Student group needs help genetically modifying foods.

[Michael_McDonald]

We are a group of 9th and 10th grade students who are doing an independent project in regards to GMOs. We need to make contact with someone who could advise our group about genetically modifying foods. Some projects that we are interested in are making our own PCR machine, and our own centrifuge. We would also like any advise is regards to how to complete this lab. If you can help us, please either respond here or reply to OP email. 

[Mega [Andreas Stuermer]]

Genetically modifying food? What could possibly go wrong? I would love to help. Do you want to make it fluorescent? If so, I have the seqeunce of a heat stable gfp. And monellin...

[Mega [Andreas Stuermer]]

What you could also do: some vitamin biosynthesis pathways have been identified and reconstructed in yeast. You could make beer in which the yeast produces vitamin C and E for example.. I immagine bber would be ideal for C because it's anaerobic and doesn't get heated so vitC does not decay a lot

[Cathal Garvey]

Lack of oxygen is not the same thing as "not oxidising", so while I'd be
interested to know whether yeast were more "anti-oxidised" to begin
with, I wouldn't expect so.

With vitamins in general, the systems to make them can be assumed to be
large and unwieldy. If the systems were trivial, then most species would
just make their own.

Take a look at the biosynthetic pathway for B12 for an extreme example.
It's more like a biosynthetic odyssey.

On 05/02/14 21:03, Mega [Andreas Stuermer] wrote:
> What you could also do: some vitamin biosynthesis pathways have been identified and reconstructed in yeast. You could make beer in which the yeast produces vitamin C and E for example.. I immagine bber would be ideal for C because it's anaerobic and doesn't get heated so vitC does not decay a lot
>

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[Michael McDonald]

Yeah... what could possibly go wrong. Fluorescence... huh... had not thought of that. We will take that into consideration.  

[Michael McDonald]

This is a great idea! Thank you! Although we cannot make beer because we are not old enough to consume it, but we could try with another substance with VitC in it. I would assume that Orange Juice could be done. What are your thoughts?

[Michael McDonald]

Thank you, I will take that into consideration and begin research on the biosynthetic pathway for B12.

[Alex]

I was under the impression that the suggestion was to create a yeast that produces vitamin c & e instead of alcohol. I could be wrong though.

That should also make it possible for you guys to produce it without raising as many eyebrows.

Alex

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[Michael McDonald]

I spoke with my group about this idea, and we really like the idea of creating a yeast that produces vitamin c & e. Can you help us get started on that process? What would we need to do to begin? To give you a perspective of our restrictions, we will be conducting our experiment in our high school's lab. 

[leaking pen]

I would heartily recommend starting with the basics and working with the standards like e coli first to get your basic techniques down.  The chemicals and reagents you will use can cost as well, beyond the equipment.  I would A. partner with any fabrication/shop teacher you have to see about doubling up with another group on the actual building, and B. see if there is a local uni that would be able to give your group lab time and or direction! 

On Wed, Feb 5, 2014 at 8:35 AM, Michael_McDonald <mmcdon...@mountvernonschool.org> wrote:

We are a group of 9th and 10th grade students who are doing an independent project in regards to GMOs. We need to make contact with someone who could advise our group about genetically modifying foods. Some projects that we are interested in are making our own PCR machine, and our own centrifuge. We would also like any advise is regards to how to complete this lab. If you can help us, please either respond here or reply to OP email. 

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[Mega [Andreas Stuermer]]

First you have to find the metabolic pathway that leads to vitamin C formation. Make sure to find out where it starts from. This starting substance must already be present in yeast. 

Then gene1 turns starting substance into metabolite1. 

Gene2 turns metabolite1 into metabolite2. 

....

Gene6 finally turns metabolite5 into vitaminC. 

Then you have a list of all genes needed and you are sure it will work like this. Then you would have to synthesize the genes (in this hypothetical example 6 genes, each of which likely has 1200 base-pairs). Synthesis costs 0.30$ per base pair roughly.  Have them synthesized under control of a yeast promoter plus kozak sequence (copy plus paste the sequence from a commercial yeast plasmid :D ),  and terminator. If you have it synthesized right into a yeast integrative plasmid, all you have to do is put the plasmid into yeast and select for it. Very easy to design the sequence, no efforts to synthesize. You just have to pay for synthesis, and that may be ~2000-3000$.... 

