Bioluminescent yogurt (Again!)

[medminus9]

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! :)

[Nathan McCorkle]

googling "electroporation acidophilus" gives lots of results, the first says electroporation yieled "7.4 × 10⁶ transformants per μg of plasmid DNA under optimized conditions"... so basically you should have no problems transforming small circular or linear DNA.

I'm also on the genomic integration is better" boat. Once its in there, as long as the genes are toxic or draining on the cell. constitutive promoters could 'drain'/strain the cell because of a constant amino acid demand... so maybe you want a low expression constitutive, or something thats only induced. Maybe lac promoter could work, so induction at least tapers off as incubation time increases.

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[elegans]

Problems:

 

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?

-The main difference here is going to be permanence. Plasmids must be selected for to be maintained. Typically, researchers return to a frozen stock of bacteria that they know contains their plasmid each time they want to use it. Your purpose requires many generations of bacteria, possibly over days or weeks, giving ample time for the bacteria to recombine or "spit out" the parts of the plasmid that aren't benefiting it, aka luciferase. The linear transfer is often referred to as "recombineering" when using recombinogenic viral proteins such as lambda Red E/T to enhance linear transfer effeciency, not sure if similar viruses have been studied in acidophilus. Check out Stewart or Murphy for the best recombineering literature.

I am searching for a constitutive promoter for the purpose and would really appreciate any help with this.

-Seems the bacteria will be in lactose, so the lac promoter may be preferred. 

Is there anything which I am missing or might have not looked over. Your suggestions please! :)

-dont forget positive / negative selections during cloning, they will make or break this procedure. I'm also not sure how much H202 will be floating around in the yogurt itself if that's where you're hoping the bioluminescence will happen.

[Nathan McCorkle]

On Sun, Apr 1, 2012 at 3:55 PM, elegans <tas...@gmail.com> wrote:
>The linear transfer is often referred to as "recombineering" when using recombinogenic viral proteins such as lambda Red E/T >to enhance linear transfer effeciency, not sure if similar viruses have been studied in acidophilus. Check out Stewart or >Murphy for the best recombineering literature.

He's not making a library or something like that, so will low
recombination efficiency even be a problem?

[elegans]

In my hands, recombination effeciency remains very low without Red proteins in E. coli. The main problem is going to be selecting against nonspecific transformants who have uptaken the linear DNA without inserting it into the genome in your preferred orientation. It is possible for the bacteria to use the DNA as a plasmid, which is why you'll need negative selection after transformation. If your electroporations are around 10^7 to 10^8 then you can expect 1-10 recombineered colonies in E. coli using red proteins to bring in a 3kb construct. Point mutations may work better, but it sounds like you'll be needing a larger integration. 

Also, I dont think this experiment would be able to get enough photons out of bioluminescent yogurt. When is it supposed to fluoresce?

-e

[Cathal Garvey]

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! :)
>
>

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

[medminus9]

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

[Cathal Garvey]

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

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[medminus9]

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

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[elegans]

"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"

Yes, exonuclease activity is required for recombination to occur. The viral beta protein will protect the single stranded fragment and promote insertion of the construct as an okazaki fragment. Here is a good recombineering guide from Stewart's lab.

http://www.biotec.tu-dresden.de/research/stewart/group-page/recombineering-guide.html

[elegans]

Exonuclease activity is required for recombineering to succeed. Exo will chew back one strand. Beta will protect the other strand. Gama will promote insertion of the linear single stranded fragment into the genome during lagging strand replication, mimicing an okazaki fragment. Here is a recombineering resource I found from Stewart's lab.

http://www.biotec.tu-dresden.de/research/stewart/group-page/recombineering-guide.html

[Nathan McCorkle]

On Tue, Apr 3, 2012 at 2:34 AM, elegans <tas...@gmail.com> wrote:
> "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"
>
> Yes, exonuclease activity is required for recombination to occur. The viral
> beta protein will protect the single stranded fragment and promote insertion
> of the construct as an okazaki fragment. Here is a good recombineering guide
> from Stewart's lab.

not sure why you keep mentioning the requirement of viruses... clearly
NEB is making $ and happy customers with their virus-free kit to
transform K. lactis with linear DNA via homologous recombination:
http://www.neb.com/nebecomm/products/producte1000.asp

