Re: [DIYbio] Re: Random gene?

You might also check out the mechanisms of mosaicism that already
exist in day to day life, like with barr bodies:
https://en.wikipedia.org/wiki/Barr_body
https://en.wikipedia.org/wiki/X-inactivation

On Thu, Apr 17, 2014 at 10:44 PM, Kevin C. Ma
<kevinchenma@college.harvard.edu> wrote:
> I agree - I think recombinases are the way to do it. You may want to also
> check out papers from the Lu Lab and the Endy Lab where they used
> recombinases to construct logic gates:
> https://microbiology.okstate.edu/faculty/prade/rprade/MICR4263P2_files/SB%20p4.pdf
>
> Specifically they used unidirectional ones, so the issue with Cre (repeated
> inversion) as Salva suggested wouldn't be a problem - after an initial
> "population splitting" event, the various sub-populations are locked into a
> single state. There are clever ways to go about arranging recombination
> sites which will give you a desired result. As is done in Brainbow (I think)
> you can have multiple sets of recombination sites, some of which may be
> excised leading to a fluorescent protein (or a combination of fluorescent
> proteins) being expressed. Another interesting approach is to overlay these
> sites which may give you more control over which states or sequences of
> states you might want to have....
>
> On Thursday, April 17, 2014 1:21:16 PM UTC-4, Salva wrote:
>>
>> There are different versions of brainbow and, if I recall correctly, there
>> is at least one that involves inversions of DNA not excisions (so there is
>> no loss of information). Cutting it short , you need to control the cre
>> expression with a promoter that only activates during division. This way you
>> force to rearrange DNA in the division events and the color should stay the
>> same for as long as the cell doesnt divide.
>>
>> El 17/04/2014 18:46, "Olivia Gallup :)" <olivia...@googlemail.com>
>> escribió:
>>>
>>> Sorry for being ambiguous; I want each cell to activate only one of the
>>> genes for the duration of its whole life cycle, and I do understand that
>>> nothing in biology is random, which is why I posted this question to ask you
>>> guys for ideas :) To choose the gene to be activated also has to be
>>> completely up to the cell without any outside genetic manipulation, so no
>>> CRISPR or Lox/Cre. In every "newborn" cell, the process for choosing the
>>> gene that the cell will express for its whole life has to restart, so that a
>>> daughter cell does not look exactly like its parent cell.
>>> The brainbow technique for marking cells like this from @Salva would be a
>>> great solution actually, except that it would only work for one generation,
>>> and in each new generation you would have to redo the coloration of the
>>> cells. To make my question more understandable; if you had a culture of
>>> bacteria that had a successful/working version of this system and each cell
>>> picked one out of say 5 colors to express, every neighboring cell would have
>>> more or less a different color.
>>>
>>> On Tuesday, April 15, 2014 7:33:11 PM UTC+2, Josiah Zayner wrote:
>>>>
>>>> Your question is interesting but not quite clear could you be more
>>>> specific? What do you mean by pick one of the genes at random?
>>>> If a transcription factor is always picking one of the genes at random
>>>> then the cell will not produce only one pigment, which I think is what you
>>>> are after?
>>>> If you want each cell to randomly activate one and only one of the genes
>>>> this becomes much more difficult.
>>>>
>>>> Understand that _nothing_ in biology is _random_ what this means is that
>>>> choosing your gene needs to be regulated by _something_. Temperature, light,
>>>> pressure, amount of a transcription factor.
>>>>
>>>> Next, once a genetic change is made, say by using Lox/Cre or CRISPR,
>>>> there is no going back(or it is very very complicated) so the "randomness"
>>>> would only be good for one generation. i.e. you transform a bacteria with
>>>> the original plasmid and it will randomly be a color but each generation
>>>> after that will be the same color.
>>>>
>>>> However, if that is all you want then the simplest approach would
>>>> probably best be done by creating a library of plasmids with promoters that
>>>> allows a constitutively active transcription factor to activate only one of
>>>> the genes at a time in the cell. Then transforming your bacteria with all of
>>>> them and each will contain a random gene that they activate for one
>>>> generation or maybe multiples to create different combinations of pigments.
>>>>
>>>>
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--
-Nathan

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