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checking on GFP expression

Following up on yesterday’s results, we came up with an experiment plan to see how the GFP expression is turned on by the addition of arabinose to the bacteria. What we want to investigate is:

  • in what stage of bacteria growth does the arabinose have the most effect
  • what amount of arabinose is needed
  • what starting amount of bacteria yields the best results

All this in the context of the bacteria being grown inside the agar medium itself, not on top.

Thus we came up with creating the following:

  • LB agar, 20 ml /plate
  • carbenicillin, 50mg/μl concentration
  • bacteria liquid cultures at:
    • 0.55 ml / plate
    • 1.1 ml /plate
    • 2.2 ml / plate

We put these plates into an incubator, and each our we get a different plate, and drop different concentration of arabinose on them, making three 5μl drops each:

  • 1000μg
  • 125μg (1/8 of the original)
  • 15.625μg (1/64 of the original)
  • 1.95μg (1/512 of the original)

So now, every hour we’re taking a set of plates, and dropping different concentrations of arabinose on them. We’re also checking to see of course if last hour’s drops have made any effect already. After the first hour, there was no effect yet. Maybe GFP expression is turned on slower than that…

But then again, we might have killed all the bacteria by mixing them into too hot LB agar (though we could hold it in our hands, so it was surely colder than 50 °C). And we didn’t really dilute the LB agar either.

results from yesterday

img_7891.jpg

Looking at the results of yesterdays experiment in the morning brought about a better feeling. It seems that things are working, though differently than expected.

For the samples in the microplate, the results show overnight that all of the bacteria that we put arabinose on became fluorescent, even the ones with the lowest concentration. If there’s a difference in fluorescence with regards to the arabinose concentration, it’s not visible to the naked eye. Only the wells which were not added arabinose at all (but simply water instead) remained non-fluorescent.

One startling but very good result is that the arabinose doesn’t dissipate in an agar gel that easily. Thus, can create drawings in the bacteria on the agar by selectively dropping arabinose on different locations - and only those locations are going to turn fluorescent. This is a great result.

The problem is that the level of fluorescence in bacteria that grows inside the agar gel (as opposed to on top of if) is much lower. This might be that the arabinose doesn’t reach too many bacteria, as it is not dissipating well in the agar. It might also be that there are fewer bacteria growing this way as compared to colonies, which are quite dense.

I also grew a liquid culture with arabinose in it, just to see a glowing flask of bacteria :)

safety meeting

This morning we had a meeting with Mark Fluttert, responsible for lab safety at the Gorleaus Lab. The topic was how to make the final installation safe and legal. After all, we’re planning to show and move around genetically modified organisms in public space.

It turns out that to be able to do that, one has to designate the area as level ML 1 - whatever that means. Well, among others it has to be certified by municipality officials as such. It has to have a water sink to wash hands. And there has to be a way to disinfect the area if there’s some spillage of material.

As for the idea to hand out the pictures made by the visitors via the installation - pictures made by fluorescent bacteria in fact - the sad news is that they have to be killed before handed out. So somehow we have to have the bacteria create the picture, and then killed. Of course, the fluorescent protein would remain there, so the image would still be visible.

Talk about abusing living creatures for our own wanton purposes…

an experiment with microplates

Yesterday we prepared an experiment with Maarten de Smit to try out if bacteria grown overnight in an agar loan, but without the arabinose (thus non-fluorescent) could be triggered to become fluorescent by adding some arabinose to it. We prepared agar with different concentrations of bacteria in it (10, 5 and 2.5μl / 200μl). We also created a series of arabinose concentrations even, to see the rate of change depending on the concentration.

The expectation was that GFP expression should start within about 15 minutes of the arabinose added to each well. (The small compartments on the microplate are called wells.)

The result is: nothing.

There could be several things wrong here. One is that the bacteria in the agar gel is already in stationary stage, having had a day to grow and multiply. The other is that they are just too stretched out in the agar - after all, previously we were looking at the effect in colonies.

So the next thing to do is to take a young (2 hour old) liquid culture, where all the bacteria is still active, and add arabinose to some parts of that - and see.

using microtiter plates as canvas

OLYMPUS DIGITAL CAMERA

Maarten de Smit came up with the wonderful idea that we could use microtiter plates as a canvas for drawing the images on GFP bacteria. These plates contain small ‘wells’ close to each other, so it’s easy to put a small amount of material in them - like the bacteria and their agar base. And we can conveniently treat each well as a single pixel, either dropping some arabinose on it so that it becomes fluorescent, or not.

The reason we can’t use one big plate of bacteria is that the arabinose dissipates in the agar, thus would spread around a large part of the plate. This is not desired when separate pixels need to be created beside each other.

We’ll grow some bacteria in these tomorrow, and try them out on Wednesday to see if they really work. Could have our first 12 x 8 = 96 pixel bacteria image as soon as Thursday!

(image courtesy of Jeffrey M. Vincour, originally published under CC-by)

the amount of arabinose makes a difference in fluorescense

Last Friday we took some bacteria from plates that were about a month old, but were stored in the cold room. These were bacteria that had the pGLO plasmid in them, and thus being brightly fluorescent. To our luck, they were still living dandily on the agar gel they were on - thanks to the cold they were slowed down considerably.

