cheaper/better transformation - using ultrasound (sonoporation)

[JonathanCline]

All,
I haven't seen reference to this method on openwetware however it
seems like it should be a standard protocol for GEM's. Can anyone
comment?

Listed under standard biobrick protocols are chemical transformation &
electroporation (recently discussed). Both have drawbacks. From my
perspective, chemical transportation is costly (reagents) and
inefficient; electroporation is expensive (equipment) or if built DIY,
is dangerous (fatal voltages). And both are stressful to the poor
bugs themselves, in different ways.

Sonoporation also seems possible, it uses acoustic standing waves to
force plasmids into cells, requiring only ultrasonic transducers (with
programmable signal time, such as via function generator or
microcontroller) and specific buffers. This method seems intensively
studied recently, for targeted gene therapy (drug therapy) in human
cells, though it is easily applicable to bacteria & yeast for
transformation. Also applicable to cyanobacteria.

Many of the articles aren't open science, so can't access them. Search
for "sonoporation" or "ultrasound plasmid" for related papers.

Here is one full text article link below, with a small exerpt to
describe what I'm referring to.


Ultrasound-mediated DNA transfer for bacteria
http://nar.oxfordjournals.org/cgi/content/full/35/19/e129

QUOTE:

We report here that a standard low frequency (40 kHz) ultrasound clean
bath can be used to successfully and efficiently deliver plasmid
pBBR1MCS2 into Pseudomonas putida UWC1. Optimized conditions gave a
delivery efficiency of 9.8 ± 2.3 x 10–6 transformants per cell, which
was significantly higher than the results of conjugation and even
electroporation.

We propose a mechanism of ultrasound-mediated plasmid transfer of
bacteria in which plasmid transfer is enhanced in the presence of
CaCl2 (Figure 4): Plasmid DNA and bacteria are initially well-mixed in
an aqueous solution (Figure 4A). The addition of CaCl2 causes changes
in the conformation of plasmid DNA or cellular membrane structures
that promote transformation (Figure 4B). As low frequency 40 kHz
ultrasound is applied to the solution the transmitted energy causes
temporary porosity in the cell membrane, which enables the plasmids to
enter through the pores (Figure 4C). When the ultrasound is switched
off the cell membrane repairs itself and the transformed cell retains
the plasmid DNA (Figure 4D). Bacteria acquire new functions, such as
growing on a selective medium, after taking up plasmid DNA (Figure
4E).


## JonathanCline
## jcl...@ieee.org
## Mobile: +1-805-617-0223
########################

[cory....@gmail.com]

> Listed under standard biobrick protocols are chemical transformation &
> electroporation (recently discussed).  Both have drawbacks.  From my
> perspective, chemical transportation is costly (reagents) and
> inefficient;

It's really not that expensive. All you need is calcium chloride and
magnesium chloride, the same stuff they sprinkle on icy roads.

Make your competent cells w/ 80mM MgCl2 and 20mM CaCl2. Re-suspend
the cells in 0.1M CaCl2. Add up to 50ng of your plasmid to 200uL of
competent cells. Put the tube in a 42C water bath for 90 seconds,
then put it on ice for a half hour. I have never had any efficiency
problems using this protocol. I can send you a complete protocol if
you want.

> And both are stressful to the poor
> bugs themselves, in different ways.

Sure, but so is sonoporation. They are all just different ways of
making holes in bacterial membranes.


-Cory Tobin

[Tito Jankowski]

Jonathon,

I like that you're looking for alternatives to accepted technologies -- sonoporation is worth some further reading.

For anyone who needs access to publications, but doesn't have them, send an email and the name of the article to:

getar...@googlegroups.com

You'll receive a PDF of the article from another member with access to the publication - I've gotten articles back in about 10 minutes.

Tito

[Mega]

There once was a sonic cleaner in the store for some 20€. 

I thought about buying it, but won't it damage the plasmids while transforming????? 

[Eugen Leitl]

Do you want to sonickate DNA? Why on earth?

[Patrik D'haeseleer]

On Sunday, February 24, 2013 3:56:56 AM UTC-8, Mega wrote:

There once was a sonic cleaner in the store for some 20€. 

I thought about buying it, but won't it damage the plasmids while transforming????? 