[Alex]

This was a really great explanation! I wouldn't have known where to start either!

Thanks Andreas!

Alex

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[SC]

Hi Alex,

 

Was this project intended to take a week, a month, a year?  As your group is comprised of high school students with a high school lab, I'd like to see you all be able to complete the project in the allotted time frame.  Engineering a complete biochemical pathway (neat though it is), may be too advanced for a first time in genetic engineering.  The last thing any of us would want is for students to get discouraged because something didn't work and then you ran out of time. 

 

Stacy

[Alex]

Michael is the only one, that I'm aware of, doing this for school. I just have been watching and appreciated Andreas's explanation haha

Alex

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[Mega [Andreas Stuermer]]

Yeah, that method I learnt from the lux operon and from kanamycin biosynthesis (which interestingly starts with glucose, so you can be sure E coli has the precursor substance).

If they would want to spend 2000-3000 Dollar on synthesis, they could have the construct within few weeks.

If you have that much money to burn in one single project I would be happy to help you with the DNA design!

[Patrik D'haeseleer]

For something as simple as Vitamin C biosynthesis in yeast, chances are someone has already tried this. In this case, a simple google search led to this paper:

Biosynthesis of Vitamin C by Yeast Leads to Increased Stress Resistance

in which they show that you only need three genes to produce L-ascorbic acid (vitamin C) in yeast, although they eventually added two more to avoid a bottleneck in the tail end of the pathway.

Engineering five genes into yeast from scratch is still beyond what can be done with a highschool team (and Vitamin E looks even harder). Luckily for you, the 2011 Johns Hopkins iGEM team already made a plasmid with the three key enzymes, although I'm not sure they got it to work:

http://2011.igem.org/Team:Johns_Hopkins/Project/VitC

Instead of Vitamin C or E, you could focus on Vitamin A (aka beta-carotene) instead. The same Johns Hopkins team also made a 3-gene plasmid for Vitamin A biosynthesis, and demonstrated that it worked in yeast (and baked a bread with it!):

http://2011.igem.org/Team:Johns_Hopkins/Project/VitA
http://2011.igem.org/Team:Johns_Hopkins/Vit/Results

You (or one of your teachers) may be able to get the plasmid from this group. At that point, it would simply be a matter of transforming it into yeast, and testing it out - something that should be well within reach of a high school team. If you get that done, you can thenn modify the pathway further to produce any of a range of other brightly colored carotenoids, such as zeaxanthin or canthaxanthin.

Patrik

[Nathan McCorkle]

That's a great find Patrik! Really cool!

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[Cathal Garvey]

> Instead of Vitamin C or E, you could focus on Vitamin A (aka
beta-carotene)
> instead. The same Johns Hopkins team also made a 3-gene plasmid for
Vitamin
> A biosynthesis, and demonstrated that it worked in yeast (and baked a
bread
> with it!):

Boom: Much more important, considering the relative prevalence of Vit
A/C deficiencies. And, while everyone bleats about Golden Rice, "pirate
yeast" could be rapidly passed hand-to-hand by people in need to make
vitamin enriched breads/biscuits/beers.

Bonus feature; in either case, the vitamin is probably secreted or
otherwise not retained within the cell..? And "naked" vitamin C has a
very short half-life in our oxygen-rich environment. I think Vit A has a
better lifespan.

Downside: Vit A can be overdosed on, whereas Vit C effectively can't.

