My first successful CRISPR experiment on E. Coli ?

[Hugues]

Hi guys,

i think i have successfully CRISPRed an E. Coli bacteria,

I have used the kit sold here:

http://www.the-odin.com/diy-bacterial-gene-engineering-crispr-kit/

I followed the protocol here:

https://docs.google.com/document/d/19RNlo-psr_kTeJtKOZK6ruFW18Uu7SIX4zZtHSc9dzQ/edit

The gene that was targeted is  Escherichia coli 30S ribosomal subunit protein S12 (rpsL) gene. One base pair was changed to render the bacteria resistant to streptomycin.

The picture attached shows my result. That plate contains a gel with streptomycin. The top part shows growth on the CRISPRed strain, the bottom part shows not growth on the normal strain.

What you guys think ?

I'm thinking of having the DNA of that gene sequenced, just to check if the edit really happened only where expected.

[Sebastian S Cocioba]

Picture shows both grew on LB Strep, are you sure your wild type is not resistant? The Wild Type looks far more resistant...or cell density at streaking point was higher. If you have access you may want to PCR to confirm. Also use a high fidelity polymerase since you are looking for a single nucleotide change. Either way glad you conducted the experiment. I would suggest doing a repeat or two just to be sure :)

Sebastian S. Cocioba

CEO & Founder

New York Botanics, LLC

Blog: ATinyGreenCell.com

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<IMG_20161218_091257.jpg>

[Hugues]

thanks for your feed-back,

i'm definitely not an expert on growing bacterial colonies, at least i'm trying to take a shower before they become to obvious LOL

i'm attaching a better quality picture. If you zoom in, you will see round dots on the CRISPR side, dots you do not see at the bottom. These are the kind of dots i had on the first plate i used to grow my normal strain (on a gel without strep). For me, these whitish streaks do not seem to be growing colonies, the streak appeared rather rapidly after i streaked both strains, once they were dried, but then they stayed like this. After 15h, i started to see the little dots on one side.

Don't know if this helps.

[Koeng]

It appears to me that you have not gotten editing. How old are the plates?

-Koeng

[Hugues]

They were 15h old at the moment of the picture.

What makes you think it did not work ? I did not have any dots at the beginning, now there are, so something is growing.

[Nathan McCorkle]

The odd thing about what you said is "For me, these whitish streaks do

not seem to be growing colonies, the streak appeared rather rapidly
after i streaked both strains, once they were dried, but then they
stayed like this."

So if you followed the instructions, you'd have dropped 100uL onto the
plate, then spread it around. From my memory, and the last time I
spread cells was probably 4 years ago, 100uL is reasonably wet... so I
would not recommend mixing strains on the same plate/dish.

Continuing, if the appearance of the agar hasn't changed ("but then
they stayed like this"), seems like you might have over-dried to begin
with. A spread of culture, assuming you don't scrape/scratch the agar,
should look like a dirty/oily window, rather than a window with
mucus/paste obviously spread over it (what I'd call yours).

A question I have, aside from why you tried to plate so much of two
cultures (100uL each) onto a single plate, is, did you mix the culture
before plating? I don't think this really matters experimentally, but
just thinking about reasons why your dry streaks look so thick (i.e.
maybe you didn't mix, sucked up the cell-pellet that settled at the
bottom of the tube).

On Thu, Dec 22, 2016 at 12:30 PM, Hugues <lalibe...@gmail.com> wrote:
> They were 15h old at the moment of the picture.
>
> What makes you think it did not work ? I did not have any dots at the beginning, now there are, so something is growing.
>

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[ukitel]

I'm afraid there is no streptomycin on the plates.
Can you describe in detail how you made the plates?

[Bryan Jones]

It definitely looks like you have growth in both sides of the plate. The bottom doesn't have dots because the growth is too thick to see gaps between the colonies.

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[Hugues]

thanks for all the feed-back guys, this is kind of a peer review of my first paper published on the DIY Biologist, LOL.

The next few days are a bit busy, but i'll come back next week with specific feed-back and potential extra test to validate my hypothesis, before i shed a couple hundred bucks on having the little critters sequenced.

I don't have all the consumables to run another experiment but I still have normal Agar plates and some with Strep, so if someone has an idea how i can use these plates to further validate (or falsify) my hypothesis then your proposals are mostly welcomed.

thanks

On Thursday, 22 December 2016 20:20:47 UTC+1, Hugues wrote:

[Simon Quellen Field]

This hypothesis should not be too difficult to test.