I just bought one of these to clean the cartridges  for the BioPrinter project:

http://www.amazon.com/Sonic-CD-2800-Ultrasonic-Jewelry-Cleaner/dp/B001DKDAVW

$25.50 with free shipping is quite a deal! I've looked for these in the past, and I don't think I've ever seen them for less than $50-$60. Guess we'll have to give the sonoporation a try as well....

[Patrik D'haeseleer]

On Sunday, February 24, 2013 3:56:56 AM UTC-8, Mega wrote:

There once was a sonic cleaner in the store for some 20€. 

I thought about buying it, but won't it damage the plasmids while transforming????? 

"Ultrasound treatment for 5–60 s results in plasmid transfer to P. putida UWC1 (P < 0.05). Exposure time of 10 s gives the highest transfer efficiency. No plasmid transfer occurred in the absence of ultrasound treatment. (B) Ultrasound reduced P. putida UWC1 survival following 60 s exposure time, but shorter treatments had little effect on bacterial survival (P < 0.05). (C) Plasmid DNA was prone to cleavage following ultrasound exposure times longer than 30 s"

So yes, you can definitely damage the plasmids and the cells themselves with longer exposure times. But you only need a 10s exposure to get optimal DNA transfer.

[Cathal Garvey]

-----BEGIN PGP SIGNED MESSAGE-----
Hash: SHA256

Does the paper include information on frequency and wattage? I've not
yet tried sonoporation but I'd love to see it tested using
commercially available jewellery cleaners, because there's a lot of
"who knows?" around the issue. If the frequencies are significantly
different, it:
A) Mightn't work at all
B) Might need more or less time to work
C) Might have a different spectrum of useless/useful/murderous activity.

Give it a go! :)

On 02/25/2013 07:27 AM, Patrik D'haeseleer wrote:
>
>
> On Sunday, February 24, 2013 3:56:56 AM UTC-8, Mega wrote:
>

> There once was a sonic cleaner in the store for some 20�.

>
>
> I thought about buying it, but won't it damage the plasmids while
> transforming?????
>
>

> "Ultrasound treatment for 5�60 s results in plasmid transfer to
> /P. putida/ UWC1 (/P/ < 0.05). Exposure time of 10 s gives the

> highest transfer efficiency. No plasmid transfer occurred in the

> absence of ultrasound treatment. (B) Ultrasound reduced /P. putida/

> UWC1 survival following 60 s exposure time, but shorter treatments

> had little effect on bacterial survival (/P/ < 0.05). (C) Plasmid

> DNA was prone to cleavage following ultrasound exposure times
> longer than 30 s"
>
> So yes, you can definitely damage the plasmids and the cells
> themselves with longer exposure times. But you only need a 10s
> exposure to get optimal DNA transfer.
>

> -- -- You received this message because you are subscribed to the
> Google Groups DIYbio group. To post to this group, send email to
> diy...@googlegroups.com. To unsubscribe from this group, send email
> to diybio+un...@googlegroups.com. For more options, visit
> this group at https://groups.google.com/d/forum/diybio?hl=en Learn
> more at www.diybio.org --- You received this message because you
> are subscribed to the Google Groups "DIYbio" group. To unsubscribe
> from this group and stop receiving emails from it, send an email to
> diybio+un...@googlegroups.com. To post to this group, send
> email to diy...@googlegroups.com. Visit this group at
> http://groups.google.com/group/diybio?hl=en. To view this
> discussion on the web visit
> https://groups.google.com/d/msg/diybio/-/8_-Z_wwWKE4J. For more
> options, visit https://groups.google.com/groups/opt_out.
>
>

- --
Please note my new email: cathal...@cathalgarvey.me
PGP Key: 988B9099
Bitmessage: BM-opSmZfNZHSzGDwdD5KzTnuKbzevSEDNXL
Twitter: @onetruecathal
Code: https://gitorious.org/~cathalgarvey
Blog: http://www.indiebiotech.com
-----BEGIN PGP SIGNATURE-----
Version: GnuPG v1.4.11 (GNU/Linux)
Comment: Using GnuPG with Thunderbird - http://www.enigmail.net/

