Ciro Santilli @cirosantilli 37 

If enough people use it, we could let people sell knowledge content through us.

Teachers have the incentive of making open source to get more students.

Students pay when they want help to learn something.

We take a cut of the transactions.

However this goes a bit against our "open content" ideal.

Forced sponsorware would be a possibility.

Would be a bit like Fiverr. Hmmm, maybe this is not a good thing ;-)

OurBigBook.com / Stack Exchange Updated 2025-07-16

Stack Exchange solves to a good extent the use cases:

I have a very specific question, type it on Google, find top answers
I have an answer, and I put it here because it has a much greater chance of being found due to the larger PageRank than my personal web page will ever have

points of view. It is a big open question if we can actually substantially improve it.

Major shortcoming are mentioned at idiotic Stack Overflow policies:

Scope restrictions can lead to a lot of content deletion: closing questions as off-topic
This greatly discourages new users, who might still have added value to the project.
On our website, anyone can post anything that is legal in a given country. No one can ever delete your content if it is legal, no matter their reputation.
Although you can answer your own question, there's no way to write an organized multi-page book with Stack Exchange due to shortcomings such as no table of contents, 30k max chars on answer, huge risk of deletion due to "too broad"
Absolutely no algorithmic attempt to overcome the fastest gun in the West problem (early answers have huge advantage over newer ones): meta.stackoverflow.com/questions/404535/closing-an-old-upvoted-question-as-duplicate-of-new-unvoted-questions/404567#404567
Native reputation system:
- if the living ultimate God of C++ upvotes you, you get 10 reputation
- if the first-day newb of Java upvotes you, you also get 10 reputation
Randomly split between sites like Stack Overflow vs Super User, with separate user reputations, but huge overlaps, and many questions that appears as dupes on both and never get merged.
Possible edit wars, just like Wikipedia, but these are much less common since content ownership is much clearer than in Wikipedia however

Bibliography:

dev.to/codemouse92/has-stackoverflow-become-an-antipattern-3icb (archive)

OurBigBook.com / Wikipedia Updated 2025-07-16

you don't get any/sufficient recognition for your contributions. The closest they have to upvotes and reputation is the incredibly obscure "thank" feature which is only visible to the receiver itself: en.wikipedia.org/wiki/Help:Notifications/Thanks
deletionism is a tremendous problem on Wikipedia, for two main causes:
- tutorial-like subjectivity
- notability
The stuff you wrote can be deleted anytime by some random admin/opposing editor, examples at: Section "Deletionism on Wikipedia".
This also possibly leads to edit wars in the case of sub-page content (full page deletion is more clearly arbitrated).
Scope too limited, and politics defined. Everything has to sound encyclopedic and be notable enough. This basically excludes completely good tutorials.
Insane impossible to use markup language-base talk pages instead of issue trackers?! Ridiculous!!! That change alone could make Wikipedia so much more amazing. Wikipedia could become a Stack Exchange killer by doing that alone + some basic reputation system. Some work on that is being done at: www.mediawiki.org/wiki/Extension:DiscussionTools, already in Beta as of 2022.
Edit wars

Overdetermination of Maxwell's equations Updated 2025-07-16

As seen from explicit scalar form of the Maxwell's equations, this expands to 8 equations, so the question arises if the system is over-determined because it only has 6 functions to be determined.

As explained on the Wikipedia page however, this is not the case, because if the first two equations hold for the initial condition, then the othe six equations imply that they also hold for all time, so they can be essentially omitted.

It is also worth noting that the first two equations don't involve time derivatives. Therefore, they can be seen as spacial constraints.

TODO: the electric field and magnetic field can be expressed in terms of the electric potential and magnetic vector potential. So then we only need 4 variables?

Bibliography:

physics.stackexchange.com/questions/20071/do-maxwells-equations-overdetermine-the-electric-and-magnetic-fields

Oxford Nanopore MinION Updated 2025-07-16

One of the sequencers made by Oxford Nanopore Technologies.

The device has had several updates since however, notably of the pore proteins which are present in the critical flow cell consumable.

