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>> [Inaudible] are Syracuse University's 2012 Beckman Scholars, they study the properties
of proteins and investigate the potential as biological sensors
but they approach the issues from different perspectives.
Mack is a senior biochemistry major
and [inaudible] is a junior biophysical science major.
Both participate in leading edge research under the tutelage of their respective professors.
>> My name is [inaudible], I'm a junior biophysical science major
and I work in the [inaudible] laboratory.
Today I'll be showing you how to make a biological membrane
and then how to insert a protein into it.
So I'm going to first establish the membrane in the chamber so the first step
to this process is taking your thousand microliter pipette, setting it to 750
and then adding your 1 molar KCL to it.
So [inaudible] we'll add hexadecane to the chamber
and what the hexadecane does is it provides organic lattice for lipids to attach themselves
on to the Teflon, add a little bit here as well.
After this, you want to add your lipids and the lipids provide the barrier
for your actual cell membrane, usually you want to add two drops per site.
After doing this, you want to wait about five minutes for your hexadecane
and lipids to settle in the chamber.
After waiting five minutes, you want to take your 1 molar KCL and you want
to top off the chamber, you do this by adding the KCL to these small holes.
So now we're ready to take the cell membrane and put it in our Faraday cage.
The Faraday cage blocks out any environmental noise from the inside system.
We take our membrane and we place it in the little pedestal inside.
Next, you want to take two electrodes and you want to add them to the cage.
You connect them to the receiving device
and then you insert the electrode tips into the actual chamber.
So now that everything's set up, we want to turn on our machinery, this is the head stage,
it provides an electrical current
and also records any data coming in from the cell membrane.
After these devices are turned on, you can open up your software.
In this lab we use [inaudible] 10.3 this is previous data that we've collected in the lab,
as you can see from this data, we don't have a baseline of 0, instead we have a baseline
of around 90, which is what we expect when we see a protein.
In this data, we see that there's a baseline of 0 and that there's a current
that we detect of 150 picoamperes.
At 150 picoamperes, the pore establishes itself at a certain voltage.
The voltage that we added to the system was 40 that means
that when we add 40 millivolts the pore sends through about 150 picoamperes of ion current.
When we see 150 picoamperes that means that we have a pore established in the membrane.
After the membrane is established, we want to the nanopore inside the chamber
so using a smaller pipette, you take your pipette tip, you set your pipette
to about .25 microliters and then you add your protein, in this case fhuA to the [inaudible]
or grounded side of your chamber.
So now that the pore is established, we want to send DNA through it.
DNA is an [inaudible] that we want to detect in the pore.
If we put DNA inside the pore, we expect to see a current blockage
or we expect to see the current go down to 0.
If the current goes down to 0, we reverse the voltage that we applied to the system,
if the voltage goes back up to 150, that means that we had something inside the pore itself.
>> Based on this technology, [inaudible] and his colleagues can identify
and study individual molecules with an end goal being
to create new screening tools for early cancer detection.
With Professor Ivan Korendovych, [inaudible] uses biochemistry tools to study a class
of proteins that binds to metals.
>> Hi, I'm [inaudible], I'm a senior and I'm a biochemistry major and my project is
to design sensors for different types of metals and their proton based sensors
so today I'm going to show you how I am putting my substrate into my 96 well plate
because when the -- my protein binds the metal,
it produces a really nice yellow colored substance so now I'm going
to show you how I add this and put it in our plate reader.
So I've already added in my buffer and my actual protein in the plate
and this is just adding three different concentrations
of the substrate that's going to produce the yellow color.
I'm bringing this over to our plate reader that's going to show us the different types
of activity for the metals so basically just press play
and it will give you your readout on this screen here.
So if our protein sensor binds metal, it should turn yellow and we take it
out of this plate reader so on the plate reader you can see the different levels
of substrate that's going to turn yellow that I added, the different concentrations.
So in the top three rows, you can see that there's no --
none of our sensor added it all so there's no activity but when you start
to see greater slopes, it means that it's binding the metal and it's going to turn
that yellow color that we're looking for.
>> Ultimately, [inaudible] work will lay ground to our ability
to introduce the proteins she made into a live bacteria that can sense metals by the virtue
of producing protein, so essentially the bacteria will do all the hard work,
we don't need a plate reader and the only thing to do is you plate your bacteria on medium
and will tell you whether you have potentially metal toxic, toxic metal in the environment.
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