Anyone have a method of synthesizing thermococcus gammatolerans?

[Finn Daffron]

I am trying to create a batch of thermococcus gammatolerans. This is because I want to use CRISPR to inject thermococcus gammatolerans into rats in order to create radiation resistance with possible implications being people working in nuclear power plants, astronauts, etc. Does anyone have any ideas on how it would be synthesized? Please get back to me.

[Dennis Oleksyuk]

Thermococcus gammatolerans is probably relying on its extremely efficient chromosome reconstruction mechanism to repair DNA damage caused by radiation. Considering that it is an Archaea with a puny DNA it is almost certain that whatever mechanism it is using will be completely incompatible with mammals cell physiology.

In case you still want to try, I suggest starting from reading this paper.

http://www.ncbi.nlm.nih.gov/pubmed/19558674

On Thu, Jan 28, 2016 at 3:11 PM Finn Daffron <finnd...@gmail.com> wrote:

I am trying to create a batch of thermococcus gammatolerans. This is because I want to use CRISPR to inject thermococcus gammatolerans into rats in order to create radiation resistance with possible implications being people working in nuclear power plants, astronauts, etc. Does anyone have any ideas on how it would be synthesized? Please get back to me.

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[Finn Daffron]

Thank you for your help and the link. I have, based on information provided in the paper and by you, determined a method that does not involve live thermococcus gammatolerans but instead the recombinant proteins that provide DNA repair in thermococcus gammatolerans.

[Koeng]

Identification of exactly what proteins (with references) confer to this resistance is the first step. 

Since biological systems for the most part are not modular between different domains (lots of exceptions), you will have to verify that these do, indeed, confer to resistance to radiation. What will your controls be for that? How will you prove statistical significance in mice? Are all proteins needed, some, or just 1?

I agree with Dennis, it will probably not work first try around. Perhaps it will with extreme modification, but that is out of the realm of DIYbio.

-Koeng

[Koeng]

"This proteogenomic study also indicates that the high radiotolerance of T. gammatolerans is probably due to proteins that remain to be characterized"

Direct injection of proteins will likely do more harm than good, and they don't even know the exact proteins that confer to their resistance. Finding the *exact* genes is the first task

On Thursday, January 28, 2016 at 3:59:23 PM UTC-8, Finn Daffron wrote:

[Dennis Oleksyuk]

Now this become curious. How would you test radiation resistance? What would be the most cost efficient way to expose cell culture to ionizing radiation? 

I bet that radioactive isotopes is off the table for majority of places. Can x-ray radiation used? Is it different in its DNA damage properties from radiation from radioactive isotopes? If so, then some old x-ray machine probably good the job. 

Is there any alternative?

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[Bryan Jones]

It depends on the radioactive isotope used. Some isotopes emit alpha or beta particles, but some emit gamma photons. The most cost effective way to expose cells to ionizing EM radiation would be to leave it outside in the sun. You get a relatively small dose of ionizing UV from sunlight, might not be enough for these experiments, but direct sunlight tends to be fairly damaging to DNA (hence melanoma). Would the damage from gamma rays be the same as UV? They are both ionizing EM radiation, but gamma rays have a lot more energy.

Another, more controllable source of UV would be tanning bed lights.

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[Gavin Scott]

On Fri, Jan 29, 2016 at 2:38 PM, Dennis Oleksyuk <ma...@dennis-o.com> wrote:
> Now this become curious. How would you test radiation resistance?

Unless thermococcus gammatolerans actually encounters unusually high levels of radiation in its environment (seems unlikely) then as suggested it's probably really just a very good DNA repair mechanism of some sort which it uses to survive some other threat. I think it would be interesting to test its resistance to chemical mutagens and other ways of damaging its DNA which might well give you an effective way of evaluating it without actually using ionizing radiation.

I'd look at papers on DNA repair, because likely many of them will have needed a convenient way of damaging DNA.

I wonder if it would be hard to use CRISPR-derived tools on such a species. It might repair itself as fast as you're trying to cut it with your Cas9 :)

G.

[Cathal (Phone)]

D.radiodurans is so radiotolerant because of mechanisms it evolved to resist dessication-induced double-stranded breaks (and deamination/depurination?). So, let's generalise basic radiotolerance to repair of or avoidance of DSBs and specific classes of SN-mutations.

