Sorry, I have a habit of overexplaining. :)
My issue isn't that the amounts of acetaldehyde will be dangerous. It's a combination of:
- Without substrate regeneration, I don't think you'll get much output
- You can't add external acetaldehyde and still suggest it's "Glowing Yoghurt" because it's a toxic additive. Inedible yoghurt is disingenuous, and fence-sitters will take this as "evidence" that genetic modification involves toxicity.
In contrast, tetradecanal is unlikely to significantly affect the safety and food suitability of yoghurt, even if you'd still be advising against eating it on common-sense grounds for now.
medminus9 <harshsethia1989@gmail.com> wrote:
>Cathal,
>
>I am a 4th year Medical student. :)
>
>On Tuesday, 3 April 2012 17:26:30 UTC+8, Cathal wrote:
>>
>> It's a normal metabolite of alcohol dehydrogenase, yes..but usually
>it
>> it's immediately converted to acetic acid by aldehyde dehydrogenase,
>the
>> second enzyme in alcohol detox.
>>
>> If you block aldehyde dehydrogenase, using artificial factors
>(antabuse)
>> or natural toxins (such as the one found in the ink cap mushroom),
>the
>> result is that you rapidly get violently sick on drinking any
>ethanol.
>>
>> The amounts of acetaldehyde in yoghurt aren't going to be big enough
>to
>> worry about, but I'm concerned that if bioluminescence levels
>disappoint,
>> you can't add extra substrate and still say "it's edible". Not that
>eating
>> engineered probiotics for novelty alone is a good idea..
>>
>> medminus9 <harshsethia1989@gmail.com> wrote:
>>
>> >Mmm, my bad. I didn't check about the toxicity of tetradecanal. But,
>> >yes
>> >acetaldehyde is a metabolite formed in the liver during alcohol
>> >metabolism.
>> >It is also found in beer in small amounts. Then again, the final
>> >product
>> >should be acetic acid i.e vinegar, which is again non-toxic but I
>can't
>> >
>> >find the comparison data.
>> >
>> >Thanks,
>> >Harsh
>> >
>> >On Tuesday, 3 April 2012 04:28:29 UTC+8, Cathal wrote:
>> >>
>> >> Are you sure about that? Acetaldehyde is extremely toxic, and is
>> >classed
>> >> as a human carcinogen and probable teratogen. It causes organ
>damage
>> >by
>> >> adducts to proteins on organ tissue, and is thought to be
>responsible
>> >for
>> >> hangovers, to boot.
>> >>
>> >> Meanwhile, tetradecanal, also known as myristic acid, is the main
>> >> constituent fatty acid in nutmeg butter among other common dietary
>> >fats:
>> >> https://en.wikipedia.org/wiki/Myristic_acid
>> >> Its LD50 for oral doses is in grams-plus; a shorthand way of
>saying
>> >it's
>> >> easier to drown the animal with the chemical than induce a toxic
>> >response.
>> >> Mild skin irritation may result from 24 hours of skin exposure,
>but
>> >that's
>> >> true of almost any liquid or solvent other than water.
>> >>
>> >> For acetaldehyde's health effects information, jump to section 11
>> >here:
>> >>
>> >
>>
>http://www.chemcas.com/AnalyticalDetail.asp?pidx=1&id=86&cas=75-07-0&page=2
>> >> For tetradecanal's, see here:
>> >> http://www.chemcas.com/msds/cas/msds129/124-25-4.asp
>> >>
>> >> On 2 April 2012 19:45, medminus9 <harshsethia1989@gmail.com>
>wrote:
>> >>
>> >>> I was considering acetaldehyde because it would help reduce the
>size
>> >of
>> >>> the linear construct (-luxCDEG) and hopefully might increase the
>> >>> transformation efficiency (this is what i thought, I might be
>> >absolutely
>> >>> wrong!). Also at the same time peak output will also benefit, but
>> >then I am
>> >>> just a novice, so?
>> >>>
>> >>> I was thinking of using a heterofermentative species for the
>> >purpose, as
>> >>> that would provide a long lasting supply of acetaldehyde, given
>that
>> >I can
>> >>> knock out the gene/enzyme responsible for conversion of
>> >acetaldehyde->
>> >>> alcohol. Also, thermophilus gives a continuous supply of
>> >acetaldehyde .
>> >>>
>> >>> 8/9 carbon chain groups with polar head need some work, but then
>> >>> acetaldehyde is much less toxic then tetradecanal, as it is
>already
>> >formed
>> >>> in metabolic pathway of alcohol.
>> >>>
>> >>> FMN and NADPH are present in some species, also one of the
>species
>> >>> L.fermentum happen to have the NADPH.FMN oxidoreductase enzyme
>> >required for
>> >>> the pathway.
>> >>>
>> >>> For the linear transfer, I am scared if the exonucleases would
>just
>> >chew
>> >>> of the construct. Is there any literature on how to avoid that,
>also
>> >won't
>> >>> the low temperature in ice bath inactivate the enzymes?
