Phage assisted DNA distribution

[Koeng]

Hey guys!

I've been working on a few things lately, one of which is an easy transformation protocol, mainly for distributing DNA easily. One of the problems I saw was that every time I recieved DNA, I had to make comp cells, which as you probably know takes some time and I was pretty lazy, mostly since my comp cells are only really good once, albeit a little less efficient. Not only that, but most people send DNA in centrifuge tubes or on paper, and for the fliter paper method I've had some problems in the past recovering plasmid. In addition, if I ever wanted to ship someone a plasmid I'd have to miniprep to make sure I give them "fresh DNA"

Off of the topic of distributing DNA, I was also thinking about starter kits. I've noticed the #1 thing beginners  mess up on is making competent cells. Whenever I've taught classes, here is were people mess up. When doing it at home, it's easy to get frustrated and quit because the first transformation with a start plasmid failed (like pGlo)


So, I came up with a possible alternative. Packaging a plasmid that expresses GFP into M13 phage particles. When I got M13KO7 from a neighboring lab (it was for a different project) they told me to just mix it with bacteria that have the F plasmid. (SS320 in my case). I simply mixed the phage and bacteria that were growing, and recovered (its a kan plasmid) and plated. Got tons of colonies. All you need for the M13KO7 to package your selected DNA instead of itself is a plasmid with the F1 origin, which is super common. (Even in pGlo!)

If this was adapted for ampicillin plasmids, all you'd have to do is mix with the phage with bacteria for a few minutes and plate. In fact, if you just put the phage on a filter disk and added that into some bacteria it'd work (there was a story of a guy who had really needed a secret phage from a lab, and they wouldn't give it to him, so he sent them a letter pleading for it, and they sent a letter back saying no. He dissolved the letter and recovered intact phage from the paper!). So, in general, recovery would be super easy, even for any DIYbiologist.

Next is distributing the actual DNA. Since E coli are constantly giving off phage (if infected with the helper plasmid, M13KO7) all you have to do is centrifuge the E coli and put the supernatant on filter disks. The entire registry of standard biological parts *could* be distributed like this for a fraction of the current cost (their standard plasmid doesn't have the F1 origin :( ). The entire library of parts could then be restored and saved since the amount of competent cells is no longer a problem. I am currently going to test this, I have pGLO and M13KO7 inside of Mach1s and some SS320 cultures going for testing this week.

However, I'd like some discussion on this, mainly with the disadvantages-

Advantages
Cheap
Easy to recover
Easy to distribute

Disadvantages
Possibly mutates quicker? (single stranded intermediate?)
Size limitation*
Lab receiving has to have F plasmid E coli
Contaimination issues***
(There would probably be a ton of contaimination since the phage are so infectious )

Anyone with experience have any comments on this? Or anyone in general, I'd like to know what you think about this system/idea of distributing parts to DIYbiologists
-Koeng

[Dakota Hamill]

Sounds pretty interesting, though I guess I'm trying to figure out exactly how it works.  You're saying you can use phage's to transfect bacteria (without having to do a standard transformation protocol / use competent cells) as long as the cells you are trying to transfect are already expressing F factor / have a F plasmid inside them?

It sounds pretty cool to be able to mix in some phage particles, plate your cells, and you're good to go.  And then, bypass a miniprep to recover plasmid, as the cells are naturally excreting the phage particles with your plasmid/DNA of interest into the growth media.

I'd be semi-worried about the single stranded DNA aspect of it.

And the other thing I was wondering, does the phage contain JUST your gene of interest (as ssDNA) with a way to incorporate it into the F plasmid already found inside the E. Coli?    Or, does the phage have an entire plasmid, containing both the F factor, AND your gene of interest, which you want to put into the cell.

If it's the prior, wouldn't that mean that in order to be of any use, you'd still have to do a normal transformation with an F plasmid into your cell, prior to the phage transfection being of any use (since it requires and F plasmid inside your cell already?)

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[Mega [Andreas Stuermer]]

Just my thought but wouldn't kanamycin selection be better? 

If you have fungal contaminants, they will happily grow on ampicilin, but kanamycin will hinder their growth or even kill them.

Or some kind of self-selection. How much payload can the phage carry?

[Koeng]

@Dakota
The plasmids have the pUC origin and the F1 origin, they only get replicated as single stranded when you add the helper phage, M13KO7, into the cells (which is replicated on the p15A origin). Pretty much, once inserted the DNA will act just like a normal plasmid except that they have a ~500bp fragment of the F1 origin inside of them. The inserts in pGLO, for example, would be F1 origin, ColE1 origin, ampicillin resistance, and GFP.

