Yuriy's fundraiser on experiment.com
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John Griessen
Jul 19, 2014, 9:30:48 AM7/19/14
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On 07/19/2014 12:37 AM, Yuriy Fazylov wrote:
> I am running a fundraiser, on experiment.com. Not doing a good job of it I am afraid. Someone told me lack in detail is doing
> me in. Plus a video would help. You and anyone interested is welcome to poke holes in it. For example gene expression stability
> always seems to be on your mind. I haven't thought of it yet.
OK. here's a new subject header for it. Please tell us the name of it, and a link.
> I am running a fundraiser, on experiment.com. Not doing a good job of it I am afraid. Someone told me lack in detail is doing
> me in. Plus a video would help. You and anyone interested is welcome to poke holes in it. For example gene expression stability
> always seems to be on your mind. I haven't thought of it yet.
OK. here's a new subject header for it. Please tell us the name of it, and a link.
Yuriy Fazylov
Jul 19, 2014, 9:57:45 AM7/19/14
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Will Sutton
Jul 19, 2014, 6:49:13 PM7/19/14
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So, I really like this concept as a DIYbio-geek, but I don't think that's who the majority of Experiment.com donors are. I think it's more "the discovery channel" folks.
My advice: Try to think how this would be written up in Scientific American or other pop-sci publications...maybe a picture/story about an astronaut farming?
Message has been deleted
Yuriy Fazylov
Jul 19, 2014, 9:22:57 PM7/19/14
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Thanks for the input and contribution.
Nothing I am working on right now is picture worthy. I am doing most work in literature and in silico. I will try to do the things you mentioned.
Josiah Zayner
Jul 22, 2014, 12:42:10 PM7/22/14
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Hey Yuriy,
Usually people use the term "IR" to refer to InfraRed radiation. I don't think I have ever heard it referred to as Ionizing Radiation. You might want to clear that up. Also, what type of ionizing radiation due you plan to use? UV? How due you plan to generate the low wavelength UV? If alpha, betta, gamma, how do you plan to generate these wavelengths? So what is your goal? Why do we need plants that are resistant to ionizing radiation?
I think you should clear these things up. Being as specific as possible really helps people understand what you are doing.
Also, putting alot of genes in plants is no easy task. Do you have experience with this? If so, mention that.
Yuriy Fazylov
Jul 22, 2014, 5:25:45 PM7/22/14
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>Usually people use the term "IR" to refer to InfraRed radiation. You might want to clear that up.
I am aware of its usual usage and I did clear that up but papers referencing ionizing radiation often times abbreviate it as such, IR. What drove them to usage of IR (Ionizing Radiation) instead of the commonly known IR (InfraRed) is beyond my understanding. One can only suppose how that happened. Perhaps someone once submitted a paper mentioning “Ionizing Radiation” repetitively and the editor of the journal wrote back condense it. Or maybe someone decided to be a tReNd SeTtEr ;)
Plus there is a limit on how many characters can be used in a section (usually 2K/section) on that website.
>I don't think I have ever heard it referred to as Ionizing Radiation.
I didn’t either, until I started looking reading studies/literature on ionizing radiation.
>Also, what type of ionizing radiation due you plan to use? UV? How due you plan to generate the low wavelength UV?
I tracked down a UV-B sensitive response enhancer/promoter system, takes 24 hours to plateau the expression of its protein – 8 hours for its first signs. That means I’ll have to grow it, expose it to UV-B, and wait 8-24 hours for it to be tested.
I have yet to look into choline synthase promoters.
I would like to engineer a promoter with promoterCAD with faster response time. I just need to get back to the article on how it runs and see if light response elements in the promoter can just be thrown together.
Plus if I throw in an untested response element into synthesis construct, well let’s just say it’s not very scientific. I wonder if Project Cyborg is trained to test for something so obscure.
I have yet to look into choline synthase promoters.
