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[Music]

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hi everyone so I'm Lauren and I'm from

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Anna Lang's Lab at University of Sydney

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in Australia and today I'm going to tell

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you a bit about the work I do on model

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so I'm sure many of us here are trying

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to answer the question of how did life

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on earth begin now I'm focusing on a

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very very specific point in time when

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life had maybe begun to transition from

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super simple primitive cells perhaps

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composed of a fatty acid bilayer

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membrane it's a more complex protocells

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or primitive cells more akin to Modern

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Biology at this point in time we'd

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started to see the emergence of

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phospholipids and some simple enzymes

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but we hadn't yet thanks but we hadn't

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yet seen the emergence of all the

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complex cellular cellular Machinery that

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exists in our cells so things like

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membrane transport mechanisms for

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example that transport nutrients and

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waste molecules out of the cell maybe

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they hadn't come around yet so that begs

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the question how are these protocols

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actually able to feed themselves how

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were they able to grow how are they able

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to divide

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so I'm trying to answer this question by

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building a propagating synthetic cell

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what that basically means is it's an

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artificial cell that is able to feed

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itself can grow it can divide and I'm

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trying to achieve this using the

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simplest components possible to try and

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understand how life transitioned into

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what we know it as today

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so this involves a pretty large

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collaboration and with my collaborator

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in Japan yutatsu he's been able to build

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a preliminary synthetic cell composed of

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a phospholipid bilayer membrane also

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known as a vesicle and this vesicle is

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composed of a mixture of popc and prpg

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two types of phospholipids and within

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this vesicle we have a system that can

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be used to actually synthesize fatty

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acids and eventually synthesize

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phospholipids that can then be

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incorporated into the bilayer membrane

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actually allowing it to grow

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now one of the big problems with this

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system is that you can only encapsulate

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a small number of nutrients actually

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within the synthetic cell within the

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vesicle there's only room for so many

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molecules and if you encapsulate as many

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as you can you only really get one to

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two percent membrane growth which isn't

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really sustainable for a cell that we

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want to make grow and divide

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so what we really need is some sort of

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regular nutrient Supply so we need an

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external feedstock of nutrients that can

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actually permeate the lipid bilayer

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membrane to make its way from the

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outside of the cell into to the inside

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of the cell that way we can actually

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achieve continual membrane growth

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so that's my role in this project I'm

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trying to figure out a way that we can

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actually get the nutrients that we need

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for the synthetic cell to function to

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make its way across the lipid bilayer

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membranes the interior synthetic cell

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so in order to do that I've needed a

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technique that can be used to monitor

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solute permeability or the permeability

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of different nutrients and to do that

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I'm using what's known as a shrink soil

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assay basically that involves preparing

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vesicles encapsulating a fluorescent dye

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known as calcine

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when you mix these vesicles with your

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solute or your nutrient interests the

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difference in osmolarity actually causes

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the vesicles to shrink because water

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rapidly exits the vesicle and we kind of

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see this these shriveled up sort of

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shrunken structures

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and because we've had water exit the

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vesicles we've actually had an increase

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in concentration of the diet that's on

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the inside of these vesicles and that

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actually causes the fluorescent signal

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to decrease because calcium is a

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self-quenching diet about certain

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concentrations so at high concentrations

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of fluorescence actually goes down

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rather than going up so it's a little

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bit counter-intuitive

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but over time if we have a permeable

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solute so a solute that does actually

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enter the vesicle you'll start to see

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the vesicles slowly swell up again and

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it'll roughly get back to its original

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size and with that we have a decrease in

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calcium concentration so we have an

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increase in the fluorescence roughly to

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what it was to begin with

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and we can basically monitor that whole

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process using some sort of fluimeter or

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something that can measure fluorescence

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and we can monitor the changes intensity

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over time so we can see initially the

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intensity is somewhere up here and then

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with the addition of the solute the

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intensity drops down and then if we have

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a permeable solute that signal will

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start to recover and we start to see the

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original intensity kind of values there

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now because these intensities are kind

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of a direct measure of the calcium

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concentration we can convert

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concentration to volume so the plots

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I'll be showing you today show the

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changes in volume over time

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and it's just worth noting that the

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initial stage where the vesicles shrink

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and swivel up shrivel up actually

