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you

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[Music]

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thank you

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I actually I would like to thank the

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four new lab members also for doing a

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very good job in opening how we opening

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the argument on how we can use

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phylogenetic reconstruction to travel

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back in time and understand the early

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events that are related to perhaps in

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shape to earth life coevolution this is

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the first time I'm presenting the

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results of this work and and I termed

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this work as is the term I use is the

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paleo phenotype reconstruction I'm a

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cell biologist and an evolutionary

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biologist and I do reconstruct genes and

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then study the behavior of these

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proteins in using modern using modern

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cells and then link the behavior that I

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measure to two questions related to deep

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life you guys hear me now in my mind

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okay good all right so let's go the

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first work that we did focused on the

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carbon isotope fractionation values so

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there are various bio signatures and

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carbon isotopes are that they were

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interesting to us because they they can

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relate all the way back to the origins

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of life and there was the most pervasive

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one and what is really interesting here

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is that just just a belief or review is

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that what we have here is a biochemical

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process if if this carbon fractionation

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is linked to a biological behavior a

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biochemical process that can cause a

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fluctuation in the amounts of isotopes

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of carbon ratios that are incorporated

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into the cell and there are different

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carbon isotopes that are available and

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then there are carbon isotopes that are

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present in the cell and the ask okay

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what are the biological components that

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are responsible inside the cell that

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caused this biochemical dependent

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fractionation that we observe in the

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rock record and the environment at least

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in the case of carbon isotope

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fractionation what was called Rubisco so

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the answer was its straightforward in

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this case because the behavior of this

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enzyme rubisco is directly associated

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with the carbon isotope records that we

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measure in the rock record in the car

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isotope data attributes to to live

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organism or organism that survive in the

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past the dubis Co converts oxidized

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carbon dioxide to reduce organic carbon

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and it's going to prevent add the

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carbon-12 isotope over carbon 13 given

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that carbon-12 which hits the enzyme

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it's a pretty interesting enzyme and

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also has been a focus of various studies

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that won't improve the photosynthetic

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ability or carbon fixation ability of

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car of cyanobacteria but our interest

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was in related to questions that are

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interesting to us the origins of life in

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astrobiology

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it's the Rubisco is the most abundant

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protein on earth it is it is rather slow

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and confused it cannot readily

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differentiate carbon dioxide with from

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oxygen which leads to a lot of big most

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of energy in the cell because the cell

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does not end up preceding the

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carboxylation reaction but then precedes

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it oxygenation reaction and thus cannot

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proceed and complete the photosynthesis

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as efficiently as it would otherwise do

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with the carbon dioxide so we simply

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asked can we resurrect

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early life bio signatures at least in

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the case for in this case four carbon

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isotope discrimination related proteins

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using Rubisco and our test was to go

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back in time by using phylogenetic some

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molecular evolution and resurrect the

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key notes using the phylogenetic tree

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and study the behavior of this ancient

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environment in this case is going to the

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ancient ruby screen van so let's come

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back to the future today we have four

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different types of Rubisco that is

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present inside the cell for our

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convenience these are named at group 1 2

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3 & 4 viscose and they at a glance at

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the monomer level they are similar but

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at the older grammar level it's the

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multiple protein domain level they may

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have some differences but at large they

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are thought to be pretty identical at

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least it's a structural level and group

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1 is the most abundant protein that we

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have right now and it's also the one

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that is used by cyanobacteria and in

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following up on the previous circuit we

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also you

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phylogenetically construction in our

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case we use different alignment tools

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and different inference tools in order

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to generate multiple trees and then

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among these trees that we created we

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picked the one that support that has

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high branch support value and that also

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led to a multiple actually a sequence of

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representation to risk operating so just

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to run you through we start with the

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currently available sequences for the

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case of Rubisco we scanned all of these

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group one two for a group one two for

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Rubisco and then performs multiple

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alignments and then from there use

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maximum likelihood phylogeny algorithm