>
> http://www.biotec.tu-dresden.de/research/stewart/group-page/recombineering-guide.html

>
>
>>
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[Cathal Garvey]

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 <harshse...@gmail.com> wrote:

>>>> > 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

--
Sent from K-9 Mail on Android

[Cathal Garvey]

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 <harshse...@gmail.com> wrote:

>>>> > 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

[medminus9]

Cathal,

I am a 4th year Medical student. :)

[Cathal Garvey]

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 <harshse...@gmail.com> wrote:

[elegans]

Hi again Nathan, 

Briefly: K. lactis is a yeast, not bacteria. Homologous recombination is easy in yeast, you can transform directly with PCR product and do not require viruses. As far as I recall, you can only hope to obtain pt mutations in bacteria using this procedure directly... feel free to try it in yogurt yeast though. Think about it. If you could just PCR amplify anything and put it into bacteria without having to worry about cloning plasmids, then why isn't everyone doing it?

Regards,

-e

On Tuesday, April 3, 2012 1:03:56 AM UTC-6, Nathan McCorkle wrote:

not sure why you keep mentioning the requirement of viruses... clearly
NEB is making $ and happy customers with their virus-free kit to
transform K. lactis with linear DNA via homologous recombination:
http://www.neb.com/nebecomm/products/producte1000.asp

Nathan McCorkle

[medminus9]

Hello Nathan and Elegans,

Thanks a lot for  your input. I would like to follow you guys on twitter, could you please leave your twitter id?

Regards,

Harsh

[Nathan McCorkle]

On Apr 3, 2012 3:45 PM, "elegans" <tas...@gmail.com> wrote:
>
> Hi again Nathan,
>
> Briefly: K. lactis is a yeast, not bacteria. Homologous recombination is easy in yeast, you can transform directly with PCR product and do not require viruses. As far as I recall, you can only hope to obtain pt mutations in

I haven't heard of that personally, can't see how it would only result
in point mutations either... seems like all or nothin

bacteria using this procedure directly... feel free to try it in
yogurt yeast though. Think about it. If you could just PCR amplify
anything and put it into bacteria without having to worry about
cloning plasmids, then why isn't everyone doing it?

Because you can't do a miniprep on genomic DNA during cloning operations.

Using the old googlulator, I found a paper transforming lactobacillus
acidophilus NCFM via homologous recombination using no viruses which
showed integration with as little as 169bp of homology on either end
of the insert, and stability over 35 generations. They used 500ng
plasmid DNA (from their cloning operations) 12.5kV/cm electroporation
and some special buffers... which could probably be done without/DIYed

http://www.scribd.com/doc/87924827/Appl-Environ-Microbiol-2011-Douglas-7365-71

[Nathan McCorkle]

On Tue, Apr 3, 2012 at 11:10 PM, medminus9 <harshse...@gmail.com> wrote:
> Hello Nathan and Elegans,
>
> Thanks a lot for  your input. I would like to follow you guys on twitter,
> could you please leave your twitter id?

I don't use twitter, sorry.

>
> Regards,
> Harsh

--

[medminus9]

On Wednesday, 4 April 2012 14:31:25 UTC+8, Nathan McCorkle wrote:

Using the old googlulator, I found a paper transforming lactobacillus
acidophilus NCFM via homologous recombination using no viruses which
showed integration with as little as 169bp of homology on either end
of the insert, and stability over 35 generations. They used 500ng
plasmid DNA (from their cloning operations) 12.5kV/cm electroporation
and some special buffers... which could probably be done without/DIYed

http://www.scribd.com/doc/87924827/Appl-Environ-Microbiol-2011-Douglas-7365-71

I am not sure, but doesn't the upp-counterselectable gene replacement system requires a plasmid? Or can we just insert a linear construct?

Thanks! 

[Nathan McCorkle]

On Wed, Apr 4, 2012 at 4:24 AM, medminus9 <harshse...@gmail.com> wrote:
> I am not sure, but doesn't the upp-counterselectable gene replacement system
> requires a plasmid? Or can we just insert a linear construct?

