Biotechnology and Bioengineering Thread

[Toxn]

AKA Toxn and Oedipus' club house.

 

This is the place for discussing molecular biology, gene constructs, lab preps and other biotechnology.

 

[Toxn]

So first up, my training is in plant biotech but my interests run very much to biomaterials.

 

So this shit is right up my alley.

[Oedipus Wreckx-n-Effect]

Harvard engineers have started using bacterial strains to produce isopropanol via cyanobacteria photosynthesis. 

 

http://hms.harvard.edu/news/bionic-leaf

 

It's interesting. I'm a big fan of going the bio diesel route with cyanobacteria. 

[Toxn]

Harvard engineers have started using bacterial strains to produce isopropanol via cyanobacteria photosynthesis.

http://hms.harvard.edu/news/bionic-leaf

It's interesting. I'm a big fan of going the bio diesel route with cyanobacteria.

One of my super secret plans is to produce high-oil strains of floating pond weeds - you then get high-protein animal feed ond oil at once.

[Oedipus Wreckx-n-Effect]

One of my super secret plans is to produce high-oil strains of floating pond weeds - you then get high-protein animal feed ond oil at once.

That's assuming you want to kill off your colony when extracting lipids. You can extract the lipids used for bio diesel without having to kill the organisms. It's more cost effective that way.

[Toxn]

So, in line with the title image I thought I'd share some of the grunt-work steps of molecular biology - we're all molecular biologists now, after all.

 

The list below covers 99.99% of what biologists do in the lab these days:

- DNA assembly and transfection

- Culturing, isolation and purification of DNA/RNA/Protein

- PCR

- Running gels

- Other analysis techniques (DNA sequencing, Mass Spec and so on)

- Data collection and analysis

 

These things tend to lead into each other, so a small project will go somethng like:

- Liquid cultures of plasmid-containing E.coli

- Isolate and purify plasmid DNA from cultures

- PCR to check identity

- Cut and ligate new plasmid from isolated stock

- Transfect plasmids into more E. coli

- Streak cultures of transfected cells onto selection medium

- PCR of picked colonies to check identity

- Liquid culture of selected colonies

- etc.

 

As you might guess, this also leads to a lot of favourite lab techniques that get carefully recorded and passed on to the newbies lest the fire go out and the lab forgets how to maxi-prep JM109 containing the super's preferred pUC variant (which he worked on as a post-grad and has been making iterations of ever since). When I was working in a lab these special techniques were kept in a seperate book with notes scribbled in the margins - our glossed codex of halal techniques. I also kept a private lab book with my own notes and collected materials drawn from websites and lab forums - my own heretical bible. 

[Toxn]

Anyway, one of these techniques which I used quite a bit was large alkaline lysis preps. This was partly to save money (kits are crazy expensive), and partly because I needed a lot of DNA but didn't need it to be particularly pure. The latter was because I was doing microprojectile bombardment, which hoovers up DNA. Finally, I'm lazy and would rather do one gigantic prep once than have to do a bunch of them every week.

 

I used something like this approach in 20ml centrifuge tubes (I was in charge of the large centrifuge, so scheduling was easy) and ended up with a bunch of gargantuan, ugly pellets (creamy white is not a sign of amazing purity). These were then resuspended in nanopore H2O, collected togething into two tubes and rotovapped to produce DNA at a ridiculous concentration. For the next two years I did all of my bombardments with this construct using only these two motherlode solutions.

[Toxn]

That's assuming you want to kill off your colony when extracting lipids. You can extract the lipids used for bio diesel without having to kill the organisms. It's more cost effective that way.

The nice thing with this approach is that you can run a continuous system and just harvest off of one end of your growing pools the whole time. As these things are clonal and have very slow lifecycles compared to algae or bacteria, you also don't have to worry too much about weird strain selection effects.

 

But then, I'm innately attracted to slightly off-kilter approaches. There is definitely something to be said for setting up a culture and just creaming off the oil which floats to the top.

