
Attempting to modify e-coli with CRISPR in my bathroom - Schiphol
https://benjamin.computer/posts/2018-04-20-e-coli.html
======
klmr
The post mentions CRISPR in the title and at several points but I can’t
actually discern at what step the CRISPR gene editing would have been
performed. As described, this mostly resembles a classical transgenic
experiment where a plasmid with an antibiotic resistance gene is introduced
(transfected) into the bacteria. This has nothing to do with CRISPR, which
allows more targeted modifications of a host genome than simple plasmid
transfection.

At any rate it’s a fun experiment that is performed, in similar form, in
biology in French high schools. Contrary to the tone in some of the comments
it’s not particularly dangerous (the bacteria can’t survive outside their agar
plates), though local regulations in some countries still might forbid it from
being performed at home.

Unfortunately the description is too imprecise to be sure what exactly the
author did.

~~~
RosanaAnaDana
Near as I can tell the only real reference to CRISPR is that he purchased a
plasmid that had been modified with CRISPR. Note: This is not what-so-ever
like using the CRISPR method to modify DNA. Modifying bacteria with a plasmid
as he is demonstrating here is no where near its equivalent. Every
introduction to biology course that I've ever seen performs the identical
procedure (and generally with much higher rates of success).

~~~
klmr
I think I got it: the plasmids (plural) contain the Cas9 and the gRNA
targeting _rpsL_ in the host organisms [1] (plus the DNA template for the
homology directed repair).

[1] [http://www.the-odin.com/crispr-bacterial-guide/](http://www.the-
odin.com/crispr-bacterial-guide/)

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Obi_Juan_Kenobi
Some issues and general observations:

* Making plates is hard. You really need an autoclave, and a laminar flow hood is also very helpful. For a DIY approach, you need a pressure cooker and a bunsen burner (in a relatively still room, the bunsen burner creates a radius of sterile space around it. Yes, this is legitimate lab technique, if a bit old-school). Those plates were clearly contaminated, and without proper sterile technique, nothing whatsoever is going to work. The kit's instructions, in my view, are unlikely to produce sterile plates. They provide sterilized agar, but need to be poured in a sterile area, but make no provision for this.

* The instructions for the kit are just barely sufficient. There are a million different ways you can do things incorrectly, so very clear and thorough steps are needed for a novice to have a chance at success. A lot of troubleshooting material should be included as well. This is a setup for failure in my view. For instance, an un-inoculated control plate is a bare minimum control. For a HN-friendly analogy, it would be like debugging software without any error reporting, print statements, etc.

* Lab equipment is mostly fridges, water baths, plastic disposables, and moderately specialized stuff like tabletop centrifuges and thermocyclers. It seems relatively accessible, which gets people excited about DIY microbiology, but it's deceptive. Temperatures, and the stability of temperatures, can matter a lot. Reliable equipment is very important. Someone seriously looking to do this (it's certainly possible!) needs to be able to devote a fair amount of space and resources to their home lab.

* The #1 difference between a 'real' lab and a home lab is access to troubleshooting resources. Shit goes wrong all the time, and experienced post-docs, lab techs, colleagues, etc. are what make research possible. You also very often need access to quality materials for positive and negative controls. It's not uncommon for entire labs to grind to a halt for weeks or months until some tricky bit of troubleshooting is resolved.

Some people are commenting that this isn't 'CRISPR'. It is. The transformation
provides a plasmid that will not convey resistance on it's own (unless they're
being dishonest about what's in the kit). Only when a stable transformation of
the genome occurs will resistance be seen.

~~~
dekhn
I spent a year in a lab learning how to clone genes into E.Coli. it's amazing
how easy it is to screw up. Once, we got a bottle of broth back from the
autoclave (it should be completely sterile) and within a week or so of sitting
on the shelf, unopened, there was already a cloudy membrane growing inside.

What I learned from the process is that it's all about control experiments and
troubleshooting. To make a real discovery today, or even just convince
yourself you succeeded in a process, you need to be outright paranoid about
every component in your system. I wasted a lot of time because I started with
only negative controls, and learned the hard way that you need positive
controls.

That doesn't even begin to describe all the problems I had extracting reliable
data from gels. I'm convinced that all the dark secrets in biology are in the
gels.

Even when I worked for a major biotech company, when i talked to the guys who
ran the continuous fermentors said they always saw strange fluctuations in
things like bulk metabolic rate, drug production, etc, that they couldn't
explain, and they were _the_ experts in reliably growing liters of e.coli.

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roel_v
What worried me most about those pictures is that there is a can in that
fridge that says 'breakfast pizza'. Is that a real thing or is this very
subtle trolling?