Official documentation: nanoporetech.com/products/minion (archive)

The following images of the device and its peripherals were taken during the experiment: Section "How to use an Oxford Nanopore MinION to extract DNA from river water and determine which bacteria live in it".

Oxford Nanopore MinION software channels pannel on Mac.

Source.

How to use an Oxford Nanopore MinION to extract DNA from river water and determine which bacteria live in it / Biochrom SimpliNano spectrophotometer Updated 2025-07-16

biochromspectros.com/spectrophotometers/simplinano-cat/simplinano-spectrophotometer.html (archive)

Manual: biochromspectros.com/media/wysiwyg/support_page/support-simplinano/Simplinano-UM.pdf (archive)

How to use an Oxford Nanopore MinION to extract DNA from river water and determine which bacteria live in it / Bioinformatics Updated 2025-07-16

Because Ciro's a software engineer, and he's done enough staring in computers for a lifetime already, and he believes in the power of Git, he didn't pay much attention to this part ;-)

According to the eLife paper, the code appears to have been uploaded to: github.com/d-j-k/puntseq. TODO at least mention the key algorithms used more precisely.

Ciro can however see that it does present interesting problems!

Because it was necessary to wait for 2 days to get our data, the workshop first reused sample data from previous collections done earlier in the year to illustrate the software.

First there is some signal processing/machine learning required to do the base calling, which is not trivial in the Oxford Nanopore, since neighbouring bases can affect the signal of each other. This is mostly handled by Oxford Nanopore itself, or by hardcore programmers in the field however.

After the base calling was done, the data was analyzed using computer programs that match the sequenced 16S sequences to a database of known sequenced species.

This is of course not just a simple direct string matching problem, since like any in experiment, the DNA reads have some errors, so the program has to find the best match even though it is not exact.

The PuntSeq team would later upload the data to well known open databases so that it will be preserved forever! When ready, a link to the data would be uploaded to: www.puntseq.co.uk/data

How to use an Oxford Nanopore MinION to extract DNA from river water and determine which bacteria live in it / Conclusions Updated 2025-07-16

against all odds, the experiment worked and we got DNA out of the water, despite a bunch of non-bio newbs actively messing with random parts of the experiment
PuntSeq and Biomakespace people, and all those tho do scientific outreach, are awesome!
biology is hard
creating insanely media rich articles like this is also hard, but the following helped enormously:
- Wikimedia Commons to store large media files out of Git
- Asciidoctor extensions to easily include those media files. The lessons learnt in this article were then an important motivation for Ciro's OurBigBook Markup, to which this article was later migrated.
- Nomacs to give Google Photos photos meaningful names and to edit people's faces out of pictures ;-)
some scientific Wikipedia pages may or may not have been edited with better pictures during the course of writing this article

How to use an Oxford Nanopore MinION to extract DNA from river water and determine which bacteria live in it / Filtration with vacuum pump Updated 2025-07-16

The first thing we did was to filter the water samples with a membrane filter that is so fine that not even bacteria can pass through, but water can.

Therefore, after filtration, we would have all particles such as bacteria and larger dirt pieces in the filter.

From the 1 liter in each bottle, we only used 400 ml because previous experiments showed that filtering the remaining 600 ml is very time consuming because the membrane filter gets clogged up.

Therefore, the filtration step allows us to reduce those 400 ml volumes to more manageable Eppendorf tube volumes: Figure 1. "An Eppendorf tube". Reagents are expensive, and lab bench centrifuges are small!

Since the filter is so fine, filtering by gravity alone would take forever, and so we used a vacuum pump to speed thing up!

For that we used:

Figure 3.
Peeling the vacuum pump filter protection peel before usage
. Source.

Figure 4.
Placing the vacuum pump filter
. Source.

Pouring the water sample into the vacuum tube and turning on the vacuum pump

. Source.