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and then we constructed ancestors and we

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use this currently available online tool

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called final but it's a friendly robot

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that automates your phylogenetically

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constructions for you I'm not paid to

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say that with free fuels and and if you

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have a favorite gene that you want to

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reconstruct you can also use this tool

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it's called Philo bot comm so here

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you're looking at the evolutionary

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history of Rubisco protein we are

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starting with our interest with soil

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bacteria because cyanobacteria is a

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first of all as I said it has the

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highest abundant of Rubisco protein

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today and also we can culture

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cyanobacteria in the lab and even

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engineer its genome and that is of

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interest to us because we are interested

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in alternately understanding what these

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ancient sequences will do inside the

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cell and hopefully measure the isotopic

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fractionation values that will be

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generated by these ancient proteins and

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then see if we can match the isotope

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fractionation values that we generate to

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the rock record so just to walk you

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through here your starting oh my

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goodness alright well designers we have

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the 1b cyanobacteria and you're the

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ancestor of 1a and 1b one in one being

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very walking backwards in time and going

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back to the ancestor one two three this

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is the whole ancestor of all the groups

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so we can walk back in the branches of

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trees and and then reach to a time in

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the past and then reconstruct the

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sequences of these enzymes and all these

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sequences for the first time are

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available for the use of any community

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really and hopefully the geobiology

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musi so by going into this address you

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can download this tree and also click in

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h and every note and not only see the

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most likely sequence but all the

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possible ancestors so if you have a ruby

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story an organism that you I don't you

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extracted in the field and you think

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that there is an interesting Rubisco

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sequence there please go ahead and use

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our tree to compare these sequences and

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see if you can find some interesting

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stories and we have some members in our

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community they are using art that are

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using our Trina for group to riscos and

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which is great so here is the

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evolutionary history of Rubisco

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operating in a more simplified way the

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in blue is the cyanobacteria the current

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cyanobacterial group and all these um I

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will come to what all these aesthetics

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is mean in a minute but we basically ask

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what is the sequence what is the

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structure and what is the functional

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suite ancient Ruby stores and can we

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infer any information about to pass by

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using these sequences structures and

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ultimately function so let's start with

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the group one ancestor here you're

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looking at the knot sequence by the

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inferred structure of the disk operating

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this is the ancestor of Group one that

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is also you're looking at the

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cyanobacteria ancestor of Rubisco that

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may be live as old as the cyanobacteria

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ancestor did and and here is the group 1

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3 ancestor and group 1 2 3 ancestor so

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as you can see there is no drastic

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change in the structure of this protein

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that the structure of the rubik's code

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seems to be conserved across time so

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okay how about variation at the sequence

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level apple can look apple in variety of

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ways but even for apple we have

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different DNA sequences and that's what

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we've looked at we basically ask that

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the structure identity mean functional

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identity and is just that general

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phenomenon in biochemistry that that's

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not necessarily true DNA sequence can be

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pretty identical for between two

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different proteins but their function

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could be drastically different depending

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on where these mutations where these

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differences between these two sequences

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may be located and that's a property

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that we see even at the currently

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existing ruby

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even though they look pretty similar the

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our carbon fractionation values that

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they exhibit and their carbon dioxide

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oxygen specificity even can be different

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and be focused on multiple locations in

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this protein that are thought to be

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associated with the carbon fixation

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ability of this protein itself and these

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are the n-terminal domains and alpha

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beta barrels and large small and large

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large subunit interfaces and these were

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all based on previously published

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literature data in the literature that

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showed that changes in this area could

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impact the property to be function of

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the enzyme that are referred as hot

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spots because biochemists like the drama

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well yo wait okay here we go so so here

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I bitmap the the Rubisco proteins that

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we see through time you're going

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backwards in millions of years so the

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structure is conserved but let's forget

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the sequence and for this lead with some

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evolutionary change evolutionary tests

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using the DNA and protein sequence data

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to understand the substitution among