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happens very quickly and the

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experimental setup I currently have I

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can't actually see that because it just

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happens too fast so the data I'll be

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showing you is just the recovery of that

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so the basic workflow for a lot of my

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experiments is to First prepare the

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vesicles composed of an equal mixture of

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popc popg in 50 millimolar happy so

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that's just the buffer it's roughly

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buffering around physiological pH and

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these vesicles all encounter all

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encapsulate that fluorescent iron is

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calcium and then make sure all the

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unencapsulated calcium is removed and I

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mix the vesicles with the solute all the

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nutrient of interest and monitor the

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changes in fluorescence over time and

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then determine the changes in volume

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so this is what some of the data looks

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like on the left we can see some

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examples of some permeable solutes so

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our control here was the 50 millimolar

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happyes in the blue and all of this data

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is normalized to the volume of that

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control at time zero so we see in the

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blue 50mm Heavies it's just a flat line

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there not much is changing and that's

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what we'd expect it's our control it's

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what we made our vesicles in so we don't

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expect any volume change there but when

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we mix our vesicles with something like

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glucose or glycine for example so the

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red and the purple we see that

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characteristic curve telling us that the

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volume is actually increasing after the

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vesicles initially kind of shriveled up

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so that tells us that these two solutes

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are both permeable across these lipid

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bilayer membranes and glycine is

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actually more permeable than the glucose

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because it increases at a faster rate

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on that plot I also have glycerol at

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first glance it kind of looks like

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nothing happens there but glycerol is

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known to be super permeable across lipid

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bilayers so it's likely that I just

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actually missed that initial shrinkage

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and swelling stage so it just looks like

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nothing's happened right now because I

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missed the first kind of couple minutes

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of this process

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now on the other plot we have some

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examples of some impermeable solutes and

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you can see there's quite a few there so

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a lot of the solids I've looked at so

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far are quite impermeable and that's we

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can see that there's like those flat

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lines at the bottom there there's no

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increase in volume over time so over

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these this time frame none of these

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Solutions are permeable which is kind of

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disappointing because a lot of these

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solutes would be useful nutrients for a

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synthetic cell things like amp ATP for

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energy and then something like sodium

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acetate I was hoping to use as a carbon

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source to actually synthesize fatty

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acids and then the phospholipids

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but it's not particularly surprising

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that a lot of these solutes were

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impermeable It's relatively well

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established in literature child that

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phospholipid bilayers aren't the most

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permeable things especially when

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comparing them to a fatty acid bilayer

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for example so fatty acid bilayers are

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kind of useful to think about because

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they have been proposed to have made up

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the first protocell membranes before we

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saw the emergence of phospholipids and

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we can see in this plot here for

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something like glucose it's a bit less

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permeable in phospholipids than in fatty

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acid bilays

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there's something like sodium potassium

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it's significantly less permeable and

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like over this scale that that's a

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really significant margin there

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and then for something like amp and

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magnesium that's not actually on the

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scale shown here for phospholipids so

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it's way less permeable in phospholipid

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membranes than it is in fatty acid

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membranes

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so again these results aren't

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particularly surprising but I still

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wanted to try and find a way to actually

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get these nutrients to permeate the

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bilayer so today I'm going to talk about

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a couple of different strategies I've

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tried to use to actually improve

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permeability the first of which is

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modifying the membrane composition

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so as I said all the vesicles I've been

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working with so far have been composed

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of pobc and popg now these lipids are

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quite nice to work with they're

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cylindrical in shape so they pack

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together in these really nice bilayers

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they're very uniform but what would

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happen if I were to say disrupt the way

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the lit lipid molecules actually pack

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together what if I introduced a leopard

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with a different shape something like

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laser PC which has a single carbon chain

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rather than two but it still has a

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relatively bulky head group so it has

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this more conical like shape and when

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that gets inserted into bilayers it can

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cause defects to form and pores which

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would potentially allow nutrients to

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pass through the membrane so I wanted to

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explore what effect adding small

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quantities of lyso PC would have on

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permeability

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and the other lipid I looked at is oleic

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acid which is a fatty acid has a single

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carbon chain and a relatively small head

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group but because it's a fatty acid it

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has very different properties to what

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phospholipids do so I wanted to explore

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how those different properties might

