welcome to any be TV
today. We're talking about cell free protein
expression,
In our science in 60, Colby Stoddard
from NEB's Business Development Team
gives an overview of the benefits and applications
of cell-free expression.
Then we will hear from Zach, Son from Thira
Biosciences and Keith Party
from the University of Toronto, and
they will discuss some interesting applications that
use cell-free expression in their labs.
And lastly, we will hear from Paula Magnelli
here at NEB and she will discuss
the workflow and benefits of NEB's
knew any be expressed cell free
E. coli expression system.
Let's get started.
Living cells could be genetically modified to
perform novel functions, such as the production
of recombinant proteins.
However, these genetic modifications
often conflict with normal cellular processes
and result in sick cells or mutational
events that negatively impact research efforts.
These shortcomings can be overcome through removing
the cellular membrane, which leaves a lysate that
is rich and enzymes that can perform both
transcription as well as translation.
This also removes the competing interests between
the cell and the researcher and creates new
opportunities for the production of toxic
byproducts,
pointed care diagnostics as well as synthetic
biology applications.
The use of self reli states also simplifies
the research workflow, since one can directly
introduced DNA without the need to perform
transformations and clone propagation.
Life states are also readily manipulated in
high throughput automated workflows and
allow for researchers to explore more biological
signs
leading to new, deeper insights into biology.
Cell free technologies are
pretty old technology. They've been around earlier
that we could engineer cells
but is really three applications. I
see cell-free had been currently useful for. One,
is to make things so you
know, there are commercial companies that do this. A lot
of R and D labs that do this, but if you have
proteins, air, toxic or otherwise, would be hard
to make in cells
some freeze, a really good way to
not only make the product that you could then use
downstream but also understand
how to make the product. So
making stuff, I think has been a historical use,
but also to other areas where
cell-free is quite useful. One is in understanding
stuff. Well, I mean by this is
if you have a set of circuit
or protein or even a cluster
that makes a small molecule, understanding
how that system works, that genetic system,
cell-free is a great way to do that. And that's kind of what's
sparking a from cell-free biology–
really to start to decipher some of
the rules of biology and rules of these systems.
And then finally, in sensing stuff.
so you know, so free as an
application itself. If you want to buy a little
computations, say to be able to sense
something in an environment, and do a biological response,
cells are really good at that, but it's very hard
to program that into kind of keeping cells alive
for, like in-field application. But cell-
free as like a sense seen material,
you know, it's quite powerful too. It allows you to
computation without having to maintain a cell.
And so I think these three applications are pretty powerful.
Know many of the challenges I
think currently are around applications set
of self free right. It's really kind
of education. Say, you know, if
you're doing a set of work in cells,
how can so free help you speed
up that process, and how do you
kind of understand the results that
you collect in cell-free, how it might influence
what you do in cells or conversely,
maybe a result isn't applicable to cells. And so,
I think education is a big part
of this kind of understanding when self free
can come handy and come into use.
Yeah, Biosciences is a company based
out of Berkeley. We're using cell-free
to effectively do functional genomics.
When we think about cell-free, we think of it as a really
good platform for understanding how to
make things, and then making things to test.
And so
if you have a bunch of D n a right
and you need to be able to make the protein
that's not working in your cell or you have a
lot of proteins you wanna make, for us,
we've built out a system that's driven by cell-free
synthetic biology, some AI,
And automation that's able to really quickly
take digital sequences of DNA
and then say, "What does this make, and what
does it do? Understand, really, the process
that goes through transcription, translation and such.
So you know, the applications for this are really in
functional genomics and also kind
of in understanding complex systems.
name is Keith party. I'm an assistant professor
on the Faculty of Pharmacy at the University
of Toronto and the Canada Research
Chair in Synthetic Biology and Human Health.
So the main challenge with mosquito
borne illnesses is really detection,
right? If you know you have early
detection,
that there's virus in the mosquitoes,
that is soon going to translate into
infections and people,
and the sooner you can identify
the infection in people as well as in mosquitoes,
the more quickly you can bring those outbreaks under control
and reduce the spread of the infection.
and so providing
tools for diagnostics that are more
portable
and low cost. Our goal is to
help in that effort. The most
sort of the largest amount of product we use from
NEB is PURExpress,
which is their recombinant cell-
free transcription and translation
system for protein expression.
And we use this system
to run our gene circuit-based
diagnostics and also for
running the portable manufacturing of
drugs.
And what we do is we freeze dry, pure
PURExpress on embed it into paper
or make pellets out of it
so that we can run these
synthetic biology applications
outside of the lab. And the goal here is
to decentralize
medical or healthcare capacities that are usually
only in hospital settings,
out into the real world
as diagnostics and the manufacturing
of drugs.
