GFP to Create Graft

[sawdonkey]

So bare with me...this is out of my league. Last night I was reading about the rainbow splice acro and how it is just a Millie with naturally occurring GFP (green fluorescent protein). And I thought, wait a second...I’m pretty sure my wife works with GFP every day.

My wife is a microbiologist / virologist and she studies herpes viruses for a living. The tag certain proteins with GFP so they can see how the protein behaves. They put it under a specific UV light and the GFP tag allows them to see what’s going on. Her GFP comes from jellyfish, not coral, but I think it’s the same thing?

Anyway, I asked her if there is a way to “tag” one of my millies with the GFP. She said that the tag their viruses biologically and not physically, so she wouldn’t know where to start.

So, is there anyone here that is educated enough to weigh in on the possibility of creating a graft artificially in this way? I’d love to try it.

I know this might not be feasible at all, but thought it would be a good discussion.

 

[Dana Riddle]

So bare with me...this is out of my league. Last night I was reading about the rainbow splice acro and how it is just a Millie with naturally occurring GFP (green fluorescent protein). And I thought, wait a second...I’m pretty sure my wife works with GFP every day.

My wife is a microbiologist / virologist and she studies herpes viruses for a living. The tag certain proteins with GFP so they can see how the protein behaves. They put it under a specific UV light and the GFP tag allows them to see what’s going on. Her GFP comes from jellyfish, not coral, but I think it’s the same thing?

Anyway, I asked her if there is a way to “tag” one of my millies with the GFP. She said that the tag their viruses biologically and not physically, so she wouldn’t know where to start.

So, is there anyone here that is educated enough to weigh in on the possibility of creating a graft artificially in this way? I’d love to try it.

I know this might not be feasible at all, but thought it would be a good discussion.

 

[Dana Riddle]

So bare with me...this is out of my league. Last night I was reading about the rainbow splice acro and how it is just a Millie with naturally occurring GFP (green fluorescent protein). And I thought, wait a second...I’m pretty sure my wife works with GFP every day.

My wife is a microbiologist / virologist and she studies herpes viruses for a living. The tag certain proteins with GFP so they can see how the protein behaves. They put it under a specific UV light and the GFP tag allows them to see what’s going on. Her GFP comes from jellyfish, not coral, but I think it’s the same thing?

Anyway, I asked her if there is a way to “tag” one of my millies with the GFP. She said that the tag their viruses biologically and not physically, so she wouldn’t know where to start.

So, is there anyone here that is educated enough to weigh in on the possibility of creating a graft artificially in this way? I’d love to try it.
The GFP from the jellyfish Aequoria victoria is only distantly related to the GFPs found in corals (see attachment). The jellyfish GFP requires calcium as a co-factor to fluoresce. There are 4 GFP-type clades found in corals and anemones with the possibility of a 5th.
I don't have any experience with the Aequoria protein, but if it is like coral GFPs, it is water-soluble (in distilled water.) It might be possible to simply inject the jelly GFP into a coral polyp with a hypodermic needle.

 

[sawdonkey]

Thanks. I will surely be pressuring my wife into helping me try this.

 

[Dana Riddle]

Thanks. I will surely be pressuring my wife into helping me try this.

Sorry for the attachment's poor quality - it's a PowerPoint slide from one of my presentations. The jellyfish protein is at the very bottom, and is not closely related to any of the Clade D coral GFPs.
I'd be very interested in hearing how this goes.

 

[sawdonkey]

I have low hopes, but nothing to lose except a few frags and some GFP. The first hurdle will be getting my wife to play along. I’ll see if she has the other versions too. I thing they do red tagging as well.

 

[User]

I have low hopes, but nothing to lose except a few frags and some GFP. The first hurdle will be getting my wife to play along. I’ll see if she has the other versions too. I thing they do red tagging as well.

Speaking as a 22 year career microbiologist and virologist that worked with tagging mammalian cells with GFP and variants- i wish it was that easy!!

The steps are fairly simple. the devil is in the details

1) build a virus that contains a transcriptional genetic element that encodes for Green Fluorescent Protein. you cant just add GFP protein to a frag- nothing will happen.