iQIcBAEBCAAGBQJRKz7pAAoJEL0iNgSYi5CZo/oQANnd8Z8/q46UDD0AwYBkhv4A
weZ/LfmaRVLSrSAf4eBjS8rUaVYOs+6SxuGP6Y9QZne1NIqbPQd64QmKrjdbve3B
FKX1rz8X/zLqsJSTxWhdHOANuRJsOcboeUGAe3F+YCg3SEZULzVhCrRdmAtHUoGX
nF7DjrnEXSxZ/RtIMCHHaAKL0n08bs+tO4Oou68GmowpdPg/5YQdPZXFKW9Cag7h
98bCdkHsnp/U+ox4bfPOmSixvIcEe4uyrbqv76nRoP5/tIOoQes0dexJlTVvop0n
PwJ3XNQCJ8d1NYXatARxXGuUubW88O31YsS91mUvKi3I7KhPvHoPNUzMQcReP39Z
jy7KjTJDqWke26p1kAI66vDAvjY4kvKsV9d3Z80bXKlMAGKW8j8xdVRDlA8p+Sjw
mjsSUMDd+pBLRMcAIxpcINyUb7NBxsDUDzbEgNaXZLeooKmbWx2Dcmlg9AsfepND
Yu4EkKMfe1W5r211JJ6+W45RZqTsRRiFbK8vT/8hwAsIytCl80Tlz4kmnlStYlJo
7cHOFQy6nfRpR0NkaPaEvI7dFjgQVJUrTYqjGwf2tWO2eSVtzlKjbt3U250pCy8J
+ywLHcv68mdFrdT2FjfBjk5AesPak6YTW9ML1sgdEMA25EjWufAoKprpBtACISbb
Fj6by6y2UFaYThrqjNgf
=XJ5B
-----END PGP SIGNATURE-----

[Mega]

Would be interesting. 

But some of the transformants may have taken up damaged DNA, so you may get false-positives?? I just assume. 

[Patrik D'haeseleer]

Alright - sounds like we're going to give this a go at BioCurious this week, using the 35W model I got. We'll let you know how it works out...

Patrik

[Jonathan Cline]

I looked at the specifications for the commercial jewelry sonic cleaners and they are within range of sonoporation protocols -- it's been awhile, but 25 kHz sticks in my head as the spec of typical jewelry cleaners whereas lab equip uses 40 kHz -- well guess what, biology likely doesn't care +/- 50%, it's probable that the lab postdocs authored/optimized the protocol with the equipment they had on hand at the time, which was probably.... 40 kHz!     The freq of the commercial unit will be fixed.  The pressure level at the node is what's important I believe (not really wattage) and might vary quite a bit with cheap units.    But I believe a microbubble agent is required as explained in http://en.wikipedia.org/wiki/Sonoporation  (I'm rather happy of writing that wikipedia entry 'cause it's one of the few I've written that remained factual/undoctored..)

"standard low frequency 40 kHz ultrasound bath" -- doi: 10.1093/nar/gkm710


BTW remind the biohackers not to stick their fingers in the bath...


## Jonathan Cline

## jcl...@ieee.org
## Mobile: +1-805-617-0223
########################

On Monday, February 25, 2013 2:37:30 AM UTC-8, Cathal Garvey wrote:

[Patrik D'haeseleer]

Success! Here's a picture of pGLO E.coli colonies under UV light, transformed using the $25 35W 42kHz ultrasonic cleaner I bought to clean the bioprinter cartridges.

I haven't seen the exact protocols used for this - and I doubt they did much in terms of optimization for this particular ultrasonic cleaner. But definitely a clear proof of principle...

Patrik

[Mega]

Awesome!! How much ug plasmid did you use?

[Avery louie]

well done!  I am also curious.  That looks like pretty low yield for compared to what i normally see from heat shock, but I would like to know more about the protocol.

--A

On Fri, Mar 1, 2013 at 4:42 AM, Mega <masters...@gmail.com> wrote:

Awesome!! How much ug plasmid  did you use?

--
-- You received this message because you are subscribed to the Google Groups DIYbio group. To post to this group, send email to diy...@googlegroups.com. To unsubscribe from this group, send email to diybio+un...@googlegroups.com. For more options, visit this group at https://groups.google.com/d/forum/diybio?hl=en
Learn more at www.diybio.org
---
You received this message because you are subscribed to the Google Groups "DIYbio" group.
To unsubscribe from this group and stop receiving emails from it, send an email to diybio+un...@googlegroups.com.
To post to this group, send email to diy...@googlegroups.com.
Visit this group at http://groups.google.com/group/diybio?hl=en.

