Re: [DIYbio] Bioluminescent yogurt (Again!)

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
>
>    - Acidophilus: I am planning to work with acidophilus for the following
>    reasons:
>
>
>    1. Peroxide tolerance: Is more tolerant to H202 in compare to bulgaricus
>    and several other species. Since bioluminescence will require aerobic
>    conditions, the yogurt bacteria's will produce plenty of peroxide.
>    2. Research: The bacteria has been thoroughly studied.
>    3. Cost: My friend is making it available to me for free. He has been
>    doing some research before.
>    4. H202: Since acidophilus is H202 producing, the H202 can serve as an
>    oxidizing agent required to form the flavin-peroxy intermediate. (Not sure!)
>
> http://www.ncbi.nlm.nih.gov/pmc/articles/PMC84537/?tool=pmcentrez
>
>    - NAD(P)H2.FMN oxidoreductase: It is found in some species of
>    acidophilus. I can't find the literature for this, but I do happen to have
>    read it somewhere. Also most of the L.casei species do have this enzyme so
>    I believe we if required we could even add the particular enzyme to the
>    construct.
>
> *Problems*:
>
>    1. Cathal in his blog post is favoring a linear transfer, but most of
>    the literature is strongly against transformation with linear DNA. So, now
>    I am confused in choosing between a plasmid transfer with chemical/ natural
>    competence or a linear transfer?
>    2. I am searching for a* constitutive promoter* for the purpose and
>    would really appreciate any help with this.
>    3. Is there anything which I am missing or might have not looked over.
>    Your suggestions please! :)
>
>

--
www.indiebiotech.com
twitter.com/onetruecathal
joindiaspora.com/u/cathalgarvey
PGP Public Key: http://bit.ly/CathalGKey

--
You received this message because you are subscribed to the Google Groups "DIYbio" group.

To view this discussion on the web visit https://groups.google.com/d/msg/diybio/-/NRcs1V9qEvMJ.

To post to this group, send email to diybio@googlegroups.com.
To unsubscribe from this group, send email to diybio+unsubscribe@googlegroups.com.
For more options, visit this group at http://groups.google.com/group/diybio?hl=en.

--
www.indiebiotech.com
twitter.com/onetruecathal
joindiaspora.com/u/cathalgarvey
PGP Public Key: http://bit.ly/CathalGKey