The F factor isn't included because its like 80kb. It's in some common strains like SS320, so you never really transform it. It's also fairly stable, so yes you'd need a strain with it inserted as a prerequisite. I'd be happy to just send the cloning strain along with though, thats something that's easy.

@Mega
Kanamycin you'd have to recover for about 45 minutes after in a shaking incubator. When I construct my library of parts (Another project I am working on for my personal lab, just a ton of modular parts that some people might be interested in) I'd like to do a 5 minute transformation to reduce the amount of steps needed. The "next level" vectors would have kan resistance. (its a biobrick/golden braid mix, I am going for ultimate compatibility).

"However, as M13 genome size increases, the efficiency of replication declines such that while recombinant phage genomes 1-10% longer than the wild-type do not appear to be significantly disadvantaged, those 10-50% longer than the wild-type replicate significantly more slowly and above 50% increase over the normal genome length it becomes progressively more difficult to isolate recombinant phage. This property has been exploited in M13 cloning vectors. " http://ntmf.mf.wau.nl/cor/m13.htm
Pretty large payload. The native phage is about 6.5kb

[Nathan McCorkle]

On Sun, Sep 7, 2014 at 11:45 AM, Koeng <koen...@gmail.com> wrote:
> Disadvantages
> Possibly mutates quicker? (single stranded intermediate?)
> Size limitation*
> Lab receiving has to have F plasmid E coli
> Contaimination issues***
> (There would probably be a ton of contaimination since the phage are so
> infectious )

Yeah so how do you 'cure' a batch of E.coli of the phage?

I tried looking up some keywords like inhibition and 'cure' but I
didn't get too much:
http://diyhpl.us/~nmz787/pdf/RIFAMYCINS_A_GENERAL_VIEW.pdf

Couldn't get this one:
http://www.sciencedirect.com/science/article/pii/0042682272903947


Seems like having a switch where they won't synthesize, and maybe
another switch for dissolving the strands somehow would be nice. If
they're really so infectious, how do you otherwise normally keep them
under control and having some uncontaminated stock culture?

[Koeng]

The phage only get synthesized in the presence of M13KO7 and only infect in the presence of the F plasmid. The shipped phage won't have either one.

However, you'd have to make sure your stock of F plasmid E coli (SS320) don't get contaminated.

[Koeng]

The contamination is because phage originally synthesized for one plasmid can possibly cross over to another culture, and once shipped it could be amplified. Bare phage gets everywhere

On Monday, September 8, 2014 6:09:59 AM UTC, Nathan McCorkle wrote:

[Nathan McCorkle]

On Mon, Sep 8, 2014 at 5:25 AM, Koeng <koen...@gmail.com> wrote:
> The contamination is because phage originally synthesized for one plasmid
> can possibly cross over to another culture, and once shipped it could be
> amplified. Bare phage gets everywhere

So how do /you/ keep things clean? How do /you/ keep you stock culture clean?

[Nathan McCorkle]

On Mon, Sep 8, 2014 at 5:24 AM, Koeng <koen...@gmail.com> wrote:
> The phage only get synthesized in the presence of M13KO7 and only infect in
> the presence of the F plasmid. The shipped phage won't have either one.
>
> However, you'd have to make sure your stock of F plasmid E coli (SS320)
> don't get contaminated.

So do you transform with F plasmid prior to phage use every time to
prevent contamination of your untransformed stock culture?

Have you come upon any means of curing the culture of the phage? The
rifamycin seems to only stop synthesis, but the paper didn't mention
anything about catabolism/disintegration of the phage particles.


If you don't do this, do you have a lab room that is devoted to phage
work? And have a different room for your stock culture operations?

[Koeng]

Normal sterile technique works well, if you practice it a bit more stringently. No standing waste, always bleach the phage immediately after you use it. The stock culture holding the phage/M13KO7 won't have the F plasmid, so the phage physically can't infect them.

Making sure your stock culture for SS320 is pure is a bit more complex. Making sure it doesn't touch any phage is very important. Use completely different flasks, and make sure to plate negative controls.

If your SS320 stock gets contaminated, beyond control, you just have to get new cells or begin streaking plates for new single colonies. Extensive bleaching and autoclaving can get rid of phage on glassware, but that won't be needed often if you keep your stuff sterile.

I work in a lab that uses this phage, rule of thumb is to always run a negative control when transforming cells (they use SS320 for a different cloning procedure, not this reason). The only real unstable cells are the E coli with the F plasmid, the ones expressing phage should be fine. You *could* retransform it every time, but I've never actually seen anyone do that because the plasmid is 80kb~. Just get new cells

-Koeng