2) use the virus to infect and deliver the gene to your frag

3) the GFP gene then needs to be transcribed and translated into protein by the coral cell. GFP + UV light (~450nm is ideal for synthetic GFP used in the industry)

4) profit!!!!


The details-

1) you need a virus that can infect coral cells. That virus also needs to be very easily manipulable in a lab, and have a genetic structure that is mapped so that you can incorporate the GFP genetic elements. TIMESCALE- 3-10 years. Difficulty: 6-8. good for a couple grad students and a postdoc or two.

2) You need to understand the codon usage system of the coral cells, so that you can optimize your GFP gene so that it makes A LOT of it, and that it can translate it into a functional protein. Timescale 1-2 years, difficulty 4. Repetitive process work. good for a grad student

3) Viral transduction dosing, expression testing, toxicity testing and genetic stability testing. Difficulty ranges from 5-8, timescales on the order of 3-8 years.

4) since you dont have a surrogate cell system with which to test any of this, youll need to do things 'by the book' for several rounds (so, 12-15 years or run several concurrently), until you start seeing results, then make educated guesses about next steps. difficulty-7-9, timescale- ongoing. good use of a full professor or research director (im on the market if you are game!).

as a real word example- we have a suite of 6-9 viruses that can deliver codon optimized therapeutic genes to human cells in a dish (60% delivery with 50% product production) and in a patient (70% delivery to liver cells, 80% production, liver cirrhosis and failure in 2% of patients). Its taken us 40 years to get there, clinical trials still fail due to toxicity, and its cost around 125 BILLION dollars.


but if you want to pay me to set up a shipping container lab in my backyard and buy me a tissue culture suite, ill happily play along. Oh, and i get to keep IP and patents rights while you assume all financial risk. All for the bargain price of $125k/year, 10 year contract paid upfront, results not guaranteed.


seriously though- its a fun thought, and it would be amazing to have some sticks glow green, red and orange under actinics, just like hammers and softies, but its just not feasible im afraid.

Now- when do you want me to start?

 

[User]

I have low hopes, but nothing to lose except a few frags and some GFP. The first hurdle will be getting my wife to play along. I’ll see if she has the other versions too. I thing they do red tagging as well.

on a serious note- if your wife gets caught taking genetically modified herpes virus out of the lab, she'll be liable for federal prosecution, possibly under bioterrorism statutes. Im not kidding. I lost a very promising med student researcher because he took home lab supplies, including GFP and RFP producing e.coli bacteria, in order to grow 'bacterial art posters' at home. you can literally paint bacteria into images on agar plates and have them fluoresce. he posted the results on Insta and got caught. He got kicked out of 3rd year stanford medical school because of it.

image from wiki, but ive worked with members of this lab.

 

[sawdonkey]

Speaking as a 22 year career microbiologist and virologist that worked with tagging mammalian cells with GFP and variants- i wish it was that easy!!

The steps are fairly simple. the devil is in the details

1) build a virus that contains a transcriptional genetic element that encodes for Green Fluorescent Protein. you cant just add GFP protein to a frag- nothing will happen.

2) use the virus to infect and deliver the gene to your frag

3) the GFP gene then needs to be transcribed and translated into protein by the coral cell. GFP + UV light (~450nm is ideal for synthetic GFP used in the industry)

4) profit!!!!


The details-

1) you need a virus that can infect coral cells. That virus also needs to be very easily manipulable in a lab, and have a genetic structure that is mapped so that you can incorporate the GFP genetic elements. TIMESCALE- 3-10 years. Difficulty: 6-8. good for a couple grad students and a postdoc or two.

2) You need to understand the codon usage system of the coral cells, so that you can optimize your GFP gene so that it makes A LOT of it, and that it can translate it into a functional protein. Timescale 1-2 years, difficulty 4. Repetitive process work. good for a grad student

3) Viral transduction dosing, expression testing, toxicity testing and genetic stability testing. Difficulty ranges from 5-8, timescales on the order of 3-8 years.