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membrane only I found

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I prepared all the vesicles the same way

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I did before but added about 10 either

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lyso PC or oleic acid to the system and

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generally as I said didn't make much of

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a difference we have one of our example

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permeable solutes there so glucose which

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we saw was permeable earlier we can see

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in those three curves there isn't really

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much difference between them if anything

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the green curve the oleic acid is

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shifted down slightly but the shapes of

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the curves are all very similar which is

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00:10:09,410 --> 00:10:07,380
telling us that the permeability hasn't

279
00:10:11,389 --> 00:10:09,420
really changed and we see something

280
00:10:13,670 --> 00:10:11,399
simple similar with one of the

281
00:10:15,590 --> 00:10:13,680
impermeable solutes that being lysine

282
00:10:17,269 --> 00:10:15,600
they still have all those flat lines so

283
00:10:19,610 --> 00:10:17,279
we saw no improvement in permeability

284
00:10:21,650 --> 00:10:19,620
there and this was generally the case

285
00:10:23,509 --> 00:10:21,660
across a lot of the different solutes or

286
00:10:25,070 --> 00:10:23,519
nutrients that I actually tried there

287
00:10:27,769 --> 00:10:25,080
weren't any marked improvements in

288
00:10:29,630 --> 00:10:27,779
permeability except for when I tried

289
00:10:32,509 --> 00:10:29,640
sodium acetate

290
00:10:35,090 --> 00:10:32,519
so when we had the presence of 10 oleic

291
00:10:36,949 --> 00:10:35,100
acid in the system we see a significant

292
00:10:39,350 --> 00:10:36,959
Improvement in permeability compared to

293
00:10:41,750 --> 00:10:39,360
what it was originally with the popcpg

294
00:10:44,569 --> 00:10:41,760
system and also compared to adding the

295
00:10:46,069 --> 00:10:44,579
10 lyso PC so we do see that curve

296
00:10:47,750 --> 00:10:46,079
that's telling us that the vesicle is

297
00:10:49,910 --> 00:10:47,760
swelling up again and we actually have

298
00:10:54,110 --> 00:10:49,920
the solute molecules permeating into the

299
00:10:55,910 --> 00:10:54,120
vesicle which is good news we saw one of

300
00:10:57,710 --> 00:10:55,920
these solutes actually

301
00:11:00,050 --> 00:10:57,720
kind of worked and actually were able to

302
00:11:01,790 --> 00:11:00,060
permeate the membrane and as I said um

303
00:11:03,530 --> 00:11:01,800
it was great to see that it did work

304
00:11:05,509 --> 00:11:03,540
with sodium acetate because we wanted to

305
00:11:07,850 --> 00:11:05,519
use that as our carbon source for fatty

306
00:11:09,350 --> 00:11:07,860
acid synthesis then phospholipid

307
00:11:11,690 --> 00:11:09,360
synthesis and that would actually help

308
00:11:13,430 --> 00:11:11,700
getting the membrane to grow

309
00:11:15,110 --> 00:11:13,440
but as I said overall it didn't work for

310
00:11:17,990 --> 00:11:15,120
a lot of the stuff I tried so I wanted

311
00:11:19,730 --> 00:11:18,000
to try it a second strategy to try and

312
00:11:21,790 --> 00:11:19,740
improve permeability and that was

313
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through the addition of divalent cations