Perhaps viewing the non-dot areas under a microscope (perhaps with a stain for that species) would quickly find individuals, or taking a small sample of that area and streaking another plate to see if colonies form.

Cheaper than sequencing, and faster.

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On Thu, Dec 22, 2016 at 3:47 PM, Bryan Jones <bryan...@gmail.com> wrote:

It definitely looks like you have growth in both sides of the plate. The bottom doesn't have dots because the growth is too thick to see gaps between the colonies.

On Thu, Dec 22, 2016, 5:38 PM ukitel <marco.r...@gmail.com> wrote:

I'm afraid there is no streptomycin on the plates.
Can you describe in detail how you made the plates?

On Thursday, 22 December 2016 20:20:47 UTC+1, Hugues wrote:

Hi guys,

i think i have successfully CRISPRed an E. Coli bacteria,

I have used the kit sold here:

http://www.the-odin.com/diy-bacterial-gene-engineering-crispr-kit/

I followed the protocol here:

https://docs.google.com/document/d/19RNlo-psr_kTeJtKOZK6ruFW18Uu7SIX4zZtHSc9dzQ/edit

The gene that was targeted is  Escherichia coli 30S ribosomal subunit protein S12 (rpsL) gene. One base pair was changed to render the bacteria resistant to streptomycin.

The picture attached shows my result. That plate contains a gel with streptomycin. The top part shows growth on the CRISPRed strain, the bottom part shows not growth on the normal strain.

What you guys think ?

I'm thinking of having the DNA of that gene sequenced, just to check if the edit really happened only where expected.

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[Hugues]

Hi Nathan,

i'm not sure anymore if these white streaks are/were growing at some point, all new to me.

But the picture i show are from my second experiment. I thought my first one had failed because i had only white streaks on both side of the plate and no dots.

In my first experiment i followed the protocol and indeed poured 100 ul of the CRISPRed mix onto the plate, it was indeed quite wet. I thought this was the reason why it failed. But i maybe mistaken.

On the second experiment, the picture i've shown, i simply used an inoculation loop to pick up cells from my CRISPRed mix and streak them on my plate, although generously. At first it was transparent on both side. The whitish color only came after maybe an hour (max 2-3 hours) on my 37 C heat plate. Then the dots on one side appeared over night.

[Hugues]

Hi,

the normal Agar LB and the Strep LB came in powder form in 2 tubes with the kit. I just followed the instructions and dissolved the powder in water, then heated in microwave a bit until it became clear, then poured in the plate. I prepared each tube separately and labeled the plates, so impossible to mix the plates. I did not invert the plate, or else my first normal strain would not have grown.

Of course i don't know if the supplier did not mix the tubes.

[Hugues]

yes good idea

i will take a loop, scrape off the little dots, spread on normal LB plate and Strep LB plate. 

I will do the same (with another loop), scrape off the withish streaks on the wild type side and spread them on new normal LB and strep plates.

Then incubate and post the results.

But can't do this until after the 28th. In the mean time, my plate is in the fridge at 4C. Will enough bacterias survive until then ?

On Friday, 23 December 2016 15:13:44 UTC+1, Simon Field wrote:

This hypothesis should not be too difficult to test.

Perhaps viewing the non-dot areas under a microscope (perhaps with a stain for that species) would quickly find individuals, or taking a small sample of that area and streaking another plate to see if colonies form.

Cheaper than sequencing, and faster.

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On Thu, Dec 22, 2016 at 3:47 PM, Bryan Jones <bryan...@gmail.com> wrote:

It definitely looks like you have growth in both sides of the plate. The bottom doesn't have dots because the growth is too thick to see gaps between the colonies.

On Thu, Dec 22, 2016, 5:38 PM ukitel <marco.r...@gmail.com> wrote:

I'm afraid there is no streptomycin on the plates.
Can you describe in detail how you made the plates?

On Thursday, 22 December 2016 20:20:47 UTC+1, Hugues wrote:

Hi guys,

i think i have successfully CRISPRed an E. Coli bacteria,

I have used the kit sold here:

http://www.the-odin.com/diy-bacterial-gene-engineering-crispr-kit/

I followed the protocol here:

https://docs.google.com/document/d/19RNlo-psr_kTeJtKOZK6ruFW18Uu7SIX4zZtHSc9dzQ/edit

The gene that was targeted is  Escherichia coli 30S ribosomal subunit protein S12 (rpsL) gene. One base pair was changed to render the bacteria resistant to streptomycin.

