I'd like to discuss transformation methods (bacterial)

[Nathan McCorkle]

I'm assisting a professor with a Molecular Bio lab this quarter, and
he's out this week due to an unexpected meeting... so that lab is
being left in my hands. Now as long as my flight back to school isn't
terribly delayed, I need to be as prepped as I can be.

We're doing a CaCl transformation, spinning down cells, decanting,
adding CaCl, spinning down and decanting, adding more CaCl, then
adding DNA, cooling for 20 mins, then 45 secs of heat shock at 42 C,
add LB broth, plate. (all steps on ice, with some waiting in between
each step)

I honestly don't know/remember why CaCL2 is used, what does it do,
etc... I've heard some say it complexes with the DNA, some say it
coats the surface of the cell, then the heat shock causes pressure and
pushes the DNA into the cell, blah blah... but I don't want to talk
bullshit to these kids.

I'll probably also mention other transformation techniques like
gene-gun and electroporation. What do you all think I could also
mention, how do you think I should explain the theory behind the CaCl
method?

-Nate

--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics

[Jordan Miller]

as far as i can remember it's still not clear the mechanism by which CaCl2 helps transformation. But it definitely helps to transform most bacterial strains (easy to do a control experiment and see efficiency drop without it), so people almost always use it.

hopefully someone that knows better will post, but you'd probably be okay in saying the exact mechanism is still not clear.

i'd also email the prof. and ask him. let us know what he says!

jordan

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[Cory Tobin]

Check out this article:
http://www.microbelibrary.org/index.php/component/resource/laboratory-test/3152-transformation-of-escherichia-coli-made-competent-by-calcium-chloride-protocol
The "History" and "Theory" sections by be interesting to you. If you
run in to a pay-wall let me know.

Also, what Jordan said. No one knows for sure how it works.

-Cory

[kingjacob]

It reduces repulsion. DNA is small enought to fit through the pores in the bacterial membrane but 

because the phospholipids and DNA are both negatively charged, It's repulsed. The 2+ Ca of the CaCl2 make it more neutral.

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Cheers,
Jacob Shiach

[Nathan McCorkle]

I will read the above posted link soon, but can anyone else
verify/second what jacob is saying... sounds good from first glance.

[Jordan Miller]

there's definitely some charge neutralization going on, but if it were *only* due to ionic strength then any salt should work equivalently taking valency into account. But as far as I know, calcium chloride is always used, so there is likely something non-charge based going on as well, and it may be specific to calcium. I dunno what it is, exactly, though.

jordan

[John Griessen]

On 12/28/2010 09:37 AM, Jordan Miller wrote:
> as I know, calcium chloride is always used, so there is likely something non-charge based going on as well,
and it may be specific to calcium. I dunno what it is, exactly, though.

CaCl2 can be in a buffered reaction with HCl in water solution. It can go towards or
away from precipitating solids like CaO or calcium carbonate as pH changes. This
comes from my experience with CuCl2 reactions in copper etching a plating, so take with a
grain of salt, literally and figuratively.

I'd look for explanations of where the calcium ions go. If they don't get in a reaction, the idea
of just having more + ions on one side of a membrane seems plausible. If they do, then with what, and
what is the resulting molecule like? If it "complexes with the DNA", what is that like as far as charges?

Possible search terms, "CaCl2 reactions DNA", "CaCl2 reactions membrane", "CaCl2 reactions cell wall" etc.

Just some SWAGs

John

[Nathan McCorkle]

Here's (1) of (2) handouts I compiled and gave to my class this week:

Today we are getting the DNA you ligated last lab into the E. coli
host, and screening the colonies for bacteria resistant to both
antibiotics (remember you began with two separate plasmids and
recombined them to be one). I've outlined key points to the protocol
below, these relate to the steps that make the protocol work well.

I can't seem to find any literature concisely defining what the CaCl2
does for the transformation, but I found a well referenced link to a
page (1) that explains how it is thought to work. I even read the
section on chemical transformation in Molecular Cloning (Sambrook and
Russel) and they basically say "we don't know how it works, but here's
the protocol".

This is what seems to be the general consensus, and what I think is a
good summary to the protocol:
--Bacteria acquire DNA in 3 ways: conjugation(bacteria to bacteria),
transduction(phage to bacteria), and transformation(naked DNA to
bacteria)

--E. coli is gram negative, and has two membranes, with a space
between (periplasm) (2)

--CaCl2's method of action is not exactly know, but it is thought to
neutralize negative surface charges on E. coli's outer membrane, these
negative surface charges would otherwise repel the negatively charged
DNA (think magnets, opposites attract, likes repel)

--Chilling removes thermal energy and decreases molecular movement,
making cells more prone to shattering/breaking, but importantly it
stabilizes Ca++::negative-surface interactions

--Thermal shock probably causes a high pressure effect that emanates
from the microtube wall into the center of the tube, DNA close to a
cell is forced inside

----------------------------------------------------------------------------------------------------------------------------------------------

There are also 3 other ways you could transform E. coli, but we are
not performing these in lab:
--electroporation (current causes membrane to re-arrange in areas
(membrane has a charge), pores form on cell surface, DNA passes
through)
--sonoporation (sound waves force DNA through cell membrane)
--gene gun (DNA adhered to metal particles, particles shot at cells)

1 - http://www.microbelibrary.org/index.php/component/resource/laboratory-test/3152-transformation-of-escherichia-coli-made-competent-by-calcium-chloride-protocol
2 - http://en.wikipedia.org/wiki/File:Gram_negative_cell_wall.svg

[Nathan McCorkle]

Here's (2) of (2) handouts I compiled and gave to my class this week:

Overview of CaCl2 Transformation Protocol:

Pre-chill these reagents - CaCl2 solution (20-50mM), E. coli,
plasmids, (4) 15mL orange-capped tube, (3) fresh 1mL tubes

add 15ml E.coli to orange-capped tube, spin at 2700g for 10 mins

decant supernatant, a little leftover is OK

add CaCl2 solution (5-30mL)

chill up to 20 mins

spin at 2700g for 10 mins, decant supernatant, resuspend in 1mL CaCl2

chill up to 20 mins

transfer 200uL of E.coli to each of (3) fresh 15mL orange-capped tubes

spin plasmids 15-30 seconds, transfer 10uL of each to a respectively
labeled tube

chill 20-30 mins

heat shock for 45-90 seconds, then immediately transfer to ice

after 1-2 mins, add 600-800uL LB broth (SOC is better)

place in 37 degree C incubator (can be shaking but at less than 50
RPM) for 45-60 mins

plate each transformant culture on appropriate antibiotic selection media

[digitalbio]

Hey Nathan,

I don't know if you've pointed this out or not, but the stage of
growth of the E. coli is quite important as well. I don't remember
this exactly, but we used to obtain the bacteria that we made
"competent" for transforming by harvesting them during a very specific
point in log phase growth. The best point varied depending on the E.
coli strain. I think for DH5alpha this was around 10 to the 8 cells
per ml.

Sandra

[Nathan McCorkle]

I didn't stress the growth-phase, but their other lab protocol
mentioned it.... I would think as long as you begin with enough cells
total, cells which are actively growing, you would be fine if you
weren't concerned with highest efficiency (not dealing with cDNA
libraries)

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