If this bug lives in the *plume* of a thermal vent, it may have to deal with a lot of cavitation from thermal bubbles, which I'm guessing may lead to sonication-indiced DSBs. But, the same mechanisms wouldn't lead to an abnormally high level of SN-mutations. However, heat *does* increase the odds of such mutations, so it may have convergently evolved the same radio-defences as D.radiodurans despite a totally different environment.

All guesses on my part, of course. :)

On 31 January 2016 06:05:00 GMT+00:00, Yuriy <yuriy...@gmail.com> wrote:

oxidation and desiccation stresses? but it lives in thermal vents, produces hydrogen, so I have a feeling it might have that covered. 

the strain sequenced already has CRISPR

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Sent from my Android device with K-9 Mail. Please excuse my brevity.

[Yuriy]

The paper doesn't mention backups for chromosomes to template off of for repair. It does mention major rearrangements in genome of either the species or the genus reconstitution. It is still dependent on the same divalent cations and chromosome condensing proteins to keep everything in place after breaks. This limits how far two strands that were together migrate away from one another. This puts a limit for an otherwise illegitimate end joining.

It could be that the species or the strain or the like has had their evolutionary training in a natural nuclear fission reactor (as in not man made). Yeah, those environments do exist. In fact nuclear material mining companies are surprised by the low yield sources when they hit unusually high output radiation source. Perhaps it was so close to radiation, it was forced to come up with new survival strategies.

Getting back to the Cas9 topic: I read somewhere that it hangs around hours after making the cut. If it does stay on for that long, it won't give you ligation or repair for at least as long. Define quick again? In any case, it definitely doesn't have type II CRISPR. Do to lack of interest in other CRISPR systems little is known about their properties.

Water is that species' natural environment, so cells in water quench ROS much quicker than those w/o water. That's another factor.

[Finn Daffron]

Thank you for all of your answers. My idea was to use UV lights as radiation. I am glad that people believe that that is a good idea. What do you think of using PCR to insert thermococcus gammatolerans DNA into the sperm of rats as opposed to CRISPR?

On Thursday, January 28, 2016 at 3:11:22 PM UTC-5, Finn Daffron wrote:

[Koeng]

How would you use PCR to insert it? What protocol are you using? Genetic modification of animal lines is difficult and takes significant planning. 

How do you "insert" the DNA with CRISPR? CRISPR doesn't integrate anything, just prompts homologous recombination, which works better with homology, provided by a DNA you provide. How will you get the rat sperm? How will you get transfection quality DNA and then transfect the sperm? What viral vector will you use? Is it biosafety 1? How will you do a polycistronic expression cassette? Given RBS strengths predicted in said organism can you make an educated guess (yes the calculator isn't perfect) on expression levels of each gene required in your target organism given a set of promoters? Do you have the materials do undergo that cloning project? 

There is a reason there is yet to be a DIYbio project that uses animals. It is pretty rare to see a successful DIYbio that effectively expresses a multi-gene construct. The highest organism is usually plants. Do you even know if your genes confer to radiation resistance in E coli? If they do, does this function in yeast? Do you have the materials to do this in rats? I don't work with multicellular organisms, but I'm pretty sure this will take some training and some expensive equipment. 

-Koeng

[Yuriy]

No one said it was a good idea. Unless you are trolling, leave the subject be until further research is done. Do google scholar alert or something. See if the paper mentioned by Dennis has been cited in more recent publications. See if the orphaned pathways have been elucidated.

What DNA will you target? You won't know if regulatory elements or protein folding will work in a mammalian expression. You won't know if there are cryptic elements. You won't know if complementing systems end up on the same cell.

[Matt Lawes]

Hello Finn,
Havent really followed the conversation this far, so apologies if I'm repeating somebody else. ... What is your specific hypothesis? You can't just throw an entire bacterial genome into a eukaryote cell. I think radiation resistance is about very efficient / active DNA damage repair? So which pieces do you wish to edit?
>matt

Sent from my T-Mobile Android device

On Feb 3, 2016 9:15 AM, Finn Daffron <finnd...@gmail.com> wrote:

Thank you for all of your answers. My idea was to use UV lights as radiation. I am glad that people believe that that is a good idea. What do you think of using PCR to insert thermococcus gammatolerans DNA into the sperm of rats as opposed to CRISPR?

On Thursday, January 28, 2016 at 3:11:22 PM UTC-5, Finn Daffron wrote:

I am trying to create a batch of thermococcus gammatolerans. This is because I want to use CRISPR to inject thermococcus gammatolerans into rats in order to create radiation resistance with possible implications being people working in nuclear power plants, astronauts, etc. Does anyone have any ideas on how it would be synthesized? Please get back to me.

