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so right now we have a special talk in

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the schedule uh from the rfg that just

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happened this last weekend

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and so this is uh for you guys that

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don't know

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part of abroad con every year we have

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hosted this research focus group in

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which it's sort of a

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very intense uh two to three day writing

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proposal writing boot camp

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where

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a bunch of you guys show up

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just write furiously for two days

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straight

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and then we have this whole proposal

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review process you present your work and

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everybody just gets together votes on it

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and has a good time writing the

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proposals talking about the proposals

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and just getting used to

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the entire proposal writing process

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so if it's it's something very important

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for early career astrobiologists to take

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a part of and it's something that if

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you're going to future ab grad cons that

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you should consider it's very helpful

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not only for writing proposals but

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getting prepared for your candidacy

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even getting prepared for your defense

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but something as scientists that we

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typically have to do a lot of is this

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proposal writing stuff

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and so

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from this last weekend

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this is the winning proposal that was

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chosen and so you can see a highlight of

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the work that is done over this weekend

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and can see what can come out of this

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and how awesome the projects can be

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so group a

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go and present

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all right well thank you very much

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bradley thanks everyone for coming

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we're going to completely jump ship from

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what the first session was about and go

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to some talks about biology

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life on earth has come to fill almost

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every niche we can imagine

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living in marginal environments and

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extreme environments as well as well

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extremes of

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things like pressure ph salinity as well

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as temperature

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finding out how life can survive in

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these various extremes is really

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important for astrobiology to understand

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the origins and evolution of life

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but also how life might thrive elsewhere

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in the universe

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and so for our research we were studying

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or proposing to study

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an algal strain that has the capability

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of surviving at extremely cold

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temperatures

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this alga

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a species of the genus klebsormidium has

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been found in environments across the

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planet some of those marginal some of

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those extreme in extremes of extremely

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low ph

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high contaminant metal abundances

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extremes of desiccation and extremely

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low temperatures they've been found in

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some cases to survive down to negative

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40 degrees celsius

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and so we're going to talk to you about

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one strain that jessica has isolated and

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the mechanisms we're searching for in

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this organism that allow it to survive

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in the cold

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and with that jessica will tell you

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so i'll be talking a little bit about

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our where we found this alga

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and that would be in colorado so

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we have this region of hydrothermal

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alteration that's rich with sulfide

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minerals to include iron copper

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silver

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and gold so these are traditionally

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mined and they have been traditionally

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mined and this a lot all along this

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region of the

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rocky mountains so the field site where

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i collected this alga was in the

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silverton caldera

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san juan mountains in southern colorado

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and that region is also characterized by

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the heavy mining activity historically

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it's been mined so it's been about 150

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years 200 years that it's been mined

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this is the red mountain where you can

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see all the sulfide weathering so when

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sulfide minerals weather they whether to

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oxidize sulfur to sulfate and oxidize

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the metals like iron 2 goes to iron

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three which you see as the rust color

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and what happens when uh the ore is

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extracted from these sulfide minerals

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from the from the mountains

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you get mine tailing so mine waste so

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you get rock piles that are upstream of

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hydrogeologic features so what happens

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is we get groundwater runoff from

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underneath from groundwater and

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precipitation so what we end up getting

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is this runoff that's toxic to aquatic

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life and it's rich in metals here you

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see there's nothing growing where we get

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the runoff from the acidic mine drainage

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and this is the overall equation for

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acid mine

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runoff so there are four equations

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stepwise equations so where we get iron

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oxidizing to iron iron two oxidizing to

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iron three

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uh the sulfur oxidizing into sulfate we

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also uh get hydrolysis and we get a lot

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of protons uh as the iron three

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precipitates into the solid we get hype

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you know it scavenges the hydroxides so

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we get very acidic runoff uh combining

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with with the um

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with the groundwater sources

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so these are the types of environments

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where we found the alga and it's quite

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it's it makes the alga unique for a

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couple of reasons and one is being in

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sub-alpine colorado it's very resistant

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to

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in culture collections because they're

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not

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they don't freeze well if you freeze an

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elgat then we can't bring it back so

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it's very they're

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difficult to maintain so these guys are

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resistant to freezing as well as

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all this very rich metal runoff

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they are also very uh

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they're very sensitive to copper so this

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runoff also has a lot of copper in it

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and actually we use copper sulfate to

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keep algae algae out of our swimming

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pools so i we took this culture uh

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cultured it in a fish tank with its

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native conditions so with under a lot of

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metals and low ph water which was ph3

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and with this algo we can

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do our downstream experiments

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so what i proposed

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my my approach to identifying the

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mechanisms that allow it to survive

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freezing is to first we can do a

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bioinformatics approach so the genome

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has been sequenced so you can do some

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bioinformatics take some

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statistics or find open reading frames

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that would presumably encode for genes

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and

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find the ones that are similar to other

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organisms so you find homologous

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sequences and other organisms that are

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tolerant to freezing and then you can

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take that gene

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so you want to switch to the lab maybe

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okay so

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we can take the genes put it in an e

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coli host which is that's what we use in

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the lab to screen for functions

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and a positive result would uh

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be the e coli that we introduced that

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gene to would be more resistant to

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freezing so that's one approach that

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also assumes or is dependent on having