Besides wanting to re-grow them, we also did a simple experiment seeing how the level of arabinose around the bacteria effects the level of fluorescense. It seems that neither too much or too little helps the cause - a concentration of 0.1 gave the best results, with both 0.02 and 0.5 giving worse results on visual inspection.

But they do have an effect nevertheless, so we’d actually be able to create gray-scale images using this approach.

dispensing valves

As previously noted, we’re looking for valves into the modified fabber that can dispense liquid precisely. Now I got word from Andre Bosse from the Waag Fablab, suggesting to use what are called ‘dispensing valves’. For example, EFD manufacture such valves.

the arrogance of (bio-) artists

I listened to art historian Danielle Hofmans’ lecture at the Victorian Circus IV event at De Brakke Grond on Thursday, and I was really astonished at the level of arrogance and god-like posturing of established bio-artists referenced in the lecture, and of which Danielle herself is very critical.

As you can read below, my first reaction is quite emotionally charged…

For a start, Eduardo Kac says in his Book Signs of Life: “[artists] invent new entities and new relationships never seen before.” I wonder, since when is it the artist who is not reflecting on his surroundings, but shaping it? When was it ever that an artist has ever invented anything new that is beyond the realm of art itself? I wonder if Eduardo Kac can claim any invention his own, even considering his fluorescent Bunny - as he has only used technology what was available for about 8 years at that time already. I wonder about similar claims of novelty on SymbioticA’s tissue culture projects - after all, the technology of tissue culturing was invented in the XIX. century, and became a routine lab technique in the 1950’s already. One can’t really claim he has done anything new using a technology invented and perfected long before he was even born, can he?

What these artists can claim is that they have brought the above results into the public limelight. And this is a nice achievement in its own - but it seems that this is just not enough for them.

What was even more disturbing for me was Eduard Kac’s position on the importance of cooperation in evolution, and framing it so that cooperation and competition are antagonists. He seems to associate competition with a rightist / corporate / evil ideology, while competition is the nice leftist concept that is obviously close to his heart. His stance obviously shows that he does not understand evolution theory. But then again, I’ve seen articles earlier where people were trying to mix their political views with hard scientific facts and universal truth about nature itself - and these were quotes in Fashionable Nonsense: Postmodern Intellectuals’ Abuse of Science by Sokal and Bricmont. It’s sad to see that even over 10 years after the useless nature of such reasoning was pointed out so elegantly, the style still thrives on.

It seems that established bio-artists develop a god-like stance, positioning themselves as ‘grand creators of beings’. Eduardo Kac says that “[bio-artists are part of] the global network known as evolution”, while Oron Catts and Ionat Zurr refer to their cell cultures as ’semi-living’, and demand that these are regarded as a new form of being. I wonder how these claims relate to the general criticism against scientists, especially genetic engineers, which states that they are ‘interfering with life’, and that is too dangerous and ‘man should not play god’. It seems to me again that artists want to put themselves in a special position, the only ones authorized to play these roles - all on ideological grounds. I think that especially their mistaken and politically charged ideology and apparent lack of knowledge and humbleness with regards to this field makes them unqualified for the position they are so dearly aiming for.

As you can see above - my reaction is an emotional one at first indeed to the above claims. Maybe I should be much cooler and provide rational criticism instead. I guess nobody is perfect…

extracting DNA from bacteria

As we’ll need to do a lot of DNA transformation using the gene generating the pGLO GFP, we have to extract some from bacteria that we grow. The idea is to grow lots of bacteria, then use a DNA purification kit to separate the DNA plasmid from the bacteria itself. Of course, all the bacteria die a gruesome death at 20.000 g acceleration in the process - but not in vain.

We’re going to use a Qiagen Plasmid Midi Kit for this purpose next Thursday. Fortunately the protocol and all details are available online - it’s quite a tedious process…

new ideas for kitchen-grade DNA transformation

Through a very nice discussion with Maarten de Smit, we came up with additional approaches to replace laboratory-grade material with widely available alternatives in our quest for kitchen-grade DNA transformation. The results include marmite, red wine and Indonesian pudding.

LB

The medium used to grow E. Coli in is called LB, short for Lysogeny Broth. This contains nutrients that the bacteria likes, so that it grows well in it. A typical LB solution is made up of the following ingredients:

An idea is to replace the above with the following ingredients, respectively:

(well, table salt is just the common name for NaCl anyway)

Arabinose

Arabinose is the promoter used in our DNA that makes the bacteria start expressing the fluorescent protein - in a way, switching on the fluorescent protein gene. According to [del Alamo], red wine contains arabinose in concentration levels that are sufficient for our needs. One needs to evaporate the alcohol from the wine first, as that would kill the bacteria.

Agar

Bacteria is grown on an agar gel for convenience, which is coincidentally used in East-Asian cuisine as well. So one just needs to get to a proper food store to get some.

Centrifuge

The process of creating competent cells involves centrifuging the sample, so as to collect all the cells together, and that one can get rid of the liquids around it. But bacteria cells can also be collected using filters aimed at creating sterile solutions, which in fact trap the bacteria. Filters of 20μm or 45μm are sufficient at trapping bacteria.

Need to do some further investigation though on where to get such filters, like in a pharmacy, for example.