Mini genes

[Koeng]

Hello everyone! I was recently planning out a synthesis to do, and I wanted to get the best bang for my buck. I am going to use gene art strings, and I am going to get 450$ worth, 3,000 bp, but my gene is only 2338 bps long. So, I wanted some ideas to fill up the rest of the space. Right now I am thinking of putting the Hok/Sok toxin antitoxin system... but thats pretty much it. Any ideas for mini-genes that would be all around useful, or interesting? Please just post them below!

-Koeng

[Jeswin]

On Sun, Mar 24, 2013 at 2:10 PM, Koeng <koen...@gmail.com> wrote:
> Hello everyone! I was recently planning out a synthesis to do, and I wanted
> to get the best bang for my buck. I am going to use gene art strings, and I

Looks cool. Are you putting together a new plasmid?

> am going to get 450$ worth, 3,000 bp, but my gene is only 2338 bps long. So,
> I wanted some ideas to fill up the rest of the space. Right now I am
> thinking of putting the Hok/Sok toxin antitoxin system... but thats pretty

Tell me about this Hok/Sok system. I looked at the wikipedia to get an
idea of what it is. What purpose would you use it for? Just curious.

[Jeswin]

On Sun, Mar 24, 2013 at 2:35 PM, Jeswin <phill...@gmail.com> wrote:

>> am going to get 450$ worth, 3,000 bp, but my gene is only 2338 bps long. So,
>> I wanted some ideas to fill up the rest of the space. Right now I am
>> thinking of putting the Hok/Sok toxin antitoxin system... but thats pretty
>
> Tell me about this Hok/Sok system. I looked at the wikipedia to get an
> idea of what it is. What purpose would you use it for? Just curious.

Did some more reading on it. So it helps you do more efficient cell
culturing? Its a kill-mechanism, isn't it. It ensures that only
bacteria with the plasmid survive. So there are less worries about
running out of antibiotics in culture?

[Koeng]

No, I am not creating a new plasmid ( I think it is better to just pcr plasmids and Gibson them together to make minimal plasmids)
The hok/sok system is a plasmids selfish gene, once you get the plasmid YOU NEVER get RID of it >:)

Pretty much I am synthesizing it cause of the old topic on creating a plasmid with antibiotic synthesis and resistance genes, making it so you don't need antibiotics in media. Idk how it would work, so any other genes I would be interested in

[Mega]

Well, the "problem" is that the hok protein is not secreted, while if they would produce kanamycin, there would be no need for an antibiotic. 

Hok/sok just ensures that the cells don't lose the plasmid, but the proportion stays the same: 999 untransformed, 5 Transformed.

But I love Hok/Sok for it's simplicity and smallness of genes... What about another toxin/antitoxin system? I read that relBE also works in yeast IIRC, and is equivalently short. 

[Mega]

What if you add a signal peptide so it gets secreted??? 

Can the other bacteria still take it up as the entire protein? (Of course the signal peptide has to be cleaved off, else it would carry the signal again to be secreted!!)

[shamrock]

There is a nice paper describing various systems that have been designed to insure against the "escape" of genetically modified organisms. The Hok system is mentioned along with data on the number of microbes that "escape" the selection. The number of escapees seems pretty high for Hok-if your goal is insuring against escape you might want to look at some of the other systems described. Here's a link to the paper: http://www.frontiersin.org/Microbiotechnology,_Ecotoxicology_and_Bioremediation/10.3389/fmicb.2013.00005/abstract

[Koeng]

I could fuse a signal peptide to the end of hok... but then again the antitoxin still destroys hok.

Also I will look into synthesizing relBE!

I never really... hehe... try to ensure that my bacteria wouldn't escape, I mean where is K12 going to go if not onto my petri dish!

-Koeng

[Koeng]

Couldn't you just PCR relBE from E coli?????????

[Cathal Garvey]

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I think you guys are barking up the wrong tree, if you are trying to
convert in-cell toxin/antitoxins into allelopathic antibiotics to kill
neighbouring cells. There's too much work trying to get that hack to
work, for the same base-pair length you'd probably do the same with a
bacteriocin antibiotic.

As bacteriocins often encode "immunity" genes rather than
antibiotic-degrading "resistance" genes, they also sidestep the issue
of destroying-your-own-antibiotic, which would be a very costly
problem with, say, Kanamycin.

All this is a bit moot though if you choose a novel means of *initial*
selection. If you use Hok/Sok to ensure plasmid maintenance within
transformants, then your primary issue is how to initially isolate
transformants from a background of untransformed neighbours. There are
lots of clever ways you could try to achieve that; perhaps using
antifreeze proteins and freeze/thaw cycling, or a UV-resistance system
and a consumer-grade UV "sterilisation" wand, or something like that.
The goal is to have two systems working in parallel; one to allow
isolation of resistant cells, and another to ensure maintenance of the
plasmid once you've transformed it into cells. Both should be as small
as you can manage to enable larger inserts in your plasmid for other
interesting stuff.

Remember, by the way, that Restriction Enzyme systems behave selfishly
too, similarly to hok/sok. It takes lots of methylases to protect a
genome, but only one restriction enzyme to cleave up an unmethylated
site, so plasmid loss tends to lead to genome destruction as
methylases become rare and remaining restriction enzymes can start
targeting new DNA as it is made. That's behind one of the popular
lines of thought on how they became so diverse; selfish
self-maintaining behaviour allows evolution to take place.

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[Koeng]

I love the freeze thaw (Freeze bacteria, non of that membrane protector stuff).... I will find some proteins to use. For it to be effective though, how long would the incubation period need to be before the Freeze to get transformants time to create the protein? Any ideas?

Koeng

On Sunday, March 24, 2013 11:10:21 AM UTC-7, Koeng wrote:

[Koeng]

Any other ideas for something nice and small? Still got 200 bp almost...

On Sunday, March 24, 2013 11:10:21 AM UTC-7, Koeng wrote:

[Dakota Hamill]

Encode a secret message, something like, "Be sure to drink your Ovaltine"

[Koeng]

"This is, the most interesting plasmid in the world"
:)

[Koeng]

I have an idea :o

I should encode a viroid!
But then again that might not get past the biosafety check :/ any other ideas?

On Sunday, March 24, 2013 11:10:21 AM UTC-7, Koeng wrote:

[Andreas Sturm]

As long as ot is only a Coliphage, and doesn't attack mammals or plants this may not be unethically. However the synthesis company may have a different view on this ;)

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[Cathal Garvey (Phone)]

Lads, viruses are usually tens or hundreds of kilobases long! :)

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[Andreas Sturm]

True, with 200 bp you won't even get the envelope protein :D 

[Koeng]

It is not a virus... it is a viroid. They are different 

http://en.wikipedia.org/wiki/Viroid

http://en.wikipedia.org/wiki/Virus

No I didn't spell virus wrong xD

-Koeng

[Cathal Garvey]

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I stand corrected; I had assumed that viroids were likewise pretty
large, I'm surprised to see them being so small in actuality. ~400bp
for a replicative system is pretty impressive!

Why include one on your plasmid though? They appear to be mostly plant
pathogens, which rules out uses against bacteria, and plant pathogens
that target anything vaguely related to an agricultural species are
regulated almost everywhere. So, even if you were only planning to do
it for the lulz, it might be illegal.

On 03/26/2013 03:04 PM, Koeng wrote:

<https://groups.google.com/d/topic/diybio/kKmT3WHzJ7U/unsubscribe?hl=en>.

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[Koeng]

True, never wanna be illegal. Any other ideas of cool systems that you can't PCR?