[Oedipus Wreckx-n-Effect]

The nice thing with this approach is that you can run a continuous system and just harvest off of one end of your growing pools the whole time. As these things are clonal and have very slow lifecycles compared to algae or bacteria, you also don't have to worry too much about weird strain selection effects.

 

But then, I'm innately attracted to slightly off-kilter approaches. There is definitely something to be said for setting up a culture and just creaming off the oil which floats to the top.

So you want to have a lighting system for a CSTR reactor with a cooling jacket? Because you'd need the UV dialed in for the mixed reactor to optimize a specific dwell time to sync up with downstream production schedules. 

 

And slow life-cycles means that the dwell time would be weird to nail down. But doable, just I don't know if it's worth it. 

[Toxn]

So you want to have a lighting system for a CSTR reactor with a cooling jacket? Because you'd need the UV dialed in for the mixed reactor to optimize a specific dwell time to sync up with downstream production schedules. 

 

And slow life-cycles means that the dwell time would be weird to nail down. But doable, just I don't know if it's worth it. 

No, nothing so fancy

 

I'm going to be a secret squirrel and PM you on it though - gotta build that air of mystery so beloved by angel investors

 

Edit: Also, I had to look up a few things to get what you were laying down there - bioreactor design being something I know about on only a superficial level. Do you think the gains on an artificially-lit reactor are worth it for something like bio-oil production, by the way? Because I thought that natural lighting was the only way to get feedstock production at a sensible cost. For pharming, on the other hand...

[Oedipus Wreckx-n-Effect]

No, nothing so fancy

 

I'm going to be a secret squirrel and PM you on it though - gotta build that air of mystery so beloved by angel investors

 

Edit: Also, I had to look up a few things to get what you were laying down there - bioreactor design being something I know about on only a superficial level. Do you think the gains on an artificially-lit reactor are worth it for something like bio-oil production, by the way? Because I thought that natural lighting was the only way to get feedstock production at a sensible cost. For pharming, on the other hand...

 

 

A basic CSTR set up looks like the following.

 

 

 

Now, I thought you were proposing using a CSTR in the production of lipids via microalgae. My idea for lipid production would either be a CSTR or a batch reactor. I would have to crunch numbers and figure out the specific rate laws governing each for the particular microorganism I would use. Plus feed to the microorganism is important. 

 

As far as light, it would be needed for the photosynthesis. That's why I am more inclined to used a semi-batch reactor design (kind of a mix between batch and CSTR) with light-emitting rods moving into the reactor to continue the reaction and subsequent growth of the microorganisms. 

I believe Semi-batch design would allow for the addition of extra feedstock. A water jacket would be needed to keep temperature constant, and after a given growth period the reactor would be drained and the cells cracked for the lipids. 

 

The lipids in this case are fatty acids that can be turned into biodiesel via the following reaction. (A general reaction in this case)

 

[Toxn]

So far as I know nobody has managed to get a continuous process running well with microorganisms - at least, not at a large scale. So the setup you've described does sound pretty optimal.

[Toxn]

As a neat segue into bio-engineering, I should mention another bright idea of mine - which is to engineer algae to produce ethanol directly by engineering them with the correct pathways.

 

This has been done already, of course, but still represents an interesting avenue for cutting out the middleman in a lot of biosynthetic processes.

 

Which brings up the issue of biological engineeing itself. As I have said before, we're in the process of trying to understand the alien radio using limited tools, and the result is that we disassemble and tinker rather than truly engineering for the most part.

 

One way out of this bind is another pet topic of mine, which is to limit the complexity/interaction issues by using networks of well-understood components rather than trying to plug in pathways from elsewhere. Once you have something which works, you can then optimise by rational design/computational biology in a way similar to Spiber.

