Bacterial Chimerae or making photosynthetic Bacillus Subtilis

[Mega [Andreas Stuermer]]

Hi!

While waiting for my construct / primers  (pGlowroplast + yeast fluorescent protein)  I  was thinking about doing something  in the meantime…

http://www.pnas.org/content/102/44/15971.full.pdf

 

This paper describes how CyanoBacillus was made…

 

Now I was thinking if I can fuse Cyanobacteria and Bacillus Subtilis. But through natural breeding (in a legal sense). I heard Cyanos are resistant to ampicillin naturally. I read Bacillus subtilis sometimes have a natural resistance against rifampicin (but where to get such a strain?).

 

Now incubate both bacteria in PEG (do I need to make protoplasts?) to make them fuse. Plate on Amp+Rif LB so the cells are forced to keep two chromosomes. Maybe the chromosomes will fuse at some point? After some time?

Or would it even be easier to introduce a bacterial photosynthesis pathway in B.Subtilis? Have never done much research on bacterial photosystems. But in plants it's quite complex.

[Koeng]

I actually in-depth read many papers about CyanoBacillus. First of all, the bacteria probably won't just fuse. The cells are most likely going to all croak because the membranes and walls are incompatible. Next, the CyanoBacillus took more then 5 years to complete. On top of that, CyanoBacillus' cyanobacteria side doesn't really express. If they both expressed, it is going to screw up the cell. Also for strains of bacillus, i recommend the bacillus genetic stock center, it is great

I would recommend looking into Bacteriorhodopsin. It is a single protein system inside of Halobacterium that uses sunlight to pump ions across a membrane. i was going to direct the evolution of it but I have other projects I need to work on... Anyway it shouldn't be THAT difficult to put it into Bacillus

-Koeng

[Mega [Andreas Stuermer]]

Hey, you're right. Haven't thought of the differing cell wall structure!

Seemingly I need to dig into bacterial oxygenic photosynthesis

>It is a single protein system inside of Halobacterium that uses sunlight to pump ions across a membrane

Just one protein to express and it may generate energy by light? I will look that up!!

[Mega [Andreas Stuermer]]

Back to the drawboard. Wikipedia says, it is not capable of fixing C from CO2.

The reason why I would want to make B.Subtilis photosynthetic is to thrive on Mars. We don't know the carbon content of the soil there (due to perchlorate oxidation when heating in the instruments), organic carbon may be close to 0.

[Mega [Andreas Stuermer]]

http://wyss.harvard.edu/viewpage/421/

Some work to make E Coli photosynthetic...

[Koeng]

Well think about what it takes to do the cycles. It needs ATP, and that is what bateriorhodopsin produces. Possible to use both the pathways?

[Cathal Garvey]

Technically (but these details are important) bacteriorhodopsin pumps
H+ ions, establishing a proton gradient which is then used by ATP
synthase(s) to turn ADP into ATP.

While this clearly captures energy for the cell, it's generally not
considered "photosynthesis" because nothing's captured into the cell
that would otherwise have been missed; e.g. CO2 fixed into constituent
C atoms of glucose.

I used to think Bacteriorhodopsin was all the hotness in photosynthesis
for bacteria, and that it would make a nice addition to algae (look at
the absorption spectra; very complementary!). That was until I attended
a lecture the other day on bacteriochlorophylls by an expert in the
field. While bacteriorhodopsin is great *because* it's simple, the
flip-side is that simplicity means inefficiency.

The bacteriochlorophylls were mind-bendingly elegant, leveraging quantum
effects such as Heisenberg's uncertainty principal to capture a high
proportion of passing photons by "sharing" the polarised-absorption
alignments of nearby chlorophylls, etc. They also used three different
absorptive elements (two chlorophylls and a carotenoid) and downshifted
the frequency of incident photons for maximum absorbance.

They were incredible; easily as advanced as algal chlorophylls, but
evolved to take advantage of the scraps left over from algae as light
passed into deeper water. A beautiful example of evolutionary
convergence, too, though I gather they had no true homology. Structures
alike and everything.

But getting a system like that working in another species would be a
huge task, because getting energy out of the system requires a whole
other set of systems that accept and extract energy from the electrons
excited by the captured photons. You're talking not only about the
"antenna array" (which has a precisely defined, intricate structure)
but the amplifiers and signal processors that go with it.

This all put poor old bacteriorhodopsin in perspective. It's neat
because its simple, but I don't think one should expect great things
from it! After all, it tends to occur only among species that have
little competition in their ecological niches, and little other
metabolic opportunites of worth in those niches. It's the energetic
begging hat of phototrophy! :)

Still, it's only one gene, and likely to be highly transferable, so
still worth a shot.

Also, a niggly note: Bacilli can *persist* on mars, sure. But not in a
respiratory state. Spores don't photosynthesise or respire energy, and
without a gaseous or liquid environment, they never will. So you'd need
liquid water for them to inhabit, and that's before you consider their
need for CHNOPS: Carbon, Hydrogen, Nitrogen, Oxygen, Phosphorous,
Sulphur. All of which may be abundant on Mars but in a form
inaccessible to the bacteria!

It *is* possible however that you could do something sneaky and add
antifreeze proteins to the spore coat so if they land on permafrost in
good weather they might be able to dig out a little puddle to live
within?

[Mega [Andreas Stuermer]]

Wait, is that thing about the different cell walls really a knock-out criterion? If I make protoplasts, they have no cell walls, only weak membranes. Those may be able to fuse?

[Mega [Andreas Stuermer]]

Now, that looks if they could fuse :D

http://filebox.vt.edu/users/chagedor/biol_4684/Methods/bacterial.gif

[Mega [Andreas Stuermer]]

If you remove the upper layers, of course

[Koeng]

Possibly, but remember that outside and inside they are very very very different. Even the proteins regulating cell size could screw up your cell.

Anyway, I have done an experiment and lysozyme treatment for an hour won't kill all of the bacillus, just a lot in a culture. Possibly it just eats anyway a small amount of the cell wall

[Koeng]

Large amount of the cell wall***

[Mega [Andreas Stuermer]]

Maybe I need to get a microinjector :D

Will that work with such small cells?

[Koeng]

i don't think so

When I looked at bacillus under my microscope they where like little moving dots at 400x magnification

[Koeng]

Maybe I should just get a better microscope xD

On Wednesday, November 20, 2013 2:33:32 PM UTC-8, Mega [Andreas Stuermer] wrote:

[Nathan McCorkle]

It might work, but you'd need smaller needles than for eukaryotes for
sure. It would probably be an order or three of magnitude harder :P

Microinjection on big cells would probably take quite some time, I'd
think at least a year of studying and finding/building apparatus, if
not 2 3 or 4 years!

On the other hand, I think PEG is a much better first try. ;)

I've made hybridomas before, was given the cultured myeloma cells in
suspension (immortal cancer B cell line), and had to slice and dice
and extract from a mouse the spleen and then rinse and smash it to get
the white blood cells out. Then after counting the ratio of B cells to
other WBCs, used overall count to calculate the B cell concentration.
Then we knew how much of culture A and culture B to mix, to get the
desired ratio of myeloma to antibody-producing B cells. That ratio
could vary, this paper says spleen cell to myeloma cell ratio is 3:1,
but can vary from 1:1 to 10:1. (pg 393)
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2596021/pdf/yjbm00116-0074.pdf


I did some searching, found this but can't get it, will let you know
if I can get it though:
http://link.springer.com/chapter/10.1007/978-1-4684-4142-0_7