Glass bead transformation

[Mega [Andreas Stuermer]]

Hi. Just wanted to share this. Read a note about this technique in a German book, but could not succeed in finding any literature. 

In German they wrote "silica needles" so I googled that. No find. 

Google "Glass beads" and find some: 

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1203545/

[Mega [Andreas Stuermer]]

It seems pretty convenient. In the book they didn't make protoplasts at first. It seemed to penetrate cell wall anyway.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3768579/

"Glass bead transformation of Gram positive bacteria was conducted as follows. Aliquots of 0.5 ml of protoplast suspension were placed in 15 ml conical disposable polypropylene centrifuge tubes (Corning, USA). One µg of pGK12 was added to the protoplasts, followed immediately by the addition of 500 µl of 30% polyethylene glycol 6,000 (PEG 6,000) and 0.3 g of acid washed glass beads (212-300 µm in diameter, Sigma, USA). Protoplasts were agitated at the highest speed on a vortex mixer for 15 sec, and then diluted by the addition of 10 ml of transformation buffer. After the beads were allowed to settle, agitated protoplast suspension was transferred to a new 15 ml conical tube. Cells were pelleted by centrifugation at 5,000 xg for 5 min and suspended in 1 ml of BHI supplemented with 0.5 M sorbitol. After incubation at 37°C for 1 h, transformants were recovered by plating the protoplast culture onto BHI agar containing 0.5 M sorbitol and erythromycin at the final concentration of 3 µg/ml and incubated at 37°C for 5 to 7 days" 

[Brian Degger]

Constanzo and Fox5 demonstrated that intact S. cerevisiae cells can be transformed by agitation with glass beads in the presence of carrier DNA and plasmid DNA at very low efficiency, namely about 300 transformants/µg of plasmid DNA. Osmotic support (with 1.0 M sorbitol) is required in the selective solid medium. (from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3056089/)

Sounds good

300 transformants would be great....

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[Andreas Stuermer]

What I wonder. E Coli is like 7 um big. A 200 um glass bead will kill it when hit. 

Same for eukaryotic cells (10* size of bacterium)

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http://diyspartanbiotech.files.wordpress.com/2014/03/genetic-engineering-and-synbio-for-beginners-v1.pdf

[Nathan McCorkle]

Seems like it could be more like beating up all the cells so they're badly injured, then the few that naturally took in the DNA have a slight advantage over the non-recipients. Electroporation by comparison can yield a million to 10 billion transformants per ug DNA. Chemical methods are usually between 1000 (10^3) and 100-million (10^8) transformants per ug.

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[Andreas Stuermer]

Yeah. I like the idea though. Can transform any cell type with just a vortexer and glass beads! 

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http://diyspartanbiotech.files.wordpress.com/2014/03/genetic-engineering-and-synbio-for-beginners-v1.pdf

[Cathal Garvey]

I suspect cavitation in the wake of the beads is effectively sonicating
the cells, rather than direct impact being somehow transformative!

One way to test this would be to try to control for cavitation by
viscosity, agitation speed, ball size/surface finish, etcetera, and
compare results to straight sonoporation or straight vortexing.

Another way to test would be a static mixer in the tube which, when
vortexed, will create an impact-free but cavitation-rich environment.
Consider that prior art for a "static mixing eppendorf-formfactor tube
for cellular transformation". ;)

On 05/08/14 18:17, Andreas Stuermer wrote:
> What I wonder. E Coli is like 7 um big. A 200 um glass bead will kill it
> when hit.
>
> Same for eukaryotic cells (10* size of bacterium)
>
>
> On Tue, Aug 5, 2014 at 7:12 PM, Brian Degger <brian....@gmail.com> wrote:
>
>> Constanzo and Fox5

>> <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3056089/#R5> demonstrated
>> that intact *S. cerevisiae* cells can be transformed by agitation with

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>> <https://groups.google.com/d/msgid/diybio/CACc%3DpRLseKhm17HCdd-8snzpXT-2gZRuQBqW_utEvjxcavt-bw%40mail.gmail.com?utm_medium=email&utm_source=footer>
>> .

>>
>> For more options, visit https://groups.google.com/d/optout.
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>
>
>

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