthesis ......

>matt

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[Koeng]

Just being able to produce an antibiotic in a DIY situation can definitely help with learning about biology and yes, off the grid would be nice because these companies really hate shipping to houses

[Matt Lawes]

OK cool.

I got it now. You know I can help with shipping in US. I have legal biz entity and FedEx account. Can tranship or drop ship if that will help anyone. If it took off I could also bulk buy for you guys too.

Koeng <koen...@gmail.com> wrote:

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[Koeng]

Its pretty awesome to have some people like you here :)

[Hugo Correia]

I had exactly the same idea. (I work with colicins) I think I'll try using one of my vectors as a test... :D

[Mike Horwath]

CCDB/CCDA is a bacteriocin toxin/antitoxin system that's already in use.  Both are encoded in the "natural" E. coli F plasmid.  CCDB kills the bacteria by inhibiting DNA gyrase, while CCDA binds and stops CCDB.  This works as a moderate pro-plasmid selection...in my understanding, the CCDB toxin is long-lived and will cause death in cells that gained but then lost plasmid.

At least 1 group has put the CCDB gene into the genome of bacteria [http://www.ncbi.nlm.nih.gov/pubmed/15945374].  This gave them very nice stability of plasmid expressing CCDA, but it also has the problem of not being able to grow up CCDB+ strain without plasmid.

I think the issue with encoding both the toxin and antitoxin on the plasmid is the 1st stage of growth after transfection.  Only a small % of the bacteria usually pick up the plasmid.  They may be secreting antibiotic, but they are likely to be outcompeted by untransformed bacteria before antibiotic reaches a critical concentration.  This might not happen every time but it would definitely affect reproducibility. You could add some antibiotic during the first passage only, I guess.

Mike

[Mega [Andreas Stuermer]]

Yeah, the same is true also for HOK/SOK.

But problem is the proteins are not excreted into the medium. Thus it does not kill other bacteria that haven't taken up the plasmid. And that should be the goal :)


If you fuse it to a signal pepptide thatdirects it outside the cell, the problem is

a) other bacteria won't take it up easily (and it ususally inhibits mRNA translation)
b) if another bacteria would take it up, it would recognize the signal peptide and excrete it.

Antibiotics usually are more or less small carbon scaffolds, which can freely diffuse through the membrane.

[Mega [Andreas Stuermer]]

Ideally you would have an E Coli strain that carries all the Kan-synthesis genes  - but the first one! - in the chromosome.

You would then transform it with a plasmid carrying the missing Kan-synthesis gene (e.g. KanA or KanB) and a Kanamycin immunity gene. It would then start producing kanamycin and be immune against it.

So it kills all the other bacteria that haven't taken up the plasmid. The plasmid itslef would aso be selfish, because if the plasmid is lost, it loses Kanamycin immunity and gets killed.


Plasmid size would not be much bigger than usual plasmids. Usually you have (100 bp promoter? +) 900 bp kanamycin resistance, now you have 1000 bp kanamycin synthesis and 1000 bp immunity. Both expressed from the same promoter, saving bp.

[Mega [Andreas Stuermer]]

It would though be possible to try this thing with the sigal peptide.

[Koeng]

I believe that there are types of bacteriocins, such as in Bacillus, that natively get exported from the cell

[Mega [Andreas Stuermer]]

Now that would be awesome!!