314
00:11:25,970 --> 00:11:23,940
so in literature it's pretty well

315
00:11:28,009 --> 00:11:25,980
established that divalent cations also

316
00:11:29,750 --> 00:11:28,019
affect the packing of the way of the

317
00:11:31,850 --> 00:11:29,760
lipid molecules so the way the bilayers

318
00:11:32,990 --> 00:11:31,860
actually packed together so I wanted to

319
00:11:34,430 --> 00:11:33,000
see if that would make a difference to

320
00:11:36,769 --> 00:11:34,440
permeability

321
00:11:38,630 --> 00:11:36,779
and again generally I found it didn't

322
00:11:41,990 --> 00:11:38,640
make that much of a difference except

323
00:11:44,870 --> 00:11:42,000
for the presence of sodium acetate so

324
00:11:46,910 --> 00:11:44,880
with the 10 Lake acid system and the

325
00:11:49,069 --> 00:11:46,920
sodium acetate when I added magnesium

326
00:11:51,110 --> 00:11:49,079
chloride which is what we see in the

327
00:11:52,850 --> 00:11:51,120
orange there was a Improvement in

328
00:11:54,650 --> 00:11:52,860
permeability if you see that curve kind

329
00:11:56,269 --> 00:11:54,660
of increases faster than what the the

330
00:11:58,550 --> 00:11:56,279
blue line which is just the pure sodium

331
00:12:00,710 --> 00:11:58,560
acetate and the green line which is just

332
00:12:02,509 --> 00:12:00,720
the magnesium chloride so for whatever

333
00:12:05,269 --> 00:12:02,519
reason the addition of magnesium

334
00:12:07,370 --> 00:12:05,279
chloride with the sodium acetate and in

335
00:12:09,889 --> 00:12:07,380
the temps in oleic acid system did

336
00:12:12,590 --> 00:12:09,899
improve permeability overall

337
00:12:14,150 --> 00:12:12,600
but again generally I found that this

338
00:12:16,130 --> 00:12:14,160
was quite difficult to achieve in a lot

339
00:12:17,870 --> 00:12:16,140
of the solutes I tried I've also tried

340
00:12:19,009 --> 00:12:17,880
other divalent cations which I haven't

341
00:12:22,370 --> 00:12:19,019
shown here

342
00:12:25,610 --> 00:12:22,380
um in general it was pretty tough

343
00:12:27,290 --> 00:12:25,620
so what does that mean overall so

344
00:12:29,569 --> 00:12:27,300
generally I've found that permeability

345
00:12:32,030 --> 00:12:29,579
can somewhat be modulated for specific

346
00:12:34,670 --> 00:12:32,040
specific sites by changing the membrane

347
00:12:36,889 --> 00:12:34,680
composition and divalent cations can

348
00:12:39,710 --> 00:12:36,899
somewhat improve the permeability of

349
00:12:41,569 --> 00:12:39,720
specific solutes but overall it was

350
00:12:43,670 --> 00:12:41,579
pretty difficult to actually see any

351
00:12:45,170 --> 00:12:43,680
marked improvements in permeability for

352
00:12:47,930 --> 00:12:45,180
a lot of the different things I studied

353
00:12:50,329 --> 00:12:47,940
so what does that mean for origins of

354
00:12:52,190 --> 00:12:50,339
life well perhaps we needed protein

355
00:12:54,410 --> 00:12:52,200
channels to actually co-evolve with

356
00:12:56,449 --> 00:12:54,420
phospholipid synthesis as I mentioned

357
00:12:58,129 --> 00:12:56,459
earlier fatty acid membranes are much

358
00:12:59,930 --> 00:12:58,139
more permeable than phospholipid

359
00:13:02,210 --> 00:12:59,940
membranes so if they were the first

360
00:13:04,550 --> 00:13:02,220
protocell membranes they were able to

361
00:13:06,170 --> 00:13:04,560
access nutrients a lot easier than

362
00:13:07,550 --> 00:13:06,180
phospholipid membranes on their own

363
00:13:09,410 --> 00:13:07,560
would be able to so we've probably

364
00:13:12,230 --> 00:13:09,420
needed some sort of mechanism to

365
00:13:15,650 --> 00:13:12,240
actually co-evolve with phospholipids as

366
00:13:17,449 --> 00:13:15,660
they started to emerge but this is a

367
00:13:19,129 --> 00:13:17,459
very much an ongoing project I'm trying

368
00:13:21,290 --> 00:13:19,139
a lot of different membrane compositions

369
00:13:23,389 --> 00:13:21,300
a lot of different divalent cations and

370
00:13:25,129 --> 00:13:23,399
I'm still working on other methods to

371