[Oedipus Wreckx-n-Effect]

So far as I know nobody has managed to get a continuous process running well with microorganisms - at least, not at a large scale. So the setup you've described does sound pretty optimal.

https://www.researchgate.net/publication/245148180_Biological_Hydrogen_Production_in_Continuous_Stirred_Tank_Reactor_Systems_with_Suspended_and_Attached_Microbial_Growth

 

Here's the abstract,

 

"Fermentative H2 production in continuous stirred tank reactor (CSTR) system with bacteria attached onto granular activated carbon (GAC) was designed to produce H2 continuously. The H2 production performances of CSTR with suspended and attached-sludge from molasses were examined and compared at various organic loading rates (8–40g COD/L/d) at hydraulic retention time of 6h under mesophilic conditions (35°C). Both reactor systems achieved ethanol-type fermentation in the pH ranges 4.5–4.8 and 3.8–4.4, respectively, while ORP ranges from −450 to −470mV and from −330 to −350mV, respectively. The hydrogen production rate in the attached system was higher compared to that of the suspended system (9.72 and 6.65L/d/L, respectively) while specific hydrogen production rate of 5.13L/g VSS/d was higher in the suspended system. The attached-sludge CSTR is more stable than the suspended-sludge CSTR with regard to hydrogen production, pH, substrate utilization efficiency and metabolic products (e.g., volatile fatty acids and ethanol) during the whole test."

 

 

So it can be done with certain set-ups and depending on your needs.

[Oedipus Wreckx-n-Effect]

As a neat segue into bio-engineering, I should mention another bright idea of mine - which is to engineer algae to produce ethanol directly by engineering them with the correct pathways.

 

....

 

 

That's an easy enzyme swap. Switch out the gene for lactate dehydrogenase to an ethanol dehydrogenase. They have done that with Lactobaccillus strains to create a bacteria that would colonize someone's mouth and displace the "bad" bacteria that cause cavities. Instead of lactic acid, they produced ethanol, which is harmless to enamel. 

 

This, however, couldn't get passed through the FDA because they couldn't figure out a way to tax it. Think about it. You could do a quick mouth-wash of this new bacteria and it outproduces your normal mouth bacteria with no side-effects. Now you're practically immune to cavities, and it's cheap as hell. 

 

Best part (or worst if you're the GOVERNMENT) is that you can transmit this strain to others just by a little sloppy kissing. 

 

If the GOV can't tax it, they don't want it. 

[SergeantMatt]

That's an easy enzyme swap. Switch out the gene for lactate dehydrogenase to an ethanol dehydrogenase. They have done that with Lactobaccillus strains to create a bacteria that would colonize someone's mouth and displace the "bad" bacteria that cause cavities. Instead of lactic acid, they produced ethanol, which is harmless to enamel. 

 

This, however, couldn't get passed through the FDA because they couldn't figure out a way to tax it. Think about it. You could do a quick mouth-wash of this new bacteria and it outproduces your normal mouth bacteria with no side-effects. Now you're practically immune to cavities, and it's cheap as hell. 

 

Best part (or worst if you're the GOVERNMENT) is that you can transmit this strain to others just by a little sloppy kissing. 

 

If the GOV can't tax it, they don't want it. 

Got any articles about this, that sounds interesting.

[Toxn]

That's an easy enzyme swap. Switch out the gene for lactate dehydrogenase to an ethanol dehydrogenase. They have done that with Lactobaccillus strains to create a bacteria that would colonize someone's mouth and displace the "bad" bacteria that cause cavities. Instead of lactic acid, they produced ethanol, which is harmless to enamel.

This, however, couldn't get passed through the FDA because they couldn't figure out a way to tax it. Think about it. You could do a quick mouth-wash of this new bacteria and it outproduces your normal mouth bacteria with no side-effects. Now you're practically immune to cavities, and it's cheap as hell.

Best part (or worst if you're the GOVERNMENT) is that you can transmit this strain to others just by a little sloppy kissing.

If the GOV can't tax it, they don't want it.