~~~
DivisionSol
No, that probably isn't a can of breakfast pizza, just looks like a recipe
that's usually printed on cans of vegetables. Further, if you were worried
that breakfast pizza was something unusual, it's just normal pizza, not
specifically for breakfast, but using breakfast ingredients (eggs, bacon,
potatoes.) Often times cans of vegetables include a recipe to... fill space?
Provide value?

The can itself looks like a can of olives (warning at the bottom says to watch
out for pits, the ingredient line for olives is very specific as to what type
of olives to use, olives are fruits in a botanical sense.)

~~~
Jach
Is there a large part of the world I'm ignorant of which considers olives and
mushrooms to be breakfast ingredients?

~~~
dekhn
they are not uncommon in omlettes. Spanish Omlette- olives & red pepper. Also,
I would imagine that the Japanese eat miso soup with mushrooms for breakfast.

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tomcam
Intro contains two of the greatest lines ever blogged:

>Grow some bacteria

>Take some bacteria and make it competent

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cwkoss
I wonder if anyone has genetically modified microorganisms to synthesize
illegal drugs. Seem like if this happens, the DEA is going to have a really
rough time.

~~~
dekhn
You mean like this?
[http://science.sciencemag.org/content/349/6252/1095](http://science.sciencemag.org/content/349/6252/1095)
or this?
[https://www.ncbi.nlm.nih.gov/pubmed/25994576](https://www.ncbi.nlm.nih.gov/pubmed/25994576)

~~~
cwkoss
Wow! Very interesting, thanks for those links

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skynetv2
is he risking his family, and possibly neighbors, with this setup in his
bathroom?

asking with genuine concern - i have no idea how contagious e-coli would be
and what its medium of spreading is.

~~~
coryrc
Your intestines are filled with e-coli. It spreads via contaminated water (or
food, usually water).

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noworld
"The e-coli experiment you can buy is an attempt to create bacteria that can
survive on an anti-bacterial plate of agar."

That's exactly what we need!

~~~
dddddaviddddd
This is standard procedure in microbiology. Resistance is to one known
antibiotic rather than an attempt to create a MSR monster. This allows one to
quickly verify if a trait intended to be conveyed with the antibiotic
resistance was transferred, since cells without the package will just die.

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dddddaviddddd
This is so hacky compared to working in a well-equipped lab. Not having
analysis instruments also limits how much can be accomplished.

~~~
SmellyGeekBoy
You say this like it's a bad thing? I'm more amazed that anything like this is
even possible outside of a lab environment.

~~~
dnautics
I've engineered a 20kbp yac in my kitchen, but also I have a PhD in chemistry
and real molecular biology experience.

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nimbius
>Attempting to modify e-coli with CRISPR in my bathroom

This either sounds like the third sentence uttered in a 911 call, The FBI
justification for a lifetime supermax incarceration, or the cause of death and
mortality summary on a medical report.

~~~
saalweachter
It made me think, "I wonder if you could modify whatever bacteria is growing
in my bathroom to emit a fresh lemon scent, so it would always _smell_ freshly
cleaned."

~~~
dekhn
We can do minty:
[https://pubs.acs.org/doi/pdf/10.1021/acssynbio.5b00092](https://pubs.acs.org/doi/pdf/10.1021/acssynbio.5b00092)

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amaccuish
Hmm, am I the only one concerned here? This technology should be strongly
regulated. I don't want some "biohacker" accidentally creating AB resistent
bacteria in their back garden...

~~~
sannee
> I don't want some "biohacker" accidentally creating AB resistent bacteria in
> their back garden...

There are much easier ways of creating AB resistant bacteria than CRISPR. In
some countries, they even give out the necessary chemical reagents for free to
anyone with a bit of patience!

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alanfranzoni
Would somebody explain if that actually makes (sort of) sense or it's pure
trolling?

~~~
1996
The accusations of trolling can do a lot of damage.

Some people find hacking ecoli is cool - I am one of them, even if this
experiment failed it is great to see what is possible right now.

~~~
alanfranzoni
Where is the accusation? Mine was a question. And, according to other
comments, the title is actually a click-bait (no CRISPR involved). Probably I
used the word "trolling" with a bit of a lateral meaning (something like "make
fun of other people"), improperly.

~~~
klmr
> no CRISPR involved

Please see my follow-up comment: I was able to find the protocol of the kit
that the author likely used and it looks like a CRISPR experiment after all.
Its description just didn’t sound like it (the actual steps are somewhat
similar between the two experiments).

~~~
alanfranzoni
Thanks for the clarification. Mine was still an honest question.