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[Alex D]

You might avoid all the hasle but it will cost alooot. What i can sujest is. Optimize bacterial chromosome to rat and then create an artificial chromosome and integrate it into rat stem cells which will then can be converted to other tissues or adipocytes at least.

There are very many promoters and ori sites that has to be replaced so the synthesis of such project will be. Probably in 10s of thousends just for the dna itself.

I would say it is doable but super expansive and probably will require something like craig venter institute to complete

On Jan 28, 2016 3:11 PM, "Finn Daffron" <finnd...@gmail.com> wrote:

I am trying to create a batch of thermococcus gammatolerans. This is because I want to use CRISPR to inject thermococcus gammatolerans into rats in order to create radiation resistance with possible implications being people working in nuclear power plants, astronauts, etc. Does anyone have any ideas on how it would be synthesized? Please get back to me.

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[David Weichselbaum]

First you should know what you want to do in detail (on the level of base pairs) and then you can think about transforming mammals.

If you know what you're doing however, you could use a pEPito mammalian vector, which is basically a small artificial chromosome that piggybacks on native chromosomes to be mitotically stable.

Then you can gather rat sperm (maybe using a syringe and anesthetics is less stressful for both you and the rat (-: ) and use the sperm mediated gene transfer protocol. This is basically centrifuging the sperm, mixing it with the vector in the right buffer and injecting it into your rat mama (you really should read up on lab animal ethics and consult specialists to help you). The efficiency of getting transgenic offspring is 80% in pigs.

Have you considered researching radiation resistance in mammals, not in bacteria? As everyone keeps saying, it's just the level of DNA repair you're after.

Get a permit for the exclusion zone of Chernobyl, Ukraine und take some live traps with you. The red forest is one of the most radioactive places on earth and while you cannot enter it, you should be able to get some really radioactive rats in its vicinity.

Take some blood and tissue samples and do an expression analysis or amplify and sequence known DNA-repair mechanisms. I bet after 30 years and hundreds of generations they are nicely adapted to their new home. After that you should be able to reverse-engineer their resistance or even amplify it. 

[Bryan Jones]

I think David's suggestion is good. Radiation resistance would probably translate better between mammal species than from bacteria to mammals. You might want to look at tardigrades as a source. While not mammals, they are at least animals, so the mechanisms of radiation resistance would be much more likely to work in a mammal context than a bacterial mechanism. I've attached a paper about tardigrade radiation tolerance.

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[Simon Quellen Field]

Having just returned from a tardigrade collecting hike, I find this thread suddenly more interesting. :-)

The radiation tolerance is probably due to the same mechanisms that allow them to undergo cryptobiosis. Sugars, fats, and disordered proteins can all aid in reducing the damage from reactive oxygen species. This is necessary to survive desiccation, and would also make radiation exposure less damaging.

Whether these adaptations would benefit a larger organism seems doubtful to me.

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[Simon Quellen Field]

The results of my Tardigrade hunt today:

"https://www.youtube.com/watch?v=I-KTvdrB9LQ&feature=youtu.be"

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[Nathan McCorkle]

On Fri, Jan 29, 2016 at 9:12 PM, Yuriy <yuriy...@gmail.com> wrote:

"I am trying to create a batch of thermococcus gammatolerans." Some things are probably lost in translation and I didn't see anyone try to correct you. You don't create anything, if it already exists.

In this context, the meaning I perceive is the desire to create "a batch", not the species. I just created a batch of cooked bacon. It definitely didn't exist in my fridge before my efforts. 

[Sebastian S Cocioba]

This is becoming a really weird conversation. Sorry to butt in but why the passive aggressive tone, Yuriy? Last few comments were not the most neutrally worded. Everything okay? 

Sebastian S. Cocioba

CEO & Founder

New York Botanics, LLC

Blog: ATinyGreenCell.com

On Feb 7, 2016, at 9:22 PM, Yuriy <yuriy...@gmail.com> wrote:

Huh, I thought I successfully deleted that communique. Oh well.

Context and perception had you dig up a deleted comment. Given the effort you put into correcting my perception (even though I wasn't the only one on perceiving it that way, on a seemingly tapered off thread), correcting thread poster, shows that you've got nothing but time. You are very resourceful though and no. That is not euphemism.
Retrieve that from DIYBio group waste basket. I am giving it ten min. Let's see how you keep up.

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