To view this discussion on the web visit https://groups.google.com/d/msg/diybio/-/iKZNHhtAlLgJ.

[Patrik D'haeseleer]

Hold yer horses! I wasn't involved in this experiment - just forwarded the picture. I'll let you know as soon as I know any more details.

Yes, the yield looks low, but I doubt they did much (or any?) optimization of the protocol so far. Not too bad for something that worked on the first try!

Patrik

[Avery louie]

Dude, very awesome!

Do you happen to know your plasmid concentration?

--A

On Mar 2, 2013 8:53 AM, "Shashank Goel" <goel.cha...@gmail.com> wrote:

Hi Guys,
     Per Patrik's request...
    Kyle Taylor,Jay Hanson and self  performed the experiment here at Biocurious, initial results are encouraging..
Only deviations from paper were because of material at hand availibility and additional Al foil cutting in tube..

-shashank


Experiment: Transform HB101 Strain with pGlo plasmid using Ultrasound device

Equipment:   Off the shelf Ultrasonic Jewellery cleaning box with about 1l bath.
              Strains, DNA and reagent in Dark Glass flat bottom tubes 5ml with Plastic Caps

Sonicator Essentials:
        42 KHz
        35 watt Peak
        20cm sq transducer area in center of bath
                      Bath filled with Tap water at room temperature
Transform Tubes:
    Four Tranform tubes with

1. 500ul of  O.N. culture of HB101 strain
2. 5ul of pGlo plasmid prepared thru miniprep
3. 1ml of 75mM CaCl2
4. Two tubes with Al foil discs (two none)

    Two Control Tubes

1.    Same as above , but no plasmid, no Al
2.    Same as Transform, with plasmid and Al

Procedure:
      Each Tube was sonicated for 10 secs at only available setting on Sonicator (on/off)
      except 2nd control tube with plasmid and Al

1.       Test transform Tube 1 with Al placed in middle of Transducer bump, flat glass  bottom down
2.      Test transform Tube 2 without Al placed in middle of Transducer bump same way
3.      Test transform  Tube 3 with Al placed in a side of the bath away from transducer
4.      Test transform  Tube 4 without Al placed in a side of the bath away from transducer
5.      Control  Tube 1 without plasmid and Al placed in middle of transducer bump
6.      Control Tube 2 with plasmid and Al left alone

   
    Post sonication
        Add 1 ml LB solution
        incubate at 37C 1 hr on a bath-shaker
                      Plate on LB and Amp agar after hour and incubate for a day
                      Check plates for colony in UV light next day



Results:
    Test tube

1. With HB/PGlo/Al/midTransducer: 9 Colonies
2. with HB/PGlo/midtransducer: 4 colonies
3. with HB /PGlo/Al/side placed: 3 colonies
4. with HB/Pglo/side placed: 1 colony

     Control
    1. Just bacteria/midplaced: 0 colony
           2. Bacteria/Pglo/Al no sonication: 0 colony

FWIW, we are pretty happy about it...open to all ideas to improve it.

To view this discussion on the web visit https://groups.google.com/d/msg/diybio/-/WGPjM3vH07QJ.

[Andreas Sturm]

What would be fun to try: 

pGlo is relatively small, I think. Ori, AmpR and GFP. 

Why not use pVIB? If one of the LuxC,D,A,B,E genes is disturbed / damaged, it likely won't work any more. Because I imagine that bigger plasmids will rather be damaged than smaller ones. But just an assumption... 

[Jonathan Cline]

This success is one for the biohacker skeptics. Open source at it's best.
I suggested this experiment 11/2008. Random periodic discussion and
suggestions followed.
Years later, first wetlab results are posted including controls and
feedback sought; from a hackerspace.
Proof that the long tail works.
Contributions from the bazaar can definitely innovate biotech, including
this protocol.
Using $25 equipment, in fact.
Love it.
Who knows, this might turn into a microfluidics USB powered
bacto-auto-transformer in a few more years.


## Jonathan Cline