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[Music]

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[Applause]

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my name is Vincent Richie and as the

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slide says I'm going to be talking about

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investigation of embittered individual

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and combined effects of blah blah blah

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blah blah that's boring so we're gonna

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get rid of it I think a better title for

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this talk would be what works and what

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works on earth and I'm going to be

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talking specifically specifically about

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RNAi oligomerization but this can really

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be applied to any sort of biological or

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prebiotic product that we're interested

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in so I want to start out by talking

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about two different approaches to

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prebiotic chemical experiments the first

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of which is a more traditional approach

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and we start out by looking at modern

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life we take these cells we look at

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what's inside of them we break them down

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into these simpler components we sort of

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pick our favorite component and then try

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to replicate it in the lab abiotic ly

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then we try to take the parameters that

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work and apply them to the early Earth

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so we look at this and we say where

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could this have happened

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but the problem is more often than not

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we get a system that doesn't look like

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anywhere on the early Earth or very

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dissimilar to the early Earth so an

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alternative approach would be to start

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with the earth we can take the earth and

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take different parameters that describe

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the different environments that we think

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were around before life and replicate

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those conditions in the lab and see what

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comes out of it so as I said we're going

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to be talking about I'm going to be

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talking about RNA here but this can be

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proteins or amino acids or whatever you

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like

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so we have conditions that foster

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prebiotic chemical reactions and we have

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conditions that were present on the

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early earth the key to finding that the

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pathways that are feasible for prebiotic

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chemistry is to look in the overlap

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between these two sets of conditions so

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if we're applying this to RNA

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oligomerization we might be interested

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in several variables and

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temperature pressure and any catalysts

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and for the sake of this talk we're

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gonna stick with these three and we're

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going to be talking about aqueous

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catalysts so this figure shows all of

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the conditions at least prior to this

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week that have been explored in the

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laboratory setting relating to RNA

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polymerization and we can compare that

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to the PT space that we think represents

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early Earth's surface environments and

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we see immediately that the majority of

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this PT space has not been filled out

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yet we're sort of stuck at one bar and a

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sort of moderate temperatures we did a

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decent job with temperatures as a

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community and we could do a similar

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comparison for our catalysts so we have

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several sets of metal catalysts

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including uranium and the lanthanides

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that have been investigated as effective

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catalysts for oligomers ation and some

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of these work and some of them don't

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work so well but we can compare that to

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the elements that we would expect to

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find in a prebiotic early Earth

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environment such as the open ocean so in

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that case the major cations would be

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sodium magnesium calcium and iron so

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there's not a lot of overlap but there

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is some overlap we have magnesium and

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calcium we're still missing sodium and

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iron well let's take magnesium and

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calcium for a second

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those have only been explored at one

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specific concentration by the sawai

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group in the in the 70s and 80s and that

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was only at 12 and a half million more

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so we don't know what increasing or

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decreasing the concentration would

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really do so we have this large empty

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plot with one data point on it so we

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don't know what the rest of this looks

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like so what do we do well we do

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experiments in our lab these are

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relatively simple experiments they're

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done in micro centrifuge tubes they're

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100 microliters experiments and all we

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do is add our aqueous metal solution at

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the concentration that we're interested

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in and we add our nucleotide now in this

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case we're using a phosphor emitters of

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light

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inactivated nucleotide is not

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particularly realistic in its own right

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necessarily but it is useful in the lab

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for probing these reactions and seeing

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how they'll behave and respond to

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different variables like temperature

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pressure and different catalysts so we

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take that solution we wait three days

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and then we extract our products and we

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measure them with maldi-tof m/s and we

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get a mass spec that looks something

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like this where each peak corresponds to

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the addition of a single nucleotide and

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again this could be a polypeptide or

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hmm here we have a max length of six so

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this is how we're going to denote the

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success or assess the extent of the this

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reaction going forward so you got a

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preview of these results but these are

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those max lengths for different

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concentrations of calcium chloride and

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we see that there's a sort of optimal

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concentration right around one molar but

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this is a relatively broad profile here

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and and it works over a range of

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concentrations we can also compare that

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to our negative controls which are

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represented by the orange line this is

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containing no metal catalyst it's just

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water and imp a but again we don't want

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to be stuck at 1 PT point we want to

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expand this and look at a range of

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temperatures and also a range of

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pressures and if we do experiments at

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one kil bar for example perhaps we can

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then extrapolate and then fill out a

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large chunk of this PT space we can do

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our high-pressure experiments in a

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static pressure vessel by loading our

