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hello my name is george shibal and today

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i'm going to talk about

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biological complexity specifically the

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cellular differentiation within

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multicellular magnetotactic bacteria

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and the implications in the evolution of

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complex life on earth

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to begin i would like to introduce the

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idea complexity in regards to the

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evolution of life on earth using a

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simple cartesian coordinate system

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for the first cell to evolve simple

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molecules had to interact to form more

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complex molecules

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that eventually yielded the first cell

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there are many great talks at

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appgrad.com that

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discuss this first step in the evolution

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of life

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but i'm going to focus on the next step

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which was when that simple cell

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continued to evolve into a multicellular

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organism

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that eventually gave rise to life that

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we see on earth today

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of course at certain steps in this

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evolution of life in regards to

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complexity some cells plateaued and

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remain the same giving the diversity we

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see on earth today

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it helps to have criteria for

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multicellularity

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to describe what we observe as far as

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multicellularity

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organisms the first criteria being that

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the

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organism is built from several cells of

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the same species

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as a specific shape and organization as

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well as synchronized growth

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so this would exclude things like cancer

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there would be no competition between

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cells and they would

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exhibit a coordinated behavior in

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response to internal and external

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stimuli

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and they would do this using cell to

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cell signaling

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and finally there would be an existence

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of a division of

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labor something like the cells in our

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lungs and stomach are from the same

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organism but they're performing a unique

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and different

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function for the entire organism

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the organism that i study is

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multicellular magnetotactic bacteria

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or mmb for short and this organism is

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multicellular in that it's composed of

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several cells from one species

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it's magnetotactic which means it's

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capable of sensing earth's geomagnetic

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poles that it uses for navigation and

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it's a bacteria belonging to the

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diesulfo bacteriota

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in this scm image on the upper left

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i show uh two mmb that have potentially

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just undergone division and you see that

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it's a

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small consortia of cells that are

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tightly packed together

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forming this ball shaped structure

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and the uh backscatter electron image in

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the lower left

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you'll see it's uh mmv and you'll see

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these white lines within each cells

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and these are the magnetosomes a

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bacterial organelle responsible for

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magnetotaxis of the organism

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the tem is showing a slice of the mmb

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where you see individual

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cells organized around a cellular center

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in which you see these black dots in

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this cell in the upper left

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and these are the same as the white dots

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you see in this backstab

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electron image the magnetosomes and

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these are

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synthesized by the mmb it's a

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mineral called gright that has a

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paramagnetic

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dipole and they form them in these

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chains that

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essentially act as a compass needle

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allowing them to sense or geomagnetic

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poles that they use for

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taxes in the water column to find their

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desired redox state for survival

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this organism was discovered in 1983

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and we still have not yet been able to

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bring it into culture which complicates

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all the studies so you can't simply

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throw genetics at it to test really

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interesting questions

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because we cannot culture them yet so

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all the techniques used in this study

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are culture independent techniques

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the mmb are an obligate multicellular

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organism

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that have a putative life cycle shown

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here in this panel sems

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where the organism grows in size and

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then eventually divides

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and similar observations have been used

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made using microscopy

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mmb cannot survive as single cells

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either

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and here in this lower left panel you

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see a live dead stain

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where cells that are stained red have

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lost their membrane integrity and have

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died

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and so here you see two dislodged cells

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that die

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and eventually this loss of cells

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compromises the integrity of the entire

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consortium and the whole consortium dies

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in the sem panels on the right we look

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at a a healthy mmb

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and then b c and d some different

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scenarios where

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cells have become dislodged and the

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organism the entire consortia has

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the mmbi study are found at little sip

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wisdom salt marsh

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located on cape cod in massachusetts in

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here i show an aerial view of what this

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site looks like

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and it's a brackish site so there's a

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influx of

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sea water from the the bay there and

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then fresh water coming out towards the

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bay

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here's a ground level view of what the

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site looks like it's just a small

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marshy pool and the bacteria that i

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study

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are just found in the sediment of this

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pool now the video i'm going to show you

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is the mmb are in the bottom of this

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eppendorf tube

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and they're swimming up towards this

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magnet that you see here

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you see them slowly taxing in this 20

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minute time lapse video

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in the next video it's a hanging water

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droplet with the mmb

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swimming towards the edge of this

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droplet where i show a cartoon of the

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magnet that is present there

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i then flip this magnet and the

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individual mmd

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all start swimming in the opposite

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direction now thinking that the magnetic

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north is in the opposite direction

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just so to know each little dot you see

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here is a single

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mmv consortium that's swimming

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now next i switch the magnet again

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and you see the mmd switch their

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swinging direction

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moving back towards the edge of this

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now when we return to the criteria for

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multicellularity in regards to mmb

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we see that several criteria are

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actually met in that they're built

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from several cells of the same species

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they have a specific shape and