How to use an Oxford Nanopore MinION to extract DNA from river water and determine which bacteria live in it / Fisher Scientific UVP LM-26E Benchtop 2UV Transilluminator Updated 2025-07-16

www.bidspotter.com/en-us/auction-catalogues/bscsur/catalogue-id-bscsur10011/lot-c6605b41-1a14-40e5-a255-a5c5000866e0 (archive) Cannot exact same product on official website, but here is a similar one: www.fishersci.co.uk/shop/products/lm-26-2uv-transilluminator/12382038 (archive).

How to use an Oxford Nanopore MinION to extract DNA from river water and determine which bacteria live in it / Using the Oxford Nanopore Updated 2025-07-16

With all this ready, we opened the Nanopore flow cell, which is the 500 dollar consumable piece that goes in the sequencer.

We then had to pipette the final golden Eppendorf into the flow cell. My anxiety levels were going through the roof: Figure 4. "Oxford nanopore MinION flow cell pipette loading.".

At this point bio people start telling lab horror stories of expensive solutions being spilled and people having to recover them from fridge walls, or of how people threw away golden Eppendorfs and had to pick them out of trash bins with hundreds of others looking exactly the same etc. (but also how some discoveries were made like this). This reminded Ciro of: youtu.be/89UNPdNtOoE?t=919 Alfred Maddock's plutonium spill horror story.

Luckily this time, it worked out!

We then just had to connect the MinION to the computer, and wait for 2 days.

During this time, the DNA would be sucked through the pores.

As can be seen from Video 1. "Oxford Nanopore MinION software channels pannel on Mac." the software tells us which pores are still working.

Oxford Nanopore MinION software channels pannel on Mac.

Source.

Pores go bad sooner or later randomly, until there are none left, at which point we can stop the process and throw the flow cell away.

48 hours was expected to be a reasonable time until all pores went bad, and so we called it a day, and waited for an email from the PuntSeq team telling us how things went.

We reached a yield of 16 billion base pairs out of the 30Gbp nominal maximum, which the bio people said was not bad.

How to use an Oxford Nanopore MinION to extract DNA from river water and determine which bacteria live in it / PCR Updated 2025-07-16

More generic PCR information at: Section "Polymerase chain reaction".

Because it is considered the less interesting step, and because it takes quite some time, this step was done by the event organizers between the two event days, so participants did not get to take many photos.

PCR protocols are very standard it seems, all that biologists need to know to reproduce is the time and temperature of each step.

We did 35 cycles of:

94˚C for 30 seconds
60˚C for 30 seconds
72˚C for 45 seconds

This process used a Marshal Scientific MJ Research PTC-200 Thermal Cycler:

We added PCR primers for regions that surround the 16S DNA. The primers are just bought from a vendor, and we used well known regions are called 27F and 1492R. Here is a paper that analyzes other choices: academic.oup.com/femsle/article/221/2/299/630719 (archive) "Evaluation of primers and PCR conditions for the analysis of 16S rRNA genes from a natural environment" by Yuichi Hongoh, Hiroe Yuzawa, Moriya Ohkuma, Toshiaki Kudo (2003)

One cool thing about the PCR is that we can also add a known barcode at the end of each primer as shown at Code 1. "PCR diagram".

This means that we bought a few different versions of our 27F/1492R primers, each with a different small DNA tag attached directly to them in addition to the matching sequence.

This way, we were able to:

use a different barcode for samples collected from different locations. This means we
- did PCR separately for each one of them
- for each PCR run, used a different set of primers, each with a different tag
- the primer is still able to attach, and then the tag just gets amplified with the rest of everything!
sequence them all in one go
then just from the sequencing output the barcode to determine where each sequence came from!

Input: Bacterial DNA (a little bit)
... --- 27S --- 16S --- 1492R --- ...

|||
|||
vvv

Output: PCR output (a lot of)
Barcode --- 27S --- 16S --- 1492R

Code 1.

PCR diagram

.

Finally, after purification, we used the Qiagen QIAquick PCR Purification Kit protocol to purify the generated from unwanted PCR byproducts.

How to use an Oxford Nanopore MinION to extract DNA from river water and determine which bacteria live in it / PCR verification with gel electrophoresis Updated 2025-07-16

Biology experiments are hard, and so they go wrong, a lot.