00:13:26,750 --> 00:13:25,139
kind of improve permeability so

372
00:13:29,389 --> 00:13:26,760
hopefully in the future I'll have some

373
00:13:30,949 --> 00:13:29,399
more definitive results with some

374
00:13:33,470 --> 00:13:30,959
improvements in permeability for a lot

375
00:13:35,990 --> 00:13:33,480
of other solutes too

376
00:13:37,970 --> 00:13:36,000
so with that I'd like to acknowledge the

377
00:13:39,949 --> 00:13:37,980
entire Land Group and my supervisor Anna

378
00:13:41,569 --> 00:13:39,959
for all her support thank you to my

379
00:13:49,329 --> 00:13:41,579
sources of funding and thank you to

380
00:13:49,339 --> 00:13:57,230
okay we have time for a few questions

381
00:14:03,050 --> 00:14:00,590
thank you I am from Ohio University I

382
00:14:06,470 --> 00:14:03,060
study multiplayer biology I'm just

383
00:14:09,889 --> 00:14:06,480
interested in the method that you use to

384
00:14:12,470 --> 00:14:09,899
measure your membrane permeability is it

385
00:14:20,389 --> 00:14:12,480
um Arch Clump electrophysiology or what

386
00:14:25,129 --> 00:14:22,370
actually a measure of the changes in

387
00:14:27,170 --> 00:14:25,139
volume but it's reliant on the fact that

388
00:14:28,850 --> 00:14:27,180
with those changes in volume they can

389
00:14:31,430 --> 00:14:28,860
only occur because the solute is

390
00:14:33,110 --> 00:14:31,440
actually permeating the membrane so it's

391
00:14:35,389 --> 00:14:33,120
actually fluorescence-based technique so

392
00:14:37,129 --> 00:14:35,399
I use a floor a fluimeter or plate radar

393
00:14:38,810 --> 00:14:37,139
depending on the experimental setup I

394
00:14:41,150 --> 00:14:38,820
have going to actually measure those

395
00:14:43,069 --> 00:14:41,160
changes in fluorescence over time and

396
00:14:45,470 --> 00:14:43,079
from that you can calculate the changes

397
00:14:47,449 --> 00:14:45,480
in volume which you can actually then go

398
00:14:48,829 --> 00:14:47,459
on to work out permeability coefficients

399
00:14:50,269 --> 00:14:48,839
from those curves which is something I

400
00:14:52,550 --> 00:14:50,279
haven't done yet but you can actually

401
00:14:54,769 --> 00:14:52,560
fit a model to that curve and extract

402
00:14:58,310 --> 00:14:54,779
the permeability coefficients as well

403
00:15:00,970 --> 00:14:58,320
okay I'm I'm just actually thinking that

404
00:15:03,290 --> 00:15:00,980
um if you can employ the patch Club

405
00:15:06,189 --> 00:15:03,300
electrophysiology method it can give

406
00:15:08,689 --> 00:15:06,199
insight into like membrane polarity

407
00:15:11,210 --> 00:15:08,699
hyperpolarization yeah which can also

408
00:15:12,170 --> 00:15:11,220
give more insight on this yeah 100 in

409
00:15:19,550 --> 00:15:12,180
this

410
00:15:19,560 --> 00:15:22,310
makes it interested

411
00:15:25,370 --> 00:15:23,930
hi I'm Tim from University of

412
00:15:27,530 --> 00:15:25,380
Wisconsin-Madison

413
00:15:29,750 --> 00:15:27,540
um I was wondering uh do you think there

414
00:15:32,150 --> 00:15:29,760
could be an effect of physical size or

415
00:15:34,009 --> 00:15:32,160
specifically surface area uh on the

416
00:15:35,629 --> 00:15:34,019
degree of permeability and if so did you

417
00:15:38,329 --> 00:15:35,639
like control for size did you extrude

418
00:15:40,730 --> 00:15:38,339
your vesicles I sure did good question

419
00:15:42,590 --> 00:15:40,740
yeah so these vesicles for this system

420
00:15:44,810 --> 00:15:42,600
I'm working with have all been extruded

421
00:15:46,910 --> 00:15:44,820
through 50 nanometer pause

422
00:15:48,949 --> 00:15:46,920
um they're roughly

423
00:15:52,310 --> 00:15:48,959
they're probably about 40 nanometers in

424
00:15:54,949 --> 00:15:52,320
diameter for the most part it's a

425
00:15:57,590 --> 00:15:54,959
relatively even distribution added to

426
00:15:59,870 --> 00:15:57,600
DLS on pretty similar systems

427
00:16:01,850 --> 00:15:59,880
um yeah but that's why I did extrude

428
00:16:05,689 --> 00:16:01,860
because I would expect family ability to