The picture attached shows my result. That plate contains a gel with streptomycin. The top part shows growth on the CRISPRed strain, the bottom part shows not growth on the normal strain.

What you guys think ?

I'm thinking of having the DNA of that gene sequenced, just to check if the edit really happened only where expected.

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[Bryan Jones]

They will survive in the fridge for weeks.

To view this discussion on the web visit https://groups.google.com/d/msgid/diybio/cf070326-f600-423a-a746-fb1f3d1aa5b2%40googlegroups.com.

[Nathan McCorkle]

On Fri, Dec 23, 2016 at 10:22 AM, Hugues <lalibe...@gmail.com> wrote:
> Hi,
>
> the normal Agar LB and the Strep LB came in powder form in 2 tubes with the
> kit. I just followed the instructions and dissolved the powder in water,
> then heated in microwave a bit until it became clear,

As mentioned in this comment, degradation might be an issue above 75
degrees F (it says prior to some patent invention, but from reading
the patent it seems like they just found a good pH and buffer system
to help get around this issue):
https://groups.google.com/d/msg/diybio/GsPMAfyfAkU/R2NmH4XZad8J

So my point is, maybe you overcooked the streptomycin... you want to
be very gentle on the solution as you microwave it (pulses of 10 to 30
seconds at a time depending on volume of liquid, then swirl to mix,
then heat again, swirl, heat)... this is even more important if your
microwave lacks a rotating plate (you could get hotspots that are
super-heated, and thus more easily degrade unstable molecules in that
volume of liquid).

[Mega [Andreas Stuermer]]

Try not spending hundreds of bucks on sequencing a single point mutations... You can either try to sequence a PCR product (2 PCR primers each 3-7 dollars, a sequencing primer 3-7$; and a sequencing run - 7$) or try directly from extracted genomic DNA (a sequencing primer 3-7$; and a sequencing run - 7$).

If you need help designing the PCR, feel free to ask ;)

[Hugues]

Following up on my earlier experiment,

I have scrapped off "mutant" bacteria from the strep plate and spread it onto a fresh strep plate and a normal plate.

I have scrapped off wild type bacteria from the strep plate and spread it onto a fresh strep plate and a normal plate.

The first picture below shows the plates just after a spread the bacteria on them.

The second picture is of the same two plates after 2 days of incubation at around 37 C.

I guess there is growth on all 4 colonies, no real difference between strep plate and normal plate. No real difference between "mutant" and wild type on strep plate.

So i cannot conclude with this that i have successfully CRIPSR'ed that bacteria. But i cannot rule out that i have edited it either.

Puzzling to see growth of wild type on strep plate. Maybe there is no streptomycin finally on that plate. Or as it was earlier proposed, i might have deactivated the strep when preparing the LB gel. I did boil it a few seconds in the microwave.

I'll try to find a cheap place to sequence these 2 colonies, just to get the final call.

On Thursday, 22 December 2016 20:20:47 UTC+1, Hugues wrote:

[John Griessen]

On 01/01/2017 01:52 PM, Hugues wrote:
> Following up on my earlier experiment,
>

> I have scraped off "mutant" bacteria from the strep plate and spread it onto a fresh strep plate and a normal plate.
>
> I have scraped off wild type bacteria from the strep plate and spread it onto a fresh strep plate and a normal plate.

>
> The first picture below shows the plates just after a spread the bacteria on them.
>
> The second picture is of the same two plates after 2 days of incubation at around 37 C.

You have spread a thick layer of bacteria, since it is visible just after transferring it. It obviously grew.
Wouldn't it be good to use streaking technique to see the results of some individual cells growing into colonies?
That might show differences between colonies if you are starting with a mixture. What you have here looks like colonies
touching each other.

What does that mean when you say, "I guess there is growth on all 4 colonies"? Is the first picture after
enough time for growth?

The left right growth rates do look close to the same.

"Maybe there is no streptomycin finally on that plate" What is a good simple test of that?

"it controls algae in ornamental ponds and aquaria." I have no experience with this -- just found it on wikipedia, but
it might be an easy test of whether you have streptomycin that is still effective. See if your boiled streptomycin
kills pond algae faster than boiled rain water side by side.

[ukitel]

Hey Hugues,

I agree with John, there is growth but there is way too much bacteria on the plate.

Look up for streaking techniques: you really need a tiny amount of bacteria to spread all over the plate.