I would like to engineer a promoter with promoterCAD with faster response time. I just need to get back to the article on how it runs and see if light response elements in the promoter can just be thrown together.
Plus if I throw in an untested response element into synthesis construct, well let’s just say it’s not very scientific. I wonder if Project Cyborg is trained to test for something so obscure.
>If alpha, betta, gamma, how do you plan to generate these wavelengths?
From my understanding the fungi can resist up to Gamma but plant allomelanin is not as well characterized as those of the fungus.
Since you are with NASA, you tell me. Where should I solicit help for radiation exposure if the project is gets off the ground? What lab?
Since you are with NASA, you tell me. Where should I solicit help for radiation exposure if the project is gets off the ground? What lab?
>So what is your goal? Why do we need plants that are resistant to ionizing radiation? I think you should clear these things up. Being as specific as possible really helps people understand what you are doing.
Realizing the amount of real estate in space Freeman Dyson once suggested a plant that will live in space and house people. Can you imagine growing space stations? Plants have a lot of properties that can be adjusted but I have never seen dramatic engineered morphogenesis. The closest was removing knots to have finer wood grain. This goal is very conservative compared to space dwelling trees, but I did have a couple of years to think what such biology would require to accommodate itself. My idea was a little more grounded. After writing some things down into what I hoped would be a book I had some computer crashes. That’s when I decided not to try to inspire people, that what the concept is for, but to try to do it. I am sure there will be something to be gained from it. Environmental cleanup seems like a valid alternative.
>Also, putting a lot of genes in plants is no easy task. Do you have experience with this?
I wouldn’t dream of putting full 20kb synthesis product into any single plant. Although I think the length of a typical Ti vector within biotech use wasn’t narrowed down. I wouldn’t know what to account for each gene if I did put it all in one vector. Is that what my plan seems like? I will work on the details tonight. Genes and construction on it will have restriction areas and primers in mind. Only the synthesis has to be done at 20kb.
I read and keep reading the literature on these topics. Floral dipping is not as difficult as I first thought but the success rate is off-putting (1%-0.1%). That is unless someone found a better way to do it. It’s cheap and fairly easy. You can grow a whole lot on an antibiotic media, or spray seedlings while it’s in soil.
>Also, putting a lot of genes in plants is no easy task. Do you have experience with this?
I wouldn’t dream of putting full 20kb synthesis product into any single plant. Although I think the length of a typical Ti vector within biotech use wasn’t narrowed down. I wouldn’t know what to account for each gene if I did put it all in one vector. Is that what my plan seems like? I will work on the details tonight. Genes and construction on it will have restriction areas and primers in mind. Only the synthesis has to be done at 20kb.
I read and keep reading the literature on these topics. Floral dipping is not as difficult as I first thought but the success rate is off-putting (1%-0.1%). That is unless someone found a better way to do it. It’s cheap and fairly easy. You can grow a whole lot on an antibiotic media, or spray seedlings while it’s in soil.
After that its all Mendelian genetics.
Simon Quellen Field
Jul 22, 2014, 5:45:10 PM7/22/14
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On Tue, Jul 22, 2014 at 2:25 PM, Yuriy Fazylov <yuriy...@gmail.com> wrote:
>If alpha, betta, gamma, how do you plan to generate these wavelengths?
Alpha is really easy. A $5 smoke detector has an Americium 242 alpha source in it that make my Geiger counter very happy.
License-free radiation sources are readily available, although a little bit more expensive.
Simon Quellen Field
Jul 22, 2014, 5:46:29 PM7/22/14
to diybio
I meant Americium 241.
:-)
Sebastian Cocioba
Jul 22, 2014, 6:41:40 PM7/22/14
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The Ti vector for transforming plants via agro (pPZP, pGreen, pClean, etc) is about 10k in length. The Ti region that agro cuts and packs hasn't really been put through the ringer in terms of seeing how much it can incorporate. I've worked with 10kbp+ and it did fine. Transformation via agro is simple. Personally I hate Arabi floral dip.