To be fair to evil.gov, I'd be a little leery (as a lay person) about the whole 'outcompetes normal mouth bacteria', 'transmitted by kissing' and 'produces ethanol' aspects. This is the sort of thing that cries out for a long-term study before being uncorked.

[Toxn]

Which is merely to say that the precautionary principle isn't a bad idea on its own, so long as it is applied evenly and tempered with a reasonable understanding of risk.

[Oedipus Wreckx-n-Effect]

Got any articles about this, that sounds interesting.

I'll try to find something on it. I learned about it by speaking to one of the lead professors doing the work. They couldn't get it through any FDA testing due to the way FDA testing on humans is defined. Instead, they went and marketed the procedure for pets, namely dogs. 

 

But, in a sense, it would be the easiest, cheapest, most reaching global medical breakthrough since iodine in salt or fluorine in water.From a public health perspective, it is amazing. To the degree that dentists would lose a ton of money.

 

 

To be fair to evil.gov, I'd be a little leery (as a lay person) about the whole 'outcompetes normal mouth bacteria', 'transmitted by kissing' and 'produces ethanol' aspects. This is the sort of thing that cries out for a long-term study before being uncorked.

 

It out competes the other strain of the same bacteria that uses the lactate dehydrogenase enzyme, so that's all the micro-fauna it displaces. 

[Toxn]

I'm a big fan of biomaterials, so this is interesting:

 

http://www.mycoworks.com/

 

http://motherboard.vice.com/blog/houses-of-the-future-grown-out-of-mushrooms

 

http://inhabitat.com/phillip-ross-molds-fast-growing-fungi-into-mushroom-building-bricks-that-are-stronger-than-concrete/

[Toxn]

I recently spent a while chatting to colleagues during a road trip, and was reminded that my training makes my thought a bit odd.

I think evolutionary biologists and geneticists become kind of comfortable with the idea that life on earth is both wonderful (in the original sense) and spectacularly cruel. Add to that the knowledge that the universe is slowly winding down and that life appeared on earth pretty much the moment the planet ceased to be molten and its hard to escape the idea that life itself is in some way an ongoing struggle against the very grain of the universe. In this framework, mankind's achievements in eliminating human predators, beating back disease, rolling back starvation and generally holding off the ghost of Malthus (however temporarily) seem like fairly audacious acts of rebellion.

In biotechnology (along with things like physics), we finally have the tools to really start fighting back. And in this most hopeful version of our future the marvels of the industrial revolution and the nuclear age might simply be the point where mankind got up, had a good stretch and took the first few proper jabs against the order of the universe.

This is what this topic is supposed to celebrate - the latest round of blows launched by the best rebels nature has yet produced.

May we live to produce more.

[Toxn]

On that note - fuck malaria and fuck mosquitoes:

http://www.nature.com/news/gene-drive-mosquitoes-engineered-to-fight-malaria-1.18858

http://www.nature.com/nbt/journal/v34/n1/full/nbt.3439.html

[Oedipus Wreckx-n-Effect]

I only read the abstract, since I'm currently balls-deep in homework, but are they creating a population of mosquitoes that will pass on at a 90% transmittance rate a gene that turns off the fertility of female mosquitoes? 

 

Fantastic. 

 

Now, if I may, I need to design a similar system and spray the entire South of the United States with it to genetically target Kudzu.

[Toxn]

Gene drives are the shit in that regard, although for Kudzu I'd also be thinking of biocontrol:

http://canr.udel.edu/biocontrol/biological-control-of-kudzu/

This is both because people in China might actually want their kudzu, and because interspecies gene flow in plants happens far more often than in animals. A lethal gene drive is a species kill vehicle, so if it jumps to, say, soybean (which is closely related enough to kudzu to make biocontrol difficult)...

[Toxn]

Reproductive biology is now our plaything as well:

 

http://www.nature.com/ncomms/2016/160913/ncomms12676/full/ncomms12676.html

 

Cooincidentally, this means that having a child by syndicate might be possible in the near future. I'd like to think that we'd name ours Theodore.