Can you rephrase the first sentence? Part of the construct flanked by
homologous regions can have a selection gene, certainly.

If you're talking about transformant selection, Cathal has had some good ideas.
"
By the way, I have in mind a way to do it that would result in an
antibiotic-resistance-free strain at the end: start by replacing the lac
operon using an antibiotic-selection cassette containing your operon,
selecting on glucose medium instead of lactose/milk medium.

Then perform another transformation to replace the antibiotic cassette,
plating on lactose/milk medium to select for transformants. Result;
chromosomal integration of your fluorescence/bioluminescence operon, and
removal of the antibiotic resistance gene. Because your DNA is on the
chromosome rather than a plasmid, it's unlikely to be lost provided
you've ensured there's little if any DNA repetition, and the
evolutionary cost of keeping it isn't punitive.
"

From (https://groups.google.com/d/msg/diybio/HFstp-wXJqE/ozUQqAxkokIJ)

--

[Petfixer71]

Im sure you have seen this:

http://www.indiebiotech.com/?p=164#more-164

 

If anyone knows were I can get the specific detailed lab protocol I would appreciate it.

[Nathan McCorkle]

I just sent a paper regarding the lab protocol for L. acidophipus, 2 messages ago.

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[Cathal Garvey]

Problem is, acidophilus isn't a primary yoghurt culture, AFAIK. It can be part of a yoghurt culture, but I suspect only as a minority share. The two main cultures in what's officially termed "Yoghurt" are S.salivarius thermophilus and L.delbrueckii bulgaricus.

The significance being that if your transformed culture isn't a significant player, it's also not gonna delivery enough light to notice. :/

Nathan McCorkle <nmz...@gmail.com> wrote:

>I just sent a paper regarding the lab protocol for L. acidophipus, 2
>messages ago.
>On Apr 5, 2012 8:31 AM, "Petfixer71" <afris...@hotmail.com> wrote:
>
>> Im sure you have seen this:
>>
>> http://www.indiebiotech.com/?p=164#more-164
>>
>> If anyone knows were I can get the specific detailed lab protocol I
>would
>> appreciate it.
>>
>> On Saturday, March 31, 2012 10:38:12 AM UTC-4, 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 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<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! :)
>>>
>>>
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[Nathan McCorkle]

On Thu, Apr 5, 2012 at 12:15 PM, Cathal Garvey <cathal...@gmail.com> wrote:
> Problem is, acidophilus isn't a primary yoghurt culture, AFAIK. It can be part of a yoghurt culture, but I suspect only as a minority share. The two main cultures in what's officially termed "Yoghurt" are S.salivarius thermophilus and L.delbrueckii bulgaricus.
>

Chromosomal Integration via Homologous recombination of L. delbrueckii:
http://www.google.com/patents/US5747310

Electrotransformation of L. delbrueckii w/plasmid:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC126594/?tool=pubmed

Chromosomal Integration via Homologous recombination of S.
thermophilus (old name?)
http://jb.asm.org/content/175/14/4315.short

This paper uses S. thermophilus, but is a bit hard to read
http://jb.asm.org/content/192/5/1444.short

But it quotes this paper "Efficient insertional mutagenesis in
lactococci and other gram-positive bacteria.":
http://www.ncbi.nlm.nih.gov/pubmed/8550537

And another just for kicks "Increasing competence in the genus Streptococcus"
http://www.micab.umn.edu/courses/8002/Havarstein.pdf

I think I have access to all these papers, if others don't let me know
and I'll post them online somewhere.

[Nathan McCorkle]

On Thu, Apr 5, 2012 at 3:47 PM, Nathan McCorkle <nmz...@gmail.com> wrote:
> Chromosomal Integration via Homologous recombination of L. delbrueckii:
> http://www.google.com/patents/US5747310
>
> Electrotransformation of L. delbrueckii w/plasmid:
> http://www.ncbi.nlm.nih.gov/pmc/articles/PMC126594/?tool=pubmed
>
> Chromosomal Integration via Homologous recombination of S.
> thermophilus (old name?)
> http://jb.asm.org/content/175/14/4315.short
>
> This paper uses S. thermophilus, but is a bit hard to read
> http://jb.asm.org/content/192/5/1444.short
>
> But it quotes this paper "Efficient insertional mutagenesis in
> lactococci and other gram-positive bacteria.":
> http://www.ncbi.nlm.nih.gov/pubmed/8550537
>
> And another just for kicks "Increasing competence in the genus Streptococcus"
> http://www.micab.umn.edu/courses/8002/Havarstein.pdf
>
>
> I think I have access to all these papers, if others don't let me know
> and I'll post them online somewhere.
>

