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[Music]

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[Applause]

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thank you very much the organizers for

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the opportunity to speak tell you about

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some work that is ongoing so most of

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this stuff is not published but we're

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getting close so kind of getting excited

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I'll try and run you through some things

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so I think the main problem that we've

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struggled with in early evolution is

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that we used to be focusing on how can

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we take things like genomes and

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reconstruct ancestral States and the big

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problem that we've run up against is

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that now that we're awash with data the

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signal actually seems to be quite poor

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so oh sorry yeah so here's just one

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example from work that we've done which

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is it I threw this in because a lot of

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our NA stuff in the meeting but people

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have done the same thing for proteins as

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well basically what we did was we looked

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at the our fam database which is a big

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database with 15 million 15 hundred RNA

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families and asked well how many things

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appear in more than one domain and as

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you can see from this Venn diagram the

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numbers are very small when you look at

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what's actually there and likely to be

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conserved you know it's it's basically

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stuff associated with the ribosomes so

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we've obviously heard quite a bit about

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the ribosome and previous sessions and

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then our own ASP the tRNAs so this is

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sort of stuff you would you would be

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able to I think guess from without too

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much trouble there's a few other little

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details in there but the main point is

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that this very little signal there the

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signal is consistent with what people

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would get when they look at protein data

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in genomes but you know that's weaker

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sort of say well maybe we can think of

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better ways to do that or maybe we can

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rethink how we're doing stuff so if the

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signals limited what else can we do one

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of the things that people doing this is

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a little nice little paper of Aaron's is

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to look at modern stuff and say well

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some interesting analogies between

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modern biological systems and presumed

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ancient systems what we're trying to do

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is sort of say okay that's a great idea

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can we take that a little bit further

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and say do some experiments to try and

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retrace some of the steps towards

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evolution and modern systems but more

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biological perspective sort of coming

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top-down if you will so there's been a

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lot of really impressive high-tech

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presentations around methods I've seen

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some quite quite spectacular pieces of

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work and I just wanted to contribute our

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own high-tech approach to how we do this

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experiment so this is a school lunch box

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we used to pack in more and more

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multiple evolution experiments on top of

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one other so there you go there's all

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kinds of ways you can do astrobiology so

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some of the questions were trying to

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grapple with with our little lunch box

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setup are listed here and I don't have

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time to go through all of them but I

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will give you a brief overview so this

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one here instead of with understanding

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how bacterial translation initiation

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evolved and I'll talk about that one a

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little more this one I don't have time

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to talk about but I'm happy to talk to

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people about this so we've done a little

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experiment where we've applied a

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continual bottleneck to to see whether

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we get complex RNA processes and heavier

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check with that about that later on and

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then the final one is we're actually

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looking now at trying to understand how

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DNA might have evolved and I'll come

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back to that again at the end so for

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translation initiation one of the things

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that we we thought was puzzling as

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biologists is that bacteria use this

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formulation process so everybody charges

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their T RNAs so you get a charged tRNA

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so this is the initiator and then in

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bacteria you have a couple of extra

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steps that you don't find in our carrier

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you carrots the first one is edition of

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the Fulton group here through an enzyme

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called a formal transferase translation

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then proceeds but as the growing peptide

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comes out of the the ribosome exit so I

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another enzyme peptid formulae's comes

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along and what's that for mate group off

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again so this is before the proteins

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really had a chance to do anything so

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it's an odd thing that you add something

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and then remove it all prior to getting

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to a point of a functioning protein

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interestingly if you treat with a drug

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that knocks out production of

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this cofactor that's required for this

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reaction here you force cells to use the

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unmodified version here so a methanol

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tRNA you can also do knockouts which

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cells don't really like but they are

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viable where you eliminate both of these

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genes and you get the same thing

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proceeding in translation proceeding

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with a non modified version of society

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of confining and finally if we look

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across the Tree of Life this is a it's

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not a universal process you only see

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this in bacteria archaea and eukaryotes

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don't do this so we thought gee that

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looks a bit strange it sort of looks

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partly like it's not that important or

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necessary because only some parts of the

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tree use it and it's intriguing that you

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can knock something out that's being

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conserved within bacteria for possibly

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several billion years so we did an

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experiment to figure that out so what

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what Alanna rickety who's here and Ryan

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Catchpole did was to do a bunch of

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experiments and this is one of them here

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so what we're doing is we're evolving

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two lines over about 1500 generations so

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this blue line here is just a wild-type

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control and this gray one here is a

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knockout where we've eliminated both of

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those genes required for formulation and

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you can see that after about fifteen

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hundred generations their their growth

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is indistinguishable so it seems that

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you can be a bacterium quite happily

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without this process the thing that we

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thought was quite unusual about this is

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that the addition and removal looks a

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little bit like a toxin antitoxin system

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so I don't know if you guys are familiar

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with these idea they're sort of

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addiction systems so given what I know a

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lot of time I'll walk you through the

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experiment that we did to test this at

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the same time as explaining what they

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are so we think it's it this may this

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could have evolved originally virus

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elfish element so let's do this

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experiment here let's reintroduce those

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D D formulas informal formulas genes

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back into these evolved lines and so the

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way we do that is to put them onto a

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plasmid if you put a plasmid into a cell

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without any any kind of selection marker

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or whatever it will sometimes mistake

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and you can have individuals that lose