For this reason, it is wise to verify that certain steps are correct whenever possible.

And so this is the first thing we did on the second day!

Gel electrophoresis separates molecules by their charge-to-mass ratio. It is one of those ultra common lab procedures!

This allows us to determine how long are the DNA fragments present in our solution.

Since we know that we amplified the 16S regions which we know the rough size of (there might be a bit of variability across species, but not that much), we were expecting to see a big band at that size.

And that is exactly what we saw!

First we had to prepare the gel, put the gel comb, and pipette the samples into wells present in the gel:

To see the DNA, we added ethidium bromide to the samples, which is a substance that that both binds to DNA and is fluorescent.

Because it interacts heavily with DNA, ethidium bromide is a mutagen, and the biology people sure did treat the dedicated electrophoresis bench area with respect! Figure 4. "Gel electrophoresis dedicated bench area to prevent ethidium bromide contamination.".

The UV transilluminator we used to shoot UV light into the gel was the Fisher Scientific UVP LM-26E Benchtop 2UV Transilluminator. The fluorescent substance then emitted a light we can see.

As barely seen at Figure 8. "Fischer Scientific UVP LM-26E Benchtop 2UV Transilluminator illuminated gel." due to bad photo quality due to lack of light, there is one strong green line, which compared to the ladder matches our expected 16S length. What we saw it with the naked eyes was very clear however.

How to use an Oxford Nanopore MinION to extract DNA from river water and determine which bacteria live in it / Post filtration purification Updated 2025-07-16

After filtration, all DNA should present in the filter, so we cut the paper up with scissors and put the pieces into an Eppendorf: Video 1. "Cutting vacuum pump filter and placing it in Eppendorf".

Cutting vacuum pump filter and placing it in Eppendorf

. Source.

Now that we had the DNA in Eppendorfs, we were ready to continue the purification in a simpler and more standardized lab pipeline fashion.

First we added some small specialized beads and chemicals to the water and shook them Eppendorfs hard in a Scientific Industries Inc. Vortex-Genie 2 machine to break the cell and free the DNA.

Video 2. Source.

Video 3. Source.

Once that was done, we added several reagents which split the solution into two phases: one containing the DNA and the other not. We would then pipette the phase with the DNA out to the next Eppendorf, and continue the process.

In one step for example, the DNA was present as a white precipitate at the bottom of the tube, and we threw away the supernatant liquid: Figure 1. "White precipitate formed with Qiagen DNeasy PowerWater Kit".

At various stages, centrifuging was also necessary. Much like the previous vacuum pump step, this adds extra gravity to speed up the separation of phases with different molecular masses.

In our case, we used a VWR Micro Star 17 microcentrifuge for those steps:

Then, when we had finally finished all the purification steps, we measured the quantity of DNA with a Biochrom SimpliNano spectrophotometer to check that the purification went well:

And because the readings were good, we put it in our -20 C fridge to preserve it until the second day of the workshop and called it a day:

How to use an Oxford Nanopore MinION to extract DNA from river water and determine which bacteria live in it / Pre-sequencing preparation Updated 2025-07-16

One cool thing we did in this procedure was to use magnetic separation with magnetic beads to further concentrate the DNA: Figure 1. "GE MagRack 6 pipetting.".

The beads are coated to stick to the DNA, which allows us to easily extract the DNA from the rest of the solution. This is cool, but bio people are borderline obsessed by those beads! Go figure!

Then we prepared the DNA for sequencing with the Oxford Nanopore specific part: Oxford Nanopore SQK-LSK109 Ligation Sequencing Kit.

Here some of the steps required a bit more of vortexing for mixing the reagents, and for this we used the VELP Scientifica WIZARD IR Infrared Vortex Mixer which appears to be quicker to use and not as strong as the Vortex Genie 2 previously used to break up the cells:

After all that was done, the DNA of the several 400 ml water bottles and hours of hard purification labour were contained in one single Eppendorf!

Parasites tend to have smaller DNAs Updated 2025-07-16

If you live in the relatively food abundant environment of another cell, then you don't have to be able to digest every single food source in existence, of defend against a wide range of predators.