So with this information of how to get the DNA in and stable... now it
seems like the luminescence or fluorescence system needs to be focused
on.... getting enough precursors so the light is bright enough. Can
heat shock proteins repress enzymes, or only gene expression? (i.e.
the light system is present but not functional, then when they're heat
shocked the enzymes go to work chewing up all the built-up reagent)

[Cory Geesaman]

I've been watching this thread for a bit but have a couple of safety questions:

Is bioluminescent yogurt a safe thing to eat? (It probably wouldn't be as bad as the radiation you get from cigarette tar, but it still introduces [albeit a different kind] radiation into an area of the body that is typically shielded from it)

How precisely are you able to tune the output wavelengths?  (I know UV would probably be a carcinogen so you would have to avoid any modifications resulting in it as an output, I'm not aware of studies with other parts of the spectrum though)

On Saturday, March 31, 2012 10:38:12 AM UTC-4, 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.

1. Research: The bacteria has been thoroughly studied.

1. Cost: My friend is making it available to me for free. He has been doing some research before.

1. 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?

1. I am searching for a constitutive promoter for the purpose and would really appreciate any help with this.

[Cathal Garvey]

In terms of safety:
 - Light output is definitely in the "Blue" spectrum, nowhere close to UV. The light isn't remotely dangerous.
 - If you hack S.thermophilus, it generally can't survive gut transit in healthy people. I don't know about L.delbruekii, but L.acidophilus *can*.

On the papers: Awesome collection Nathan, thanks for sharing!

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[medminus9]

@Nathan: By upp-counterselectable gene replacement system, I meant the method they used for gusA3 for the insertion. I looked into the references and understand that the method requires a plasmid.

@Cathal: Acidophilus can be used a a primary yogurt culture with thermophilus, much like bulgaricus. But it has a longer generation time, I believe (my results say so). Also, this is an article in which they used acidophilus with thermophilus as the primary culture http://www.ajcn.org/content/71/3/674.full

@Nathan: Thanks for sharing those links. Some of them were really useful. :)

@Cory: The wavelength naturally should be in the blue region. But with modifications they can be altered.

As for the toxicity, we will have to check the tetradecanal-aldehyde and look for its toxicity. But if luciferase metabolizes actealdehyde as well, which is present in normal yogurt, we could expect it to be less toxic. Conclusively, it shouldn't be toxic.

Cheers,

[Nathan McCorkle]

Harsh,

On Apr 6, 2012 9:32 AM, "medminus9" <harshse...@gmail.com> wrote:
>
> @Nathan: By upp-counterselectable gene replacement system, I meant the method they used for gusA3 for the insertion. I looked into the references and understand that the method requires a plasmid.
>

While they used a plasmid, it was merely a byproduct of their prior cloning procedures, linear DNA (E.G. synthetic DNA) would have worked just the same.

> Conclusively, it shouldn't be toxic.

I don't think we can say that so easily... People complaint that the Bt protein, a single protein, gives them weird problems

[medminus9]

> @Nathan: By upp-counterselectable gene replacement system, I meant the method they used for gusA3 for the insertion. I looked into the references and understand that the method requires a plasmid.
>

>While they used a plasmid, it was merely a byproduct of their prior cloning procedures, linear DNA (E.G. synthetic DNA) would have worked just the same.

But they are rarely any articles where they used a linear construct for transforming lactobacillus species. None, that I found! :(  

[Nathan McCorkle]

Right, that's because its still cheaper to clone than synthesize DNA
(USD $0.30/bp at least for long DNA)

[elegans]

It sounds like they do this in a specific deletion mutant to aid in their selection scheme.

