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

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