So because DNA replication is a key limiting factor of bacterial replication time, you just reduce your genome to a minimum.

And likely you also want to be as small as possible to evade the host's immune system.

Power, Sex, Suicide by Nick Lane (2006) section "Gene loss as an evolutionary trajectory" puts it well:

One of the most extreme examples of gene loss is Rickettsia prowazekii, the cause of typhus. [...] Over evolutionary time Rickettsia has lost most of its genes, and now has a mere  protein-coding genes left. [...] Rickettsia is a tiny bacterium, almost as small as a virus, which lives as a parasite inside other cells. It is so well adapted to this lifestyle that it can no longer survive outside its host cells. [...] It was able to lose most of its genes in this way simply because they were not needed: life inside other cells, if you can survive there at all, is a spoonfed existence.

and also section "How to lose the cell wall without dying" page 184 has some related mentions:

While many types of bacteria do lose their cell wall during parts of their life cycle only two groups of prokaryotes have succeeded in losing their cell walls permanently, yet lived to tell the tale. It's interesting to consider the extenuating circumstances that permitted them to do so.
[...]
One group, the Mycoplasma, comprises mostly parasites, many of which live inside other cells. Mycoplasma cells are tiny, with very small genomes. M. genitalium, discovered in 1981, has the smallest known genome of any bacterial cell, encoding fewer than 500 genes. M. genitalium, discovered in 1981, has the smallest known genome of any bacterial cell, encoding fewer than 500 genes. [...] Like Rickettsia, Mycoplasma have lost virtually all the genes required for making nucleotides, amino acids, and so forth.

Paris Updated 2025-07-16

Ciro Santilli lived in Paris for a few years between 2013 and 2016, and he can confirm the uncontroversial fact that "Paris is Magic".

Not just one type of magic though. Every quarter in Paris has its own unique personality that sets it apart and gives it a different mood.

Ciro knows Paris not from its historical facts, but from the raw feeling of endless walks through its streets in different times of the year. Ciro is a walker.

Maybe one day Ciro will expand this section to try and convey into words his feelings of love for the city, but maybe the effort would be pointless. Maybe such feelings can only be felt by other free-roaming walker souls living in the city, and that is both beautiful and a shame.

Ciro had written the following in the past before he lived in smaller cities, started cycling and joined the Street reclamation movement he thought:

Paris is a friendly city to walkers, as it is not too large, and does not have too many extremely busy roads, you can basically cross all of it on foot.

Perhaps compared to São Paulo City, which is what he knew before that was true. But no, his standards have improved since. Paris has way too many cars. The noise of internal combustion engine vehicles is extremely annoying. And because there are too many personal vehicles, cars have to horn a lot to fight for space. Fuck cars. Paris has been making a big cycling push in the early 2020's, and that is great. But it is still far, far from good.

Path integral formulation Updated 2025-07-16

This one might actually be understandable! It is what Richard Feynman starts to explain at: Richard Feynman Quantum Electrodynamics Lecture at University of Auckland (1979).

The difficulty is then proving that the total probability remains at 1, and maybe causality is hard too.

The path integral formulation can be seen as a generalization of the double-slit experiment to infinitely many slits.

Feynman first stared working it out for non-relativistic quantum mechanics, with the relativistic goal in mind, and only later on he attained the relativistic goal.

TODO why intuitively did he take that approach? Likely is makes it easier to add special relativity.

This approach more directly suggests the idea that quantum particles take all possible paths.

Path to AGI Updated 2025-07-16

There are two main ways to try and reach AGI:

AI training robot: expensive, slow, but realistic world
AI training game: faster, less expensive, but possibly non-realistic-enough realistic

Which one of them to take is of of the most important technological questions of humanity according to Ciro Santilli

There is also an intermediate area of research/engineering where people try to first simulate the robot and its world realistically, use the simulation for training, and then transfer the simulated training to real robots, see e.g.: realistic robotics simulation.

Pauli-X gate Updated 2025-07-16