I'd put money towards a Vit.A yeast if anyone's planning to replicate
the Hopkins team work, assuming you can't just get the plasmid on
request! :)

On 11/02/14 00:39, Patrik D'haeseleer wrote:
> For something as simple as Vitamin C biosynthesis in yeast, chances are
> someone has already tried this. In this case, a simple google search led to
> this paper:
>

> Biosynthesis of Vitamin C by Yeast Leads to Increased Stress Resistance<http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0001092>

>
> in which they show that you only need three genes to produce L-ascorbic
> acid (vitamin C) in yeast, although they eventually added two more to avoid
> a bottleneck in the tail end of the pathway.
>
> Engineering five genes into yeast from scratch is still beyond what can be
> done with a highschool team (and Vitamin E looks even harder). Luckily for
> you, the 2011 Johns Hopkins iGEM team already made a plasmid with the three
> key enzymes, although I'm not sure they got it to work:
>
> http://2011.igem.org/Team:Johns_Hopkins/Project/VitC
>
> Instead of Vitamin C or E, you could focus on Vitamin A (aka beta-carotene)
> instead. The same Johns Hopkins team also made a 3-gene plasmid for Vitamin
> A biosynthesis, and demonstrated that it worked in yeast (and baked a bread
> with it!):
>
> http://2011.igem.org/Team:Johns_Hopkins/Project/VitA
> http://2011.igem.org/Team:Johns_Hopkins/Vit/Results
>
> You (or one of your teachers) may be able to get the plasmid from this
> group. At that point, it would simply be a matter of transforming it into
> yeast, and testing it out - something that should be well within reach of a
> high school team. If you get that done, you can thenn modify the pathway
> further to produce any of a range of other brightly colored carotenoids,
> such as zeaxanthin or canthaxanthin.
>
> Patrik
>

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[Michael McDonald]

Thank you for the information that you have given us. We will get back in touch with you once we look into our available options. 

[Michael McDonald]

Unfortunately, our deadline for this project is April 17th. Due to this time frame, what do you think would be our best course of action?

[Michael McDonald]

Thank you for mentioning this! Our group was wondering where we can start with this and you showed us exactly that. We are considering going with Vitamin A (beta-carotene) after we read up on the experiment John Hopkins and his team designed and executed. Do you know if this set up would work with Vitamin A? 

On Monday, February 10, 2014 4:14:27 PM UTC-5, Mega [Andreas Stuermer] wrote:

[Michael McDonald]

You say that this was done with E coli? How would the experiment have to be altered to fit Vitamin A (beta-carotene) if at all? 

[Michael McDonald]

Hmm... I read up on beta-carotene and the downsides that come from large doses of this only turns the skin a yellow-orange color which can be reversed. We are considering modifying the yeast so that it can be turned into many different products such as beer and breads. Thank you for this information.

[Mega [Andreas Stuermer]]

If you have the entire stuff synthesized, you could succeed until april. but downside, 2000 $

[Dakota Hamill]

http://2012.igem.org/Team:TU_Munich/Results/Biobricks

Check out Munich's page from 2012, they made yeast that spit out caffeine + other things alongside ethanol.

On Thu, Feb 20, 2014 at 1:45 PM, Mega [Andreas Stuermer] <masters...@gmail.com> wrote:

If you have the entire stuff synthesized, you could succeed until april. but downside, 2000 $

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[SC]

Hi Michael,

Since you have very little time, I would suggest working with yogurt.  A color, a glow, a smell.  Good smells you could use are banana and wintergreen, which have already been worked out. Engineering an entire pathway is too much for such a small time frame.

Best of luck, and please let us know how it goes.

 

[Patrik D'haeseleer]

On Thursday, February 20, 2014 5:17:53 PM UTC-8, SC wrote:

Hi Michael,

Since you have very little time, I would suggest working with yogurt.  A color, a glow, a smell.  Good smells you could use are banana and wintergreen, which have already been worked out. Engineering an entire pathway is too much for such a small time frame.

Disagree! You'd have to develop a construct for Lactobacillus from scratch. If this needs to be done in 2 months, then their best chance is to contact the Johns Hopkins team, and ask for a copy of their Vitamin A yeast vector. 

Patrik

[Michael_McDonald]

Hello, we have been gone a while and we would like to thank you for all the advice that you have given us this far. We have decided to move forward with Vitamin C in yeast to make bread that would contain more vitamin C and be more efficient than a standard tablet. In order to proceed with our first experiment, we need to know how much Vitamin C the GMO yeast can produce unit/mass, so if you can inform us of how to progress we will be very grateful. 

We are basing our research on a particular group that is modifying yeast. However if there is another group that you would recommend, we would be more than glad to hear about it. Link below.

Thank you,
Michael