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organization

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there's no observed competition between

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cells and they exhibit

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a coordinated response as we see by an

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external stimuli

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of a magnetic field that coordinates

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their swimming

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we still are investigating cell to cell

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signaling that we're using genomics for

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and we want to know more about a

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division of labor within mmb

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where we may potentially observe

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differences between the function of

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cells within the consortia

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so to begin we want to do whole genome

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sequencing

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so we sampled sediment from the marsh

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and

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magnet next to it

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for about 60 minutes and the mmd will

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swim

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up out of the sediment towards the

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magnetic magnet and

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form this small cell pellet you can see

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here that we can then simply just

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remove by pipetting we then took that

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sample of mmb and sorted them using

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fluorescent activated cell sorting to

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isolate the population of mmb

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and we sorted them into a 96 well plate

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where we did multiple displacement

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amplification

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in short read illumina sequencing to get

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22

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mmv genomes

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we then use the mmb genomes to address

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whether or not the

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consortium is clonal and then is the

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genome in one cell the same as the

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genome in its neighboring cell

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addressing this division of labor

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question

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and what we found where they were not

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entirely clonal so

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what this sequencing project did is we

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generated

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a several libraries from a single mmb

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in a well that was sequenced and we

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mapped those libraries back to the best

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assembly to identify

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single nucleotide polymorphism

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differences

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within a single mmb consortia

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and this work done by frederick schultz

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at jgi

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found that mmp have a higher rate of

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variance

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uh snips per genome as compared to the

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pseudomonas control

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we use as well as other environmental

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co-sorts that we had

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so what we ended up with was roughly

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50 to 110 sniff differences per genome

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within the consortia

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so between individual cells there was

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this difference

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well we're still interpreting this

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result we do know it's going to raise

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some very interesting questions and

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using the full-length 16s rna

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gene that we obtained from these genomes

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we found that there are

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five distinct populations or groups of

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energy

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existing in our site so we built this

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phylogenetic tree to show their

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relatedness

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and we compared them to other known and

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published on

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mmb existing across the world here you

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see a

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b and c and the top of this tree have

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this morphology of a

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spherical shape with these soft edge

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shaped cells within the aggregate

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and in the bottom of the tree in d e and

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f of course finding these micrographs

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you see that the mmv have an oblong

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shape to them and these this

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uh unique h4 shaped shell

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cells so we then have the question well

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do we observe these unique morphologies

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in the samples from our site

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that are shown in these colored groups

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and when we go to the fluorescent

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microscope

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just using a dna stain we see you know

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basically that the morphology

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is uniform across the different

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populations so we needed to develop a

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method to look at the

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distinct groups or populations of mmb

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from our site

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and their morphology so we developed a

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protocol for correlative fluorescent

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in-situ hybridization or fish

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scanning electron microscopy shown here

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we're going to take a sample to a

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scanning electron microscope obtain an

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image

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and then use fish to identify specific

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populations here

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group 1 and group 4 from our sample site

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and then map that back to the sem image

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to look at their morphology

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i'll discuss a little bit more of where

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this project is going at the

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in my last slide we also wanted to look

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at the

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division of labor and to do this we use

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substrate analog probing

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where you'll incubate the mmb in the

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presence of a

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methionine analog or an amino acid

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analog that has the

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azide functional group and so when new

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proteins are synthesized these new

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proteins will have an azide functional

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group

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they can then do azide alkyne click

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chemistry and

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covalently attach a fluorescent dye to

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the proteins

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so what we hypothesized was that the mmb

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consortia

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would either not incorporate this aha

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and see no fluorescent in the contortion

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or every cell in the consortia would

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incorporate this uh

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methionine analog dha or alternatively

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we'd see a division of labor where some

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cells are incorporating

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the aha and newly synthesized protein

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and would be highly active and other

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cells may not be making

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as much protein we can also use other

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substrate analogues for peptidoglycan

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synthesis

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and as well as a fatty acid for membrane

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synthesis

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we then use structured illumination

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microscopy

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the incorporation

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or the synthesis of protein and

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peptidoglycan

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between cells within the consortia and

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we did this with the peptidoglycan and

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the membrane and didn't observe any

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major differences the dark spots you see

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here are

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uh just the light block from the

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polyhydroxybutyrate uh

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granules or energy storage granules

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within the cells

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so this investigation is ongoing

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to conclude the current and future work

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of this project is to

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continue with the correlated microscopy

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where we'll use stable isotope probing

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and link that with our scanning electron

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microscopy

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to get morphology elemental composition

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as well as raman microspectroscopic

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micro spectroscopy to look at the

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chemical composition

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and link that with fish and then finally

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use nanosims to look at the localization

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of these

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substrates in individual cells within

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the consortium

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to link taxonomy physiology and

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of course i couldn't do this without all

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my collaborators in my lab as well as

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those at jgi and emsl