>> >>>
>> >>> Thanks,
>> >>> Harsh
>> >>>
>> >>>
>> >>>
>> >>> On Monday, 2 April 2012 21:44:20 UTC+8, Cathal wrote:
>> >>>
>> >>>> Some thoughts on acetaldehyde:
>> >>>> - Using Acetaldehyde instead of tetradecanal is, in my view,
>not
>> >the
>> >>>> best route. Bioluminescence isn't an ongoing degradation of an
>> >abundant
>> >>>> precursor; half the operon is devoted to regenerating the
>> >substrate. I
>> >>>> would expect that acetaldehyde would be rapidly degraded as it
>was
>> >>>> created, but not at a level that's visible to the naked eye.
>> >>>> - The paper you referenced, while pretty cool, made it clear
>that
>> >>>> bioluminescence from Acetaldehyde was poor-to-nil without a
>> >co-substrate
>> >>>> that probably isn't abundant in normal culture conditions; an
>8/9
>> >chain
>> >>>> carbon group with a polar head such as a bromide. So, you'd have
>to
>> >add
>> >>>> awkward stuff to your yoghurt to make it glow?
>> >>>> - Finally, in order to maintain the legitimacy of
>"Bioluminescent
>> >>>> Yoghurt", I'd use only one of the two yoghurt cultures. There
>are
>> >>>> transformation procedures for both of them in the literature,
>and
>> >AFAIK
>> >>>> their genome sequences are publicly available. Working from
>that,
>> >>>> there's little preventing you/us from deriving what's needed.
>> >>>>
>> >>>> So, I'd suggest going with the "wild" system, but with a heavy
>> >>>> refactoring of the bioluminescence operon. Literature suggests
>that
>> >>>> separating LuxAB from the rest can allow you to really boost
>light
>> >>>> output beyond peak natural levels by decoupling the production
>of
>> >>>> substrate from the luciferase complex. I believe the cambridge
>team
>> >did
>> >>>> this some time ago?
>> >>>>
>> >>>> To make sure that your substrates (tetradecanal + FMN+NADPH) are
>> >>>> produced or present, you may need to either include synthesis
>genes
>> >or
>> >>>> include tetradecanal separately (some spices or seasonings have
>it,
>> >I
>> >>>> recall). Ideally if you could afford it, find a system for fatty
>> >acid
>> >>>> production that yields lots of tetradecanal. It's possible that
>> >yoghurt
>> >>>> already has some level of the stuff, though?
>> >>>>
>> >>>> On the genetics end of things:
>> >>>> - You'll be able to derive a constitutive promoter by inference
>> >from
>> >>>> commonly constitutive genes. For example, rRNA genes are
>generally
>> >>>> constitutive and have good promoters. If you can find any
>> >bacteriophage
>> >>>> for the species you want, you'll find some strong viral
>promoters;
>> >the
>> >>>> immediate-early promoters are probably powered by host
>> >transcription
>> >>>> factors.
>> >>>> - For shine-dalgarno sequences, you can likewise work from
>> >information
>> >>>> in the genome; firstly, by looking at likely high-expression
>> >candidate
>> >>>> genes and using their SG-sequences, or alternatively by just
>using
>> >the
>> >>>> 3' end of the rRNA as a template for your Shine-Dalgarno, as
>that's
>> >>>> ultimately what SG's are for; binding and initiating ribosomes
>> >using
>> >>>> rRNA binding.
>> >>>> - Linear transfer can deliver one copy of a gene to a pretty
>> >stable
>> >>>> location, if successful. I would then go the extra mile of
>removing
>> >the
>> >>>> antibiotic resistance genes you'd probably use to get the genes
>> >there in
>> >>>> the first place; the ideal "press release" includes assurances
>that
>> >no
>> >>>> resistance genes remain in the strain. In fact, for iGEM this
>would
>> >be a
>> >>>> significant factor towards the public-interest/engagement end of
>> >the
>> >>>> project.
>> >>>> - However, plasmids can get *lots* of copies into the cell, but
>> >you'd
>> >>>> need something to keep them there. Rather than standard
>antibiotic
>> >>>> resistance, why not consider including a nisin
>production/immunity
>> >>>> operon, so cells that have the correct DNA not only keep it, but
>> >kill
>> >>>> any mutants that lose it?* You'd have to make both yoghurt
>strains
>> >>>> immune to nisin for this to work, but nisin is food-safe; it's
>> >often
>> >>>> used as a preservative!
>> >>>> - Delivery to S.thermophilus is, as I discovered and detailed
>on
>> >my
>> >>>> blog, pretty easy if you fork out �20 or so on inducer
>peptide.
>> >That
>> >>>>
>> >>>> renders the strains naturally competent, but that's also why I
>> >suggested
>> >>>> linear DNA; competence systems generally grab one end of
>> >double-stranded
>> >>>> DNA and digest one strand in order to import the other; circular
>> >DNA is
>> >>>> therefore much less efficient at triggering transformation.
>> >>>> - Delivery to L.bulgaricus is probably achievable through
>> >>>> electroporation, but I imagine there's a chemical transformation
>> >method
>> >>>> too. Much of the time though, difficult-to-transform species
>> >require
>> >>>> protoplasting prior to chemical transformation, and that's not
>> >worth
>> >>>> trying if you're not experienced with the cultures already.
>> >>>>
>> >>>> * Consider this idea prior art if no assholes have patented
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