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these proteins and deceased these

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substitutions can be adaptive so what we

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see is that the main changes happen

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between group 1 3 and group 1 and these

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are in the internal location of Group 1

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tree and group 1 seems to be

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experiencing mutation all over the place

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so the enzyme is evolving very rapidly

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when it comes to the ancestor of

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cyanobacteria and the group 1 ancestors

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sequence overall seem to resemble the

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modern cyanobacteria Rubisco which

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belongs to oxalate whereas the oldest

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ancestors resembled an anoxic rate

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sequence when we did multiple alignments

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and test using the currently existing

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sequence information so with that with

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that we thought perhaps the transition

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between the group one tree and group mom

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vacations that are the functionally

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important regions could maybe a

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correlate so again I have to show the

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slightest like everyone else in this

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session that revisiting the Earth's life

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history and the change in the atmosphere

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perhaps the these changes could be

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correlated with the changes in the

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most feared conditions and very wild

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when we don't see much significant

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change after the rise of the oxygen but

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what previous and even the previous

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presenters showed some cases of this

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early victim change in atmosphere so in

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fact our data seems to be suggesting

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these changes because we do see

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mutations in the protein that is even

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prior to the rise of cyanobacteria and

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our early example early data in the

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biochemical biochemical if you look at

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these enzymes we show that the early

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mutations that we seem to be changing

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the activity of the protein as much a

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little bit but the group one does seem

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to be changing the proteins activities

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which is pretty interesting that we

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think perhaps the ancient cell harboring

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the group one tree and sister may be

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preconditioning in if the atmosphere

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that this organism was living it has a

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little bit of oxygen increase or vice

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versa and our current direction is now

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to reconstruct the cellular partners

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that will be scar is interacting with

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and resurrected phenotype by measuring

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the carbon isotope fractionation of

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these genes that are present in

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cyanobacteria and for that we are

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focusing on the car boxes on this is

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also in cyanobacteria we have Rubisco

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Rubisco as many proteins in the cell is

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not functioning by itself it does rely

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on another protein I always call them as

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Batman and Robin the Batman gets all the

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credit but Robin is doing all the work

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carbonic anhydrase is actually helping

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the the car boxes on to concentrate the

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carbon dioxide inside the car box is

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also very constructed the the ancient

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sequences of carbonic anhydrase and this

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tree also will be available for you to

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use so we are currently engineering the

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cyanobacteria to not only contain

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ancient Rubisco but it is ancient

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partners to see if we can recreate an

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ancient carbon fixation system inside a

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modern organism for the first time and

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study the isotope behavior of this

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engineered organism which is exactly

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what I said here and with that

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I would like to thank our funders and

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for thinking that this project could fit

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here thank you very much Tim and thank

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you for listening thanks very much

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very nice talk for that last part and

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trying to reconstruct this in

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cyanobacteria what organism are you

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thinking of using your using snicker

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caucus oh go ahead

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super quick comment and this is also

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relevant to the previous talk to and as

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microbial ecologists I spent a lot of

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time measuring oxygen and mass and you

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saw in earlier plots that there's a lot

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of local buildup of oxygen and so you

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know it took a very long time for that

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to saturate different sinks and then

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build up in the atmosphere so as you're

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thinking about evolution is different

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either oxygen requiring enzymes or

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things that protect yourself from oxygen

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or things that are tolerant to an toxic

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toxic environment to keep in mind the

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local environment and the selection

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pressure pressure that could be provided

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there the timing of that may be very

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different from widespread oxygenation in

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that yeah exactly

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which is also why we're using libraries

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and not just one Rubisco of ancient

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Rubisco and tying that is the single

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parameter but we are using the whole

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library to see if we can look at the

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variation across different sequences

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that may represent different localities

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as well thank you yeah we'd add to that

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there's different scales of

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heterogeneity within math but also

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within certain portions of the ocean

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productive regions so it's a really good

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point we should move on but thanks you