4) since you dont have a surrogate cell system with which to test any of this, youll need to do things 'by the book' for several rounds (so, 12-15 years or run several concurrently), until you start seeing results, then make educated guesses about next steps. difficulty-7-9, timescale- ongoing. good use of a full professor or research director (im on the market if you are game!).

as a real word example- we have a suite of 6-9 viruses that can deliver codon optimized therapeutic genes to human cells in a dish (60% delivery with 50% product production) and in a patient (70% delivery to liver cells, 80% production, liver cirrhosis and failure in 2% of patients). Its taken us 40 years to get there, clinical trials still fail due to toxicity, and its cost around 125 BILLION dollars.


but if you want to pay me to set up a shipping container lab in my backyard and buy me a tissue culture suite, ill happily play along. Oh, and i get to keep IP and patents rights while you assume all financial risk. All for the bargain price of $125k/year, 10 year contract paid upfront, results not guaranteed.


seriously though- its a fun thought, and it would be amazing to have some sticks glow green, red and orange under actinics, just like hammers and softies, but its just not feasible im afraid.

Now- when do you want me to start?

This is exactly why I posted here! It was a fun thought and and I’m glad you responded and brought me down to earth. In fact, are you my wife? This is very close to what she told me, but I didn’t want to believe her. I guess I’ll just suck some juice out of a highlighter and squirt it on a coral. That should do.

 

[Dana Riddle]

This is exactly why I posted here! It was a fun thought and and I’m glad you responded and brought me down to earth. In fact, are you my wife? This is very close to what she told me, but I didn’t want to believe her. I guess I’ll just suck some juice out of a highlighter and squirt it on a coral. That should do.

Why not graft differently colored milles?

 

[User]

This is exactly why I posted here! It was a fun thought and and I’m glad you responded and brought me down to earth. In fact, are you my wife? This is very close to what she told me, but I didn’t want to believe her. I guess I’ll just suck some juice out of a highlighter and squirt it on a coral. That should do.

Didn't you see where i offered to do the work for you? Bargain price too. I just assume you'd say yes so I already ordered the shipping container. Let me know where to send the invoice.

 

[User]

Why not graft differently colored milles?

Because he wants super bright glow in the dark custom GMO milliezzzzz!!!

 

[User]

This is exactly why I posted here! It was a fun thought and and I’m glad you responded and brought me down to earth. In fact, are you my wife? This is very close to what she told me, but I didn’t want to believe her. I guess I’ll just suck some juice out of a highlighter and squirt it on a coral. That should do.

It works for knock-off 'glo-fish' - can't see why it wouldn't work perfectly. Flawless plan

 

[sawdonkey]

Didn't you see where i offered to do the work for you? Bargain price too. I just assume you'd say yes so I already ordered the shipping container. Let me know where to send the invoice.

Yeah, pretty good deal, but I’m gonna let you have all of the glory and patents etc. and just do it out of your own lab. Keep me in line for a frag though. I’ll trade you a highlighter Millie frag for one of your GMO frags. Just don’t get it wet, the color tends to wash off.

 

[sawdonkey]

Why not graft differently colored milles?

I’ve accidentally tried this plenty of times and they tend to just grow over each other and don’t mix genetics. I assume if it were this easy, others would already be all over it. That’s not to say I’m opposed to cutting up a bunch of my millies and giving it a shot!

 

[Dana Riddle]

I’ve accidentally tried this plenty of times and they tend to just grow over each other and don’t mix genetics. I assume if it were this easy, others would already be all over it. That’s not to say I’m opposed to cutting up a bunch of my millies and giving it a shot!

I've never tried it with Acros. But it seems easy enough with Montiporas. Don't have a clue as to why.

 

[StatelineReefer]

I've never tried it with Acros. But it seems easy enough with Montiporas. Don't have a clue as to why.

I know Adam at @Battlecorals has a grafted acro with a quite heavy GFP infection. Thing looks phenomenal and unique, but GFP isn't my thing.

 

[Leishman]

Happened naturally in my tank with my Setosa

 

[User]

Happened naturally in my tank with my Setosa

Does setosa have gfp or is it just green?

The bright green on the left is a good example of true gfp- although that's not really accurate, since the actual GFP protein OP is talking about (used in a research setting by his wife) is derived from jellyfish

 

[Leishman]

Does setosa have gfp or is it just green?

I cut my setosa in two years ago. The other half is pure orange and only ~3-4” away from this chunk. Green started as tiny spot and has slowly been spreading.