Colicin V (or microcin V or ColV) is a poor solution for DIYbio transformations

[Koeng]

Hello all

Recently I have gotten the chance to work with the Colicin V operon and conduct some tests with it. More than a year ago, there was a post about synthesizing the kanamycin resistance cassette, which then sparked more conversation on some of the alternatives because Kanamycin has a big operon. Cathal, with IndieBB, was going to attempt to use this specific operon to allow for antibiotic-less selection of transformed plasmids.

Instead of synthesizing ColV, I PCRed it out of a native plasmid and into pUC19, using gibson and the blue/white screen to get good colonies. I sequenced them, and discovered 2 silent mutations in one of the transport proteins cvaB. I named this new vector pColV. If anyone wants a sample of it, I'd be more than happy to send a small amount of liquid culture.

First, I did a functional analysis where I grew co-cultures of pColV and pUC19. I can go into more detail if anyone wants, but generally it showed that pColV grows much slower than pUC19. However, once a critical mass of pColV cells were reached, it was able to take over the population even if it was growing much slower, extincting pUC19 from that test. In a 1:1 ratio test, at 12 hours of shaking growth at room temperature there was ~5 times more pUC19 than pColV. However, over the next 6 hours the pColV took over, and there was ~3 times as many pColV cells as there was pUC19 cells. When there was a 9:1 ratio of pColV to pUC19 respectively, pColV was able to extinct the pUC19 population by hour 18. In a 1:9 ratio of pColV to pUC19 respectively, the ColV population stayed relatively stable, never outgrowing or taking over the pUC19 population, which expanded exponentially. 

The results give a clue to what the transformation assays will look like. pColV must achieve a critical concentration of cells to effectively destroy rival cells at a rate that compensates for the metabolic burden of the ~4.2kb operon.  For the transformation assay, I transformed pColV and pUC19 into Top10 cells. After this, I immediately transferred to liquid culture without antibiotic selection. After growing up for a night, I plated on antibiotic plates. Assumably if it had worked, I would get a plate full of ampicillin resistant cells. However, I only got 6 transformants, less than I got by immediately plating on antibiotics.

Of course, for each experiment there were more controls, but this is the general gist of what the results were. I also attempted to purify ColV from supernatant using a filter, but this supernatant no antibiotic properties, as it has been reported in literature. If DIYbiologists want to achieve an antibiotic-less selection mechanism, they will have to search elsewhere. I recommend personally a toxin/antitoxin system. The toxin will be permanently integrated into the genome, and repressed by, lets say, growth at 30c. Transform, and grow at 37. Cells without the antitoxin, (think ccdA/ccdB) die, and the others survive. Of course, this wouldn't be a very portable system by any measure, but it would work. I may tinker with that idea later since I have temperature sensitive lambda repressor and both the toxins.

Cheers,

-Koeng

[Nathan McCorkle]

Awesome post! You could write this up and post it online on scribd or
ResearchGate (on your profile) and no one would know you weren't a PhD
candidate or industry scientist. Just repeat some experiments, add the
results you said you omitted, break apart the info into the normal
journal article sections, add some graphics (molecules, growth curves
or something, tables of cell/blue-white counts), etc...


Did you use IPTG to induce expression? I wonder if you used 10 or 50X
less inducer... if they would grow faster and thus take over faster. I
don't know how many molecules of Colicin it takes to kill a non-immune
cell, and I also don't know if those molecules are multi-use or are
one-time-use only.

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-Nathan

[Koeng]

Thanks! I will definitely look into doing a formal write up on my experiment! 

So the issue with ColV is it is optimized for low-copy F-type plasmids. More than likely, I am overexpressing ColV simply by having it on the pUC origin. I have compatible sites to insert it into pBeloBac11, and I also have pBeloBac11, so that may be useful for these studies. An interesting point is that it does look like ColV expression, even below the threshold for taking over, does repress some growth of pUC19. Another issue with effectively studying it is that the operon is split so there are 2 different bicistronic cassettes. To get optimal expression, I would have to modulate 2 different gene sets (assuming the RBSs are already evolved for an optimal relationship).

Personally, I think the best way to go about studying this system is genome integration. Single stable copy, shouldn't have much effect on the cell. Some ColV operons in nature are also like this.

They've never really studied colV that in-depth, an unfortunately I don't have the resources to see how many ColV molecules it takes to kill a cell. Assumably (No evidence to back up claim), the ColV toxin is not reusable because the secretion domain is cleaved upon export from the cell. They also don't have crystal structure for it, making simple speculation a bit more difficult

-Koeng

[Mega [Andreas Stuermer]]

According to wikipedia, one molecule was speculated to be able to kill a cell. However, pUC Ori copy number is 100 to 500 per cell and so you should have tons of colV to kill other cells.

I haven't looked into the immunity mechanism but it involves blocking the TolC receptor ehere colicinV usually binds too.

You could definitely try to induce colV-immunity with IPGT to see if that increases survival.

[Koeng]

The immunity mechanism actually (hypothetically) involves blocking binding to an inner membrane protein sdaC. TolC is used in conjunction with cvaA/B for export

I have an experiment planned which I think will work for DIY transformations, will update if it works. Essentially, I want to integrate the ColV operon without the immunity protein into the genome under the rhamnose promoter. Upon activation of rhamnose, all cells should die. Or alternatively they can have the cvi protein on a plasmid to survive. This entire experiment should only take 2 cloning reactions, which is nice. Quick to complete, relatively.

-Koeng

[Dakota Hamill]

Rhamnose seems more expensive than common antibiotics for selection , or at the very least, compatible. 

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[Koeng]

It'd more be a proof of concept, since you can have toxic protein stable under the rhamnose promoter. 

If it works well enough, I'll switch it out for a cl/cro system or a flp system so you only have to induce once. 

-Koeng