I just grow agro with plasmid to OD600 = 0.5, incubate leaf disks of month old plantlets (1cm dia.) in 15mL of liquid culture, then dab on sterile cotton pad or gauze, and plate on MS BAP NAA and Agar pH 5.8 (tobacco) for 48hrs or until decent bacterial halo forms around explant. Then transfer to MS BAP NAA Agar and selection herbicide (I like kanamycin 50-100mg/L) and let nature do the rest.
Replate every two weeks till shoots form, transfer shoots to rooting media (MS NAA Agar Kana) and wait till roots form. If strong roots form and dig into media then u have transformants (verify by pcr to be sure). Transfer to soil and slowly lower humidity. Cool white lights all the way through. 100 microeinsteins or more. 16hr days. Works for me every time.
Have done well over 700 transformations and less than 0.1% were false positives. Results may vary based on experience but tobacco is easy and not as sensitive to environs as Arabidopsis. There is no rational reason why I hate floral dip. Arabi seeds are terrible to handle, Silwet is expensive, and overall a boring experience.
Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D
I just grow agro with plasmid to OD600 = 0.5, incubate leaf disks of month old plantlets (1cm dia.) in 15mL of liquid culture, then dab on sterile cotton pad or gauze, and plate on MS BAP NAA and Agar pH 5.8 (tobacco) for 48hrs or until decent bacterial halo forms around explant. Then transfer to MS BAP NAA Agar and selection herbicide (I like kanamycin 50-100mg/L) and let nature do the rest.
Replate every two weeks till shoots form, transfer shoots to rooting media (MS NAA Agar Kana) and wait till roots form. If strong roots form and dig into media then u have transformants (verify by pcr to be sure). Transfer to soil and slowly lower humidity. Cool white lights all the way through. 100 microeinsteins or more. 16hr days. Works for me every time.
Have done well over 700 transformations and less than 0.1% were false positives. Results may vary based on experience but tobacco is easy and not as sensitive to environs as Arabidopsis. There is no rational reason why I hate floral dip. Arabi seeds are terrible to handle, Silwet is expensive, and overall a boring experience.
Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D
From: Yuriy Fazylov
Sent: 7/22/2014 5:25 PM
To: diy...@googlegroups.com
Subject: [DIYbio] Re: Yuriy's fundraiser on experiment.com
>Usually people use the term "IR" to refer to InfraRed radiation. You might want to clear that up.
I am aware of its usual usage and I did clear that up but papers referencing ionizing radiation often times abbreviate it as such, IR. What drove them to usage of IR (Ionizing Radiation) instead of the commonly known IR (InfraRed) is beyond my understanding. One can only suppose how that happened. Perhaps someone once submitted a paper mentioning “Ionizing Radiation” repetitively and the editor of the journal wrote back condense it. Or maybe someone decided to be a tReNd SeTtEr ;)
Plus there is a limit on how many characters can be used in a section (usually 2K/section) on that website.
>I don't think I have ever heard it referred to as Ionizing Radiation.
I didn’t either, until I started looking reading studies/literature on ionizing radiation.
>Also, what type of ionizing radiation due you plan to use? UV? How due you plan to generate the low wavelength UV?
I tracked down a UV-B sensitive response enhancer/promoter system, takes 24 hours to plateau the expression of its protein – 8 hours for its first signs. That means I’ll have to grow it, expose it to UV-B, and wait 8-24 hours for it to be tested.
I have yet to look into choline synthase promoters.
I would like to engineer a promoter with promoterCAD with faster response time. I just need to get back to the article on how it runs and see if light response elements in the promoter can just be thrown together.
Plus if I throw in an untested response element into synthesis construct, well let’s just say it’s not very scientific. I wonder if Project Cyborg is trained to test for something so obscure.