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other organisms with the mechanisms

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already have been having been

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characterized so that's not always the

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case and uh so an alternative approach

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is to do a functional screen where we

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take the we stress out the mrn of the

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algae we stress out the alga we freeze

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it bring it back harvest the mrna which

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is or all the messages so this is the

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rna is run on a polyacrylamide gel

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we run a current through it and we

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separate the rna by size so here we have

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the ribosomal rna in between the light

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gray areas those are all the messages so

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it's only two to five percent of the

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total rna the messenger rna so what we

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can do is we can harvest that and

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convert it to c

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complementary dna using a viral

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enzyme

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and we can amplify it and turn it into

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dna so that we can use it as a gene

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so what we can do is take all the cdna

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that we have made from the rna

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and

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here we see that here's a ladder that

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will tell you the size of all this dna

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that's been run on a gel

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so all of these are the different

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transcripts different sized messages

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from all the genes that it's been

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transcribing while it's stressed for

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during stress during during freeze

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stress as well as a bunch of other stuff

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that we don't know what they are we

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don't know what the transcripts are so

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we take these bits of dna put them into

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a vector a little piece of dna that we

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can introduce to an e coli cell so this

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is the chromosomal dna that the e coli

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needs to live it also has extracellular

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pieces of dna that we can introduce

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and we can introduce the functions that

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these genes encode for

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now what we can do with e coli to select

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for the genes that confer resistance is

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we can freeze these cells and the cells

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that don't survive are the ones that

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don't have any uh dna from the algae

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that allow resistance

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so the ones that do survive will have

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presumably have the inserts that confer

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resistance and then we can take those

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separate them on a gel find the one of

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interest send it for sequencing and map

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it to the

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gene that uh on the genome because it's

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been sequenced and then follow with uh

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characterizing the gene product and uh

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the mechanism so that's one approach and

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that leads us to the protein

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characterization and the types of

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mechanisms that we can

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possibly screen for that graham will go

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okay thank you jessica uh there are

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various mechanisms that have been

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discovered already through which

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organisms survive extremes of cold these

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include increased uh solutes inside the

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cell in the cytoplasm like sugars like

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glucose

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and sucrose to help the cell survive in

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lower temperatures but one really

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interesting way that cells have learned

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to survive these kinds of low

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temperatures is by creating novel

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proteins in the case of this picture

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here they've created anti-freeze

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proteins that effectively create ice

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cages that bind around ice crystals and

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stop them from forming large structures

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to allow the cell to survive a lot of

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small ice crystals inside the cell next

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slide

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so to continue our characterization of

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this algal strain this is kind of a

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quick and easy way doing some lab work

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to assess what temperatures this these

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cells can survive down to how fast they

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can be cooled without without dying

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completely

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how fast it can be heated back up and

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the duration of time they can withstand

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freezing from days to months seasonally

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or even yearly next slide please

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and in doing our characterizations

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looking at proteins there's a lot of

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things you can do in molecular biology

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to understand proteins it's a very wide

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field but we would start off with

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something really easy doing an assay

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just to quantify the amount of protein

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within a stressed cell in the cold

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versus a non-stressed cell to see if

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there's a difference and then after that

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if we wanted to we could use other

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techniques like this image i have behind

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but can't explain through the time

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that we could use to characterize these

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so my job is to

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test the geochemistry

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condition in the water where the algae

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is will

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help or inhibit the algae to survive

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freezing so this is a picture of the

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algae we have and

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the um we are testing the concentration

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of chlorophyll um

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before freezing and after freezing they

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are the green

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pigment in those cells and the process

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is we grind it and

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we use a filter to get the juice

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and then analyze the concentration of

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chlorophyll by the

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spectrophotometer and then we

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we freeze the algae under

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three different

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geochemical conditions first is to

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mimic the real environment and then use

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the real environment then on the third

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one is the control using the pure water

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and we then defreeze it

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saw and to continue the grind filter and

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analyze it again

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so the there are three results we expect

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to see

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uh not three more than three but i'm

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listing these three simple ones and on

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the x-axis you can see

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under different conditions um

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the y-axis is the concentration of

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chlorophyll and if it looks like image

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one

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means the they are um the chemical

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conditions are helping them to

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survive freezing and second one

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means they um inhibit them to survive

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freezing and the third one means they

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have no effect

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for that

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and water

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or what if there are minerals

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participate

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in the solution

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so there comes our technology xrd so

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this is a image of xrd in my lab and

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that's a geochemistry

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geometry and that's a principle behind

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it i have no time to explain

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so just super quickly um we're talking

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about this in the context of guiding

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exploration of other planets like

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particularly mars and europa

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on the on the right i just have a plot

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here of the daily temperature

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fluctuations on mars

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but i'm just going to skip to the next

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slide

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and talk about the relevance to nasa

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objectives so the reason that we picked

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mars in europa is because

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for the planetary decadal survey for one

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reason they're the two highest priority

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flagship missions are to those two

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planets

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but also as applications to the mars

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exploration program which is explore

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habitability and also the nasa

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astrobiology roadmap

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in the last slide i just have

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description of the two goals that our

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project meets towards astrobiology

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roadmap

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including biochemical adaptation to

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extreme environments so with that thank