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experiments into syringes and these

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syringes contain our experiments and

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when we apply pressure with water we use

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this group on that pressure is

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redirected to the sample so we have our

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concentration profile we need to pick

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one or two or however many

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concentration points that we're

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interested in to do our other

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experiments so we're gonna pick one

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molar and this is what we get we have

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our 1 bar our atmospheric experiments in

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orange and our one kill bar experiments

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in white and we see that there really

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isn't a large difference between these

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two so we can say in this particular

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system not all systems that pressure

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isn't particularly significant we also

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see that there is a moderate decrease in

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oligomers lengths as you increase

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temperature so we can do this with the

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other metals as well we have calcium we

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did this with sodium and magnesium but

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what about iron we've heard a lot about

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iron today and throughout the conference

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what can I do for RNA we know it it's

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good for amino and acid synthesis and a

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variety of other reactions what about

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RNA oligomerization but this is a little

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bit more complicated but because we have

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to keep our systems and oxic otherwise

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we'll just end up with a rusty tube so

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instead of using microcentrifuge tube we

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use gas tight exit a nerve isles we load

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our nucleotide into these vials and we

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flush the headspace with pure nitrogen

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to expel any oxygen then we inject our

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anoxic iron solution again we wait three

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days extract our products and we end up

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with the mass spec so these are the

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results of our iron experiments and

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again well note first that we're on a

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log scale now just to make everything

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easier to see but again we end up with a

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local optimum concentration right around

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100 millimolar and again we have our

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negative control down here at 2 but we

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get up to about about 6 we don't want to

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be stuck so we explored this optimal

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concentration over temperature as well

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just like we did for calcium and we get

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a nice linear relationship with

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but this time all of these experiments

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are above our baseline our negative

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control here so even at our highest

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temperatures we're still seeing an

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enhancement of oligomerization however

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this concentration is outside the

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feasible limits for prebiotic iron in

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the early ocean so we have our optimal

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concentration and then this is sort of

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the range of estimates in the literature

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for prebiotic ocean iron so we have this

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other point up here this is sort of the

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maximum feasible concentration so let's

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take that instead and ask us the

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ourselves the same question well this is

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what it looks like in blue and we don't

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see that decrease in oligomerization

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with temperature in fact we beat out the

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higher concentration when we get out to

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80 or 90 so we've added some of these

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missing elements to our table we have

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our four primary metal cations in the

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early ocean what can we do with that

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well we we can start to ask ourselves

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what a more complex environment might

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look like but we're still going to keep

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it relatively simple we're going to look

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at the open ocean we have our controls

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are negative in our positive control so

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2 for no catalysts and with 1 molar

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calcium chloride which is what we use as

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our positive positive we get Penta MERS

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and then before we put everything in one

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pot let's just make sure we know what

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each thing does individually so if we

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look at calcium at modern ocean

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concentrations we see that we get

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trimers sodium sulfate we get dimers

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magnesium chloride trimers and sodium

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chloride dimers so when we put these

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together we can ask ourselves okay are

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we going to get trimers or ever again

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get timers or are we going to get three

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plus two plus three plus two turns out

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that we just get three so these aren't

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additive at least in terms of the

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lengths that

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we get we can't really say anything

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about yields because we're using multi

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and it's not quantifiable but we're

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interested in the early ocean so we add

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in iron and when we mix that with our

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other components at our other salts at

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modern concentrations and take out the

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sulfate because there wasn't any sulfate

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we can run similar experiments and we

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see that again the biggest sort of wins

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so iron which is up here at five is

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pretty close to what we get for our iron

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rich synthetic seawater let's push it a

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little bit farther because we don't know

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exactly what the Hadean look like as

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we've discussed throughout this

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conference so let's test a range of

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so here we have twice modern salinity

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half modern solidity and modern salinity

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in green and we see that as we would

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probably expect slightly higher salinity

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produces slightly longer oligomers all

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of these are above our negative control

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and as I showed before there's no

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relationship with temperature because

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we're sort of below that 10 millimolar

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level for iron so our takeaway points

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from this is that iron is very

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significant not only for RNA synthesis

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but for a variety of prebiotic reactions

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but more than that I want to emphasize

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the importance of starting with

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environmental considerations and working

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our way up from there and it doesn't

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have to be earth it can be Europa

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Enceladus or Titan or whatever we're

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interested in but we have to interpret

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our data and design our experiments with

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our environment in mind that way we're

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not shoehorning an environment on to the

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early Earth so with that I'll thank our

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funding agencies and I'll take any