>If alpha, betta, gamma, how do you plan to generate these wavelengths?
From my understanding the fungi can resist up to Gamma but plant allomelanin is not as well characterized as those of the fungus.
Since you are with NASA, you tell me. Where should I solicit help for radiation exposure if the project is gets off the ground? What lab?
>So what is your goal? Why do we need plants that are resistant to ionizing radiation? I think you should clear these things up. Being as specific as possible really helps people understand what you are doing.
Realizing the amount of real estate in space Freeman Dyson once suggested a plant that will live in space and house people. Can you imagine growing space stations? Plants have a lot of properties that can be adjusted but I have never seen dramatic engineered morphogenesis. The closest was removing knots to have finer wood grain. This goal is very conservative compared to space dwelling trees, but I did have a couple of years to think what such biology would require to accommodate itself. My idea was a little more grounded. After writing some things down into what I hoped would be a book I had some computer crashes. That’s when I decided not to try to inspire people, that what the concept is for, but to try to do it. I am sure there will be something to be gained from it. Environmental cleanup seems like a valid alternative.
>Also, putting a lot of genes in plants is no easy task. Do you have experience with this?
I wouldn’t dream of putting full 20kb synthesis product into any single plant. Although I think the length of a typical Ti vector within biotech use wasn’t narrowed down. I wouldn’t know what to account for each gene if I did put it all in one vector. Is that what my plan seems like? I will work on the details tonight. Genes and construction on it will have restriction areas and primers in mind. Only the synthesis has to be done at 20kb.
I read and keep reading the literature on these topics. Floral dipping is not as difficult as I first thought but the success rate is off-putting (1%-0.1%). That is unless someone found a better way to do it. It’s cheap and fairly easy. You can grow a whole lot on an antibiotic media, or spray seedlings while it’s in soil.
On Tuesday, July 22, 2014 12:42:10 PM UTC-4, Josiah Zayner wrote:
Since you are with NASA, you tell me. Where should I solicit help for radiation exposure if the project is gets off the ground? What lab?
>So what is your goal? Why do we need plants that are resistant to ionizing radiation? I think you should clear these things up. Being as specific as possible really helps people understand what you are doing.
Realizing the amount of real estate in space Freeman Dyson once suggested a plant that will live in space and house people. Can you imagine growing space stations? Plants have a lot of properties that can be adjusted but I have never seen dramatic engineered morphogenesis. The closest was removing knots to have finer wood grain. This goal is very conservative compared to space dwelling trees, but I did have a couple of years to think what such biology would require to accommodate itself. My idea was a little more grounded. After writing some things down into what I hoped would be a book I had some computer crashes. That’s when I decided not to try to inspire people, that what the concept is for, but to try to do it. I am sure there will be something to be gained from it. Environmental cleanup seems like a valid alternative.
>Also, putting a lot of genes in plants is no easy task. Do you have experience with this?
I wouldn’t dream of putting full 20kb synthesis product into any single plant. Although I think the length of a typical Ti vector within biotech use wasn’t narrowed down. I wouldn’t know what to account for each gene if I did put it all in one vector. Is that what my plan seems like? I will work on the details tonight. Genes and construction on it will have restriction areas and primers in mind. Only the synthesis has to be done at 20kb.
I read and keep reading the literature on these topics. Floral dipping is not as difficult as I first thought but the success rate is off-putting (1%-0.1%). That is unless someone found a better way to do it. It’s cheap and fairly easy. You can grow a whole lot on an antibiotic media, or spray seedlings while it’s in soil.
After that its all Mendelian genetics.
On Tuesday, July 22, 2014 12:42:10 PM UTC-4, Josiah Zayner wrote:
Hey Yuriy,Usually people use the term "IR" to refer to InfraRed radiation. I don't think I have ever heard it referred to as Ionizing Radiation. You might want to clear that up. Also, what type of ionizing radiation due you plan to use? UV? How due you plan to generate the low wavelength UV? If alpha, betta, gamma, how do you plan to generate these wavelengths? So what is your goal? Why do we need plants that are resistant to ionizing radiation?