And one of the largest efforts we do
is in diagnostics, molecular diagnostics,
where we use the genome of a pathogen
as a bar code for identifying
the presence of the pathogen.
and one of those projects.
That sort of our flagship project
is for the detection of Zika
and Chikungunya in Latin America.
And so this project
involves eight labs in five countries,
And our partners in Brazil,
Bogota, Colombia,
and Ecuador
are collecting samples and running
our technology down there, too.
Determine the presence of the virus and and
basically establish
the essay.
So we're speaking with Pollen Agnelli, who is an
application and development scientists here at NEB.
Hi, Paula. Hi, Deana. How are you? Good thank
you. So, could you tell us a little bit
about the challenges associated with extract-
a cell free expression?
I would say the biggest challenge
is variation. When you
are trying to express a protein
using a cell based on
vitro system, it is difficult
to obtain the same result
every time. Another
common challenge
is the fact that in a cell
free environment,
some properties have a difficult
time with falling on with
the formation of the correct
disulfide bonds.
and final challenge is
the concern that many scientists have
of whether this type of reaction
that is in
in vitro is not a life
cell, will be able to achieve
the high level off proteins expressions
that they require for there.
experiments. So you were involved in the
development of these new NEBExpress
cell-free expression kit, correct? Yes. Could
you talk a little bit about some of the advantages
of using that kit?
Yes, Uh, we,
a one off. Our main goals when we
developed this was to reduce
the variability. This is important
because in a
protein evolution experiment
in the discovery
off new protein variation,
you need to capture those
small differences in performance,
small differences in expression
levels. And if
a system has too much variability,
that will needs that you don't
really know if the differences
you observe are real
or is just error.
another feature that we
really wanted this kit to have
was compatibility with the disulfide
fiber on enhancer
it assist
in combination with the NEBExpress
a kid.
It is these properties that have difficult,
difficult kenetics, difficult time
to achieve the correct folding
to form the right
amount and the right combination off
disulfide bonds
in the plotting our sequence.
Therefore, what you obtain
is not only
a some yield off prodding the protein
that you get is the right
form, is the active form. So that
is a nice
perk. It's great to have. Great.
And when you're talking about yields, how much
protein are we talking?
A typical bra thing. I mean, not all protein
is expressed at the same level, but
I would say that a typical protein makes
about 1/2 a milligram
per mL.
So that will be the concentration that
you get at the end. That's great. And
you can also extend incubation time, right?
Yes, that is something that is a
very, very nice feature. So
many protests fold better
or express better at very
low temperatures. But they concern
with the selfless system is whether
you could extend the incubation
as you do with live cells, make
a 24 hour incubation,
a very low temperature so your
protein can express and can fold
correctly.
And that is possible.
And something that is
very interesting to us is that we
have seen for some targets that
in a short incubation time of
course, at 25 degrees for instance,
your yields a lower and that's,
you know,
typical.
But those reactions when
they are incubated at 40 actor
sorry, fo 24 hours,
the final yields can be even
higher
than than an incubation at 37
degrees for the same protein. So
that gives you a lot of flexibility.
You can choose go to short
at a higher temperature or go
long at a lower temperature and
depending on the, um,
the goal of your experiment,
the throughput of your experiment, you
can have, with one kit,
This, you know,
flexibility. And I think that
it satisfies
more different
types of experiments, scientific
projects
which has... you don't need to think, "Oh, which
one it is.
It does everything. So I mean, we are
actually very proud of that,
those features. So can you walk us through the workflow
for this product?
Yes, sure,
The workflow is very simple
and that was something that we are
on purpose. This is, you know, by the
sun. The kid provides
for components, plus a
positive control plasmid.
So the reaction set-up is very
simple.
First,
the reagents that are stored at minus
80 degrees have to be thought on
ice.
Once they are completely thought,
each reaction is assembled
by mixing
the cell. I say the extract
the protein synthesis buffer,
The T seven nominates. Paulie Marie's
Andi are amazing. He bitter on off
course, your DNA or RNA
template that is for your protein
of interest. And also,
if the user wants, the
reaction control can be performed
using our positive control plasma,
And that is incubated at the
temperature that you want, typically 37.
You can use a different temperature for 2 to
4 hours,
and then the reactions
are ready for analysis. And
now, the advantage is, once
the reaction is complete,
the samples are ready to run on a protein
gel. The components off the
express don't interfere with,
um, protein electrophoresis,
so there's no need
to do an acetone precipitation
or T C. A. Precipitation before
running the gel. Thank you so much for joining me
today. You're welcome. It was a pleasure.
Thank you so much for joining us today.
If you would like to learn more about NEB's products
available for cell-free protein expression,
you can use the link below where you
can get access to brochures, feature articles
and other informative content.
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future episodes, please let us know.