Our efforts to realize the bio.display project at the Ars Electronica BioLab have failed, due to the reason that the lab used was contaminated by Clostridium bacteria, which were also ampicillin resistant. And, well, they took over, like a young cockoo in a birdnest.
For the experiments done at the Ars Electronica Center, I’m using 12×12cm plates again. This means that each plate would take about 120mm x 120mm x 7mm = 100800mm³ = 100.8ml. This means I can pour about 10 plates / liter of LB agar with bacteria in there. As per my protocol page, each liter of LB agar with bacteria will need 55ml of overnight liquid culutre. Thus, if my aim is to create 40 plates, I need to prepare for 4 liters of LB agar with bacteria, which is 55ml x 4 = 220ml of liquid cutlure.
As they have 500ml Erlenmayer flasks here, of which a quater volume (125ml) can be used for the liquid culture, I need to use two of them, which will bring me to 250ml of liquid culture. To create the same amount again based on liquid cultures, I’ll need 2ml of liquid culture to mix with fresh LB medium.
For a 125ml of liquid culture, I’ll need:
I’ll make two of these.
I’ll also make a smaller, 100ml Erlenmayer flask control with arabinose – to see if it glows as expected. This will contain:
Jos Arents just informed me that the experiment done yesterday didn’t really work out. It seems that the transformation of pHluorin to the XL1blue and DE21(DE3) E. Coli strains didn’t seem to work out, as there’s no visible inidication of the fluorescent protein being there.
It seems that the results for the experiments done so far are not pointing in a positive direction. In some strains the fluorescence is just not visible to the human eye. In others, it seems that while the appropriate DNA is there, it’s just not expressed, so the fluorescent protein it should build is just not around.
Today we put them to a test again, to see if the two DNA sections put into the same bacteria are interfering with each other. We’ll see tomorrow.
It’s also hard to pinpoint which fluorescent protein is visible enough to the naked eye. In research, the results are measured by sensitive apparatus, so there it’s good enough if the differences in fluorescence are minuscule, but still picked up by these machines. But here we’re aiming for a display tailored at humans.
Given the fixed timeframe of this project, we don’t have the liberty to engineer the proteins and DNA we need. But it seems that the ones we’d need are just not there at the moment.
It seems that for the TorA-GFPmut3* GFP, which we’re trying to control by pH change, the excitation wavelength (color) is just too close to the emitted light for a human eye to see.
Yesterday and today with Maarten de Smit we’ve prepared an experiment that we can do tomorrow, and see if we can really turn the glow in the bacteria on and off - thus creating a bio-pixel.
Meanwhile, Jos Arents at the University of Amsterdam as preparing the first plates with the bacteria that could have the same effect - but triggered by light. He’ll have the plates grow out hopefully tomorrow as well.
The sad news is, that the pGLO bacteria, which was extremely bright after performing the original transformation, doesn’t really glow now at all. The bacteria with the TorA-GFPmut3* GFP in itdoes glow, but not really bright. And it seems it has a tendency to glow when lit with lower wavelength UV light - which is dangerous to the bacteria itself an to humans as well.
Anyway, we put the TorA-GFPmut3* GFP version into deep freeze storage (-70 °C), under the code MS 903, so that it is readily available whenever needed.
The bacteria we put out in plates all grew out today - which was kind of expected. We grew them on plates without arabinose, so the DNA that was put into them would not be expressed. This is to make sure the bacteria are in their best form (the alien DNA may interfere with it somewhat), so can we can used these specimens for long term deep-freeze storage.
But to verify that these really are the ones we need, today we’ve put them on plates with arabinose as well. We’ll see if they glow tomorrow (or, actually Friday.)
Along with the TorA-GFPmut3* E. Coli strain, we’ll also maintain the bacteria that came as a result from the BIO-RAD kit I experimented with earlier. Additionally, we also put in non-fluorescent bacteria that is resistant to ampicillin, as a control. Well, more can never be a problem, can it
So today we’ve basically put some of the bacteria on plates, which were made of LB and ampicillin. Tomorrow we’ll see what grows out, and which one will glow eventually.