I think you should clear these things up. Being as specific as possible really helps people understand what you are doing.
Also, putting alot of genes in plants is no easy task. Do you have experience with this? If so, mention that.
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Josiah Zayner
Jul 22, 2014, 7:00:09 PM7/22/14
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Yuriy,
I would add more of what you wrote in the email to the website. It makes your project much more appealing. On Tue, Jul 22, 2014 at 2:25 PM, Yuriy Fazylov <yuriy...@gmail.com> wrote:
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Will Sutton
Jul 23, 2014, 12:06:08 PM7/23/14
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Here's some write-ups an iGem team did on the subject: http://2011.igem.org/Team:Brown-Stanford/PowerCell/Background
And the astronaut farming of course: http://2011.igem.org/Team:Brown-Stanford/PowerCell/Introduction
On Saturday, July 19, 2014 9:30:48 AM UTC-4, John Griessen wrote:
Yuriy Fazylov
Jul 23, 2014, 1:02:20 PM7/23/14
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Yeah, one can only hope to be guided by Lynn Rothschild, NASA's SynBio leader.
Josiah Zayner
Jul 23, 2014, 1:26:56 PM7/23/14
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I think Lynn is considered NASA's SynBio Evangelist _not_ Leader. She has never published any peer-reviewed Science in synthetic biology, protein engineering or the likes.
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Yuriy Fazylov
Jul 23, 2014, 3:08:47 PM7/23/14
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I found this YouTube channel to be helpful for Arabidopsis.
https://www.youtube.com/channel/UCQgD6eojgUpAe4x3vmO4hOw
You are right. It is tedious to work with Arabidopsis. I could bypass Arabidopsis even though I have a ton of seeds of Col-0.
I’ll add tobacco to my dicot list.
Back in 2013 a teammate suggested to dissolve the cell walls and pour in agro with vector in it, followed by dousing everything with the antibiotic after an incubation period. The vector would carry the antibiotic. The protoplasts would yield a lot more transformants from non-transformand background. I was leaning to that until I found out about Agroinfiltration.
https://www.youtube.com/channel/UCQgD6eojgUpAe4x3vmO4hOw
You are right. It is tedious to work with Arabidopsis. I could bypass Arabidopsis even though I have a ton of seeds of Col-0.
I’ll add tobacco to my dicot list.
Back in 2013 a teammate suggested to dissolve the cell walls and pour in agro with vector in it, followed by dousing everything with the antibiotic after an incubation period. The vector would carry the antibiotic. The protoplasts would yield a lot more transformants from non-transformand background. I was leaning to that until I found out about Agroinfiltration.
I had no idea Agroinfiltration was that easy. I found some more detail on JoVe.
https://www.youtube.com/watch?v=GHc7PU_jG2M&list
http://www.jove.com/video/50971/agroinfiltration-pvx-agroinfection-potato-nicotiana
Here is a brief plant disc method Sebastian mentioned in case anyone is curious.
https://www.youtube.com/watch?v=I3fCD0uUJk0 Thank you for your advice.
https://www.youtube.com/watch?v=GHc7PU_jG2M&list
http://www.jove.com/video/50971/agroinfiltration-pvx-agroinfection-potato-nicotiana
Here is a brief plant disc method Sebastian mentioned in case anyone is curious.
https://www.youtube.com/watch?v=I3fCD0uUJk0 Thank you for your advice.
Yuriy Fazylov
Jul 23, 2014, 3:10:34 PM7/23/14
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Thank you John Griessen
On Saturday, July 19, 2014 9:30:48 AM UTC-4, John Griessen wrote:
On Saturday, July 19, 2014 9:30:48 AM UTC-4, John Griessen wrote:
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