[medminus9]

I a searching for literature on how to decide where to target you linear DNA insert. This is to understand where my  luciferase gene insert most likely work best and be stable in the bacterial protoplast. Any suggestion? :)

[Nathan McCorkle]

On Sun, Apr 8, 2012 at 1:40 AM, medminus9 <harshse...@gmail.com> wrote:
> I a searching for literature on how to decide where to target you linear DNA
> insert. This is to understand where my  luciferase gene insert most likely
> work best and be stable in the bacterial protoplast. Any suggestion? :)

I said this earlier in this thread, here it is copy-pasted:

Cathal has had some good ideas.
"
By the way, I have in mind a way to do it that would result in an
antibiotic-resistance-free strain at the end: start by replacing the lac
operon using an antibiotic-selection cassette containing your operon,
selecting on glucose medium instead of lactose/milk medium.

Then perform another transformation to replace the antibiotic cassette,
plating on lactose/milk medium to select for transformants. Result;
chromosomal integration of your fluorescence/bioluminescence operon, and
removal of the antibiotic resistance gene. Because your DNA is on the
chromosome rather than a plasmid, it's unlikely to be lost provided
you've ensured there's little if any DNA repetition, and the
evolutionary cost of keeping it isn't punitive.
"

--

[medminus9]

Cathal has had some good ideas.
"
By the way, I have in mind a way to do it that would result in an
antibiotic-resistance-free strain at the end: start by replacing the lac
operon using an antibiotic-selection cassette containing your operon,
selecting on glucose medium instead of lactose/milk medium.

Then perform another transformation to replace the antibiotic cassette,
plating on lactose/milk medium to select for transformants. Result;
chromosomal integration of your fluorescence/bioluminescence operon, and
removal of the antibiotic resistance gene. Because your DNA is on the
chromosome rather than a plasmid, it's unlikely to be lost provided
you've ensured there's little if any DNA repetition, and the
evolutionary cost of keeping it isn't punitive.

I have been thinking about the lac replacement and re-replacement, but I am searching for some articles or resources where there is some literature on how to know where your gene insert would work best.  I have this recent craving to understand that if nature had this gene and bacteria in cup of solution, where in the bacterial genome would it have inserted this gene, as that would be the most stable product. :)  

[Nathan McCorkle]

well around the lac operon is exactly the reason Cathal mentions using
this, because its required for growth in milk where lactose is the
predominant sugar... that's as good stability you can get I think

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[Nathan McCorkle]

Oh, I guess Cathal didn't actually say that for the second
transformation you would be replacing the antibiotic gene with the lac
operon/b-galactosidase.... so it would look like this

chromosomePartA----lacOperon----chromosomePartB

transform with 1kbUpstreamOfLacOperon-antibioticGene-lightSystem-1kbDownstreamOfLacOperon

to get

chromosomePartA----antibioticGene-lightSystem-----chromosomePartB

select with antibiotics

then transform the recent transformants with
1kbUpstreamOfLacOperon-lacOperon-lightSystem

to yield

chromosomePartA----lacOperon-lightSystem-----chromosomePartB

which can be selected for on lactose medium with no antibiotic (as any
antibiotic resistant non-2nd-transformants won't have lac to grow)

[medminus9]

Ok, I have some new questions (most of them probably because my background is medicine):

1. If i use/ target lac promoter, will it function anymore as a promoter to the lac-z, y, a and other genes. I understand that a promoter will promote the genes few (2-3kbs) below, therefore if I target my sequence for the lac promoter, my bacteria's might not grow on milk anymore.
2. Is it possible to have 2 lac promoters, simultaneously?  If not, then how can they have another lac promoter in the plasmid (is that OK?)
3.  I have been trying to look for constitutive, inducible promoters for Streptococcus in the literature, but there in very little to hunt (lacA, mob, com, blp and a few more, is all what I could find through my thorough search on NCBI, but all this is still unconvincing)  Am I looking in the wrong place.
   

Looking forward to your guidance and suggestions.