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the plasmid but they're not affected by

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that in our experiment what we've done

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is we forced the plasmid up by using a

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temperature sensitive one so we kick it

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out and what you see here is two

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different lines so these two one of

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these as colony forming units and the

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other is optical density so two ways of

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measuring growth and you can see this is

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the plasmid occupancy you can see the

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plasmid it gets kicked out pretty

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quickly but it has no effect on growth

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if we take a known toxin antitoxin

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system and do the same experiment so we

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take the exact same plasmid put a toxin

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antitoxin system on it and then force a

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kick out we get a very different curve

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here so there are two things that are

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that are happening here

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one is the plasmid is getting kicked out

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but the second is that both the

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measurements of growth are showing a

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reduction in growth and so what's

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happening in this system here is that

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when you get a knockout of the loss of

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the the plasmid you get cell this and

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this is sort of the cleverness of these

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addiction systems basically the way they

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work is if you get rid of one gene that

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is you're effectively your antidote to

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the toxin and both of these are carried

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here then you can no longer protect

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yourself against the toxin and you die

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so we did the same exact same experiment

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reintroducing our two genes the

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formulation the and the methyl

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transferase back on a plasmid and if we

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get this kind of result then we would

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say there's no sort of selfish element

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activity if we get this kind of result

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it would be consistent with this post

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segregation of killing phenotype

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and here's the result so we think that's

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pretty clear that once you've removed

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these two genes from this lineage and

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evolved them to sort of tolerate not

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having them and you put them back in you

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don't get sort of a complementation

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phenotype you get an addiction phenotype

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so what we think is is happening then is

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that these things are functional now and

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it's a you could I give everything's

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co-evolved you kind of need them because

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they're there but it's not obvious to us

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that this is particularly adaptive and

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there's two reasons for that one is we

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can get rid of it with no effect on

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growth after we

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sort of readapted that lineages the

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other two lineages are care and

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eukaryotes don't use this so it seems

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that this there may not be an adaptive

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function what we think might instead

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have happened is that this parasite got

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in and ensconced itself in translation

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and then the whole system is sort of

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co-evolved around that and once that's

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done that you can't actually get rid of

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it but not for reasons if it being

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advantageous okay so the second thing

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just how am I going for timer a little

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bit of time okay cool so I just used the

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last couple of slides just to tell you

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some stuff that's much earlier on in the

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process we're interested in this pathway

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here this shows how the DNA building

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blocks are made in all cells so

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basically what happens is you get the

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RNA building blocks here an enzyme

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called ribonucleotide reductase performs

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a reaction to convert these into deoxy

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ribose nucleotides but as you can see if

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you start from you your fourth deoxy is

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going to be a DLC you and there's a

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bolt-on pathway here to get to T so some

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of you may hopefully have seen a poster

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by Alana rickety but if you haven't

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please come take a look at it she's been

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addressing this these two questions here

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one can we delete these steps the answer

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is yes you can so she now has lines

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growing that don't do any of these steps

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here so they should stop at deoxy U and

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then what we've been doing is trying to

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get them to adapt to the absence of this

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pathway by supplementing them with the

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oxy T in the medium but then ratcheting

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down the amount that they have and so it

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still early days but she now has them

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growing without supplement whatsoever so

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our next question will be sort of what's

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happening there at the genome level is

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your asil accumulating we've also gone

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after arriving nucleotide reduction and

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I'll just show you some very preliminary

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results on that so question we we sort

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of had is can we eliminate this pathway

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turns out we can do a knock out of that

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and you have to supplement with the four

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deoxyribonucleotides for this cell line

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to survive so two people were being

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working on this analysis if I ever form

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an honor student and Sam heiress who's

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in the lab is the technician at the

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moment but it's probably more postdoc

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than a technician and what you can see

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is over about eight transfers that we've

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done so far we see a tenfold reduction

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in the dependency so this is sort of

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minimum concentration that these eco

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lice can survive on in terms of

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supplements so they're in a minimal

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media with a little bit of glucose and

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then a supplement of deoxyribonucleoside

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so they're clearly adapting to that this

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may be tricky to see but while here's

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some wild type e.coli and what we get is

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a very interesting sort of elongate

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stress morphology in these bacteria so

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this is a transfer one you can see that

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these Ecola much much longer than in the

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wild-type and some of these are you know

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ridiculously long by about transfer six

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other sort of some of them may be I

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think thirty to forty micrometers so

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they're huge not very heavy anyway so

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we're still working on that and of

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course one of the challenges is whether

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we we actually need to eliminate this

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before we we can get complete loss of

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dependency on this pathway here so I

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leave you with that teaser and just make

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some acknowledgments that are people to

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do the work and obviously if you ever

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want to do any research involving

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lunchboxes I can highly recommend this

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New Zealand company here that makes it's

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probably the world's best Google lunch

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[Music]

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yeah I mean we've done a bunch of other

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experiments which I didn't have time to

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present but you know we've knocked them

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into the genome and looked at whether

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there's a fitness improvement there

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isn't we we noticed that they become

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very very difficult to knock out as soon

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as you've got them in we yes we've also

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sort of evolved the the knock in lines

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again

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to see whether the system rear depth and

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it becomes very difficult to remove them

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and we definitely see that so this is a

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bunch of other data for sure okay thank

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you and so we have to move to our next

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speaker sync thanks again