When you have that many bacteria, streptomycin is not able to kill all of them.

It is a known phenomenon that if you plate too many bacteria or cells in a dish, they are more difficult to be killed by the antibiotic.

Best!

[Hugues]

ok fair,

if a good streaking technique is shown in this video:

https://www.youtube.com/watch?v=Ay2hhujTuvg

specifically at 1'54"

then yes i took too large of a sample in my streaks.

so i'll do it again and this time take a tiny bit only.

thanks

[Hugues]

so as mentioned in my last post i streaked the "mutant" and the wt onto another plate with strep,

this time i took a very small qty of each colony and spread it as much as i could,

As you can see in below pictures (0, +6h, +9h and +30h after streaking), initially i could barely see any trace, then both colonies grew, maybe a little less on the wt side, but i don't know if it is significative.

So i would think i don't really have strep on this plate. 

I guess i would have to sequence both colonies to be really sure if i managed to edit the "mutant" or not.

On Thursday, 22 December 2016 20:20:47 UTC+1, Hugues wrote:

[Abizar Lakdawalla]

If you are not sure of streptavidin, you can add sterile strepatvidin solution on the top of the plates(0.5 ml - spread by tilting the plate), let dry.

Use two separate plates, one for WT, one for mutant. On each plate streak a loopful (about 10=20ul of bacteria) using the T-method (draw a T on the back of your dish, first streak the bacteria at the top of a T (red in diagram), then sterilize the loop, and pull down a streak from the top of the T down the leg of the T (yellow in diagram), sterilize loop again, and then starting from the bottom zigzag up (green in diagram) leaving about 1 to 2 cm from the cross line of the T (a sterile zone)). Incubate, you should get single colonies.

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[Abizar Lakdawalla]

with the t streaking method image

On Sun, Jan 15, 2017 at 8:55 AM, Hugues <lalibe...@gmail.com> wrote:

--

[Hugues]

thanks for the guidance,

the CRISPR edit is for streptomycin resistance, i think i can buy streptomycin here:

http://www.sigmaaldrich.com/catalog/product/sigma/p4333?lang=fr&region=CH

On Sunday, 15 January 2017 18:44:57 UTC+1, Abizar Lakdawalla wrote:

If you are not sure of streptavidin, you can add sterile strepatvidin solution on the top of the plates(0.5 ml - spread by tilting the plate), let dry.

Use two separate plates, one for WT, one for mutant. On each plate streak a loopful (about 10=20ul of bacteria) using the T-method (draw a T on the back of your dish, first streak the bacteria at the top of a T (red in diagram), then sterilize the loop, and pull down a streak from the top of the T down the leg of the T (yellow in diagram), sterilize loop again, and then starting from the bottom zigzag up (green in diagram) leaving about 1 to 2 cm from the cross line of the T (a sterile zone)). Incubate, you should get single colonies.

On Sun, Jan 15, 2017 at 8:55 AM, Hugues <lalibe...@gmail.com> wrote:

so as mentioned in my last post i streaked the "mutant" and the wt onto another plate with strep,

this time i took a very small qty of each colony and spread it as much as i could,

As you can see in below pictures (0, +6h, +9h and +30h after streaking), initially i could barely see any trace, then both colonies grew, maybe a little less on the wt side, but i don't know if it is significative.

So i would think i don't really have strep on this plate. 

I guess i would have to sequence both colonies to be really sure if i managed to edit the "mutant" or not.

On Thursday, 22 December 2016 20:20:47 UTC+1, Hugues wrote:

Hi guys,

i think i have successfully CRISPRed an E. Coli bacteria,

I have used the kit sold here:

http://www.the-odin.com/diy-bacterial-gene-engineering-crispr-kit/

I followed the protocol here:

https://docs.google.com/document/d/19RNlo-psr_kTeJtKOZK6ruFW18Uu7SIX4zZtHSc9dzQ/edit

The gene that was targeted is  Escherichia coli 30S ribosomal subunit protein S12 (rpsL) gene. One base pair was changed to render the bacteria resistant to streptomycin.

The picture attached shows my result. That plate contains a gel with streptomycin. The top part shows growth on the CRISPRed strain, the bottom part shows not growth on the normal strain.

What you guys think ?

I'm thinking of having the DNA of that gene sequenced, just to check if the edit really happened only where expected.

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[Nathan McCorkle]

amazon and ebay also has some strep, but it's sold with additional "inert ingredients"... might be worth trying for the $14 or whatever it costs with shipping for you.

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