Thanks,

[Nathan McCorkle]

On Apr 16, 2012 7:53 AM, "medminus9" <harshse...@gmail.com> wrote:
>>
>> Ok, I have some new questions (most of them probably because my background is medicine):
>
> If i use/ target lac promoter, will it function anymore as a promoter to the lac-z, y, a and other genes. I understand that a promoter will promote the genes few (2-3kbs) below, therefore if I target my sequence for the lac promoter, my bacteria's might not grow on milk anymore.

In my last email I showed replacement of the lac operon with genes, then adding it back in.

You can definitely have multiple promoters that respond to the same signal, at different locations in DNA, controlling different genes.

If you follow my logic, and Cathals for that matter, you should understand the intermediary step you /do/ lose the ability to grow on milk, which is later restored.

> Is it possible to have 2 lac promoters, simultaneously?  If not, then how can they have another lac promoter in the plasmid (is that OK?)
>  I have been trying to look for constitutive, inducible promoters for Streptococcus in the literature, but there in very little to hunt (lacA, mob, com, blp and a few more, is all what I could find through my thorough search on NCBI, but all this is still unconvincing)  Am I looking in the wrong place.
> Looking forward to your guidance and suggestions.
>

Look into housekeeping genes, they're usually if not always on/expressed. You can usually just use 300-500bp upstream of those genes as a constitutive promoter.

[Harsh Sethia]

You can definitely have multiple promoters that respond to the same signal, at different locations in DNA, controlling different genes.

But will spacing affect them? Also, could you refer any article, using same promoters, simultaneously?

Look into housekeeping genes, they're usually if not always on/expressed. You can usually just use 300-500bp upstream of those genes as a constitutive promoter.

Did you mean 300-500bp upstream of the orf's?

Lastly, if I use a bacteriophage promoter of that particular species, will it be advantageous over lac for my casette? (earlier, you mentioned that it should not be energy draining - will that really affect a lot, any references?)

Thanks a lot, again! :)

[Nathan McCorkle]

On Apr 16, 2012 12:56 PM, "Harsh Sethia" <harshse...@gmail.com> wrote:
>>
>> You can definitely have multiple promoters that respond to the same signal, at different locations in DNA, controlling different genes.
>
> But will spacing affect them? Also, could you refer any article, using same promoters, simultaneously?

I can't think of any specific article, but the GFP beginner E.coli
kits often use the lac promoter, on a plasmid which is a completely
different piece of DNA than the chromosome. This is while retaining
the ability to use lactose thanks to the chromosomal lac operon.

>>
>> Look into housekeeping genes, they're usually if not always on/expressed. You can usually just use 300-500bp upstream of those genes as a constitutive promoter.
>
> Did you mean 300-500bp upstream of the orf's?
>

Yes, upstream of the start codon.

> Lastly, if I use a bacteriophage promoter of that particular species, will it be advantageous over lac for my casette? (earlier, you mentioned that it should not be energy draining - will that really affect a lot, any references?)

Ideally you want to control the level of transgenic protein produced,
if too much is formed inclusion bodies can form (vesicles with tons of
the excess protein contained within) and cell growth can be slowed
significantly.

Therefor I don't recommend constitutive promoters, because you really
don't need tons of protein, what you really need is tons of
luminescent precursor.

>
> Thanks a lot, again! :)
>

No prob, keep us update on what you come across!

[Harsh Sethia]

> But will spacing affect them? 

the GFP beginner E.coli kits often use the lac promoter, on a plasmid which is a completely different piece of DNA than the chromosome. This is while retaining the ability to use lactose thanks to the chromosomal lac operon.

Then again, those two lac promoters (plasmid and chromosome) will be at a significant distance. I remember you suggested to insert the gene casette close to the lac operon, as that would be among the most stable regions. But could 2 lac promoters at such little distance make the gene cassette unstable. (Is there any other way to look for a stable site based on GC/AT content, etc?)

 

No prob, keep us update on what you come across!

Sure! ( I will be more than happy to share my results with DIYbio community, as I strongly believe that this is the future of innovation) :) 

[medminus9]

Does anyone has access to these pay-walled articles? 

• Genetics of Streptococcus thermophilus: A Review
  
• Isolation and characterisation of promoter regions from Streptococcus thermophilus 
  
• Expression in bacteria other than Escherichia coli
  

Thanking in anticipation! :)

Regards,