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 it by
>now.
>>>> The same goes for any other antibiotics, bacteriocins or
>lantibiotics,
>>>> for that matter. The idea of using production and immunity to a
>killer
>>>> factor of any kind is, to my mind, "Obvious to those skilled in the
>>>> art", anyway.
>>>>
>>>> On 31/03/12 15:38, medminus9 wrote:
>>>> > Hello all!
>>>> >
>>>> > I am working as an instructor to a HS iGEM team and as a part of
>their
>>>> > project I am helping them design bioluminescent yogurt. I have
>been
>>>> through
>>>> > the previous thread and all the blog posts attached to the
>thread, but
>>>> > found that most of the ideas were lacking references and
>supportive
>>>> > literature. So, this is what i have proposed after going through
>the
>>>> > literature, your inputs and help will be much appreciated! :))
>>>> >
>>>> > - Acetaldehyde: Acetaldehyde can react with luciferase complex
>to
>>>> > produce bioluminescence. Since acetaldehyde is produced by
>>>> lactobacillus
>>>> > and streptococcus thermophilus in abundance, it will be an
>ideal
>>>> substrate
>>>> > for the luciferase to chew-on. It is also among the most
>abundant
>>>> chemicals
>>>> > on Earth and will help reducing the size of our construct from
>
>>>> luxCDABEG to
>>>> > luxABG.
>>>> >
>>>> > http://www.jbmb.or.kr/jbmb/**jbmb_files/[20-1]0204292121_**
>>>>
>02600541.pdf<http://www.jbmb.or.kr/jbmb/jbmb_files/%5B20-1%5D0204292121_02600541.pdf>
>
>>>> >
>>>> > - Acidophilus: I am planning to work with
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