and so I'm very pleased to
announce our next speaker Mary Lindstrand
we're very fortunate to have Mary here today she's
suffering from a little bit a voice problem so we're gonna turn up the
volume here a bit but
the Mary's been here a number of times before and has given talks but
so mary is many view no hails from the University of Washington where she is
vice provost for research she's also the Frank younger chair
have engineering and has appointments in biology in chemical engineering
in mary has the made many outstanding contributions to microbiology
particularly
in the area above the file a traffic organisms that are involved in
process the scene single carbon molecules and
in the past she's use the tools and genetics physiology biochemistry to
understand
the pathways that these organisms use and now her
researches evolved to using more systems level tools to understand these
processes season
a developing technologies to investigate things a
basically the single-cell level soul very
that alright so hmm
I have to apologize for my voice today those few
heard me speak before now is not my normal speaking stop
for I've discovered sense I got laryngitis
people listen very closely tonight
sup um who time very pleased to be here today
I'm really wearing two hats with my vice provost at
I'm representing the universe to Washington
at the opening office wonderful facility
but I'm also wearing my faculty member at
and talking about a very interesting
feel we funded project were in the second year of his
it's a work in progress up but I think you long
find a spirit interesting 100 pick up on
this concept of functional redundancy
that alan talked about and this project is about
a very challenging problem which is finding p
I'm now when it comes to functional redundancy okay
saw I take a minute and explain you
what's the Department of Energy interested
in the cycling one carbon compounds
I've actually at funding from the to-ing
since nineteen eighty so has been a lot of investment in this
why is that well actually there's a number of reasons
call issue of carbon cycling C-one cycling turns out
to be very important in all environments
global warming there are many
one carbon compounds in plated compounds
better key elements I'm
in that issue in the whole detoxification by remediation issue
these organisms are important in the degradation of halogenated solvents
and finally the area that I'm most interested in myself
his in cream chemistry krissi001 compounds
as part of it stops right so it kid
to the score question that this particular project is about
we know and we heard
today that most picture the fireman
have never been either cultural are studied
so how to killing cells in their genomes
to functions in the environment when
most fun we don't know anything about so
this area has importance both in biogeochemical cycling
and in the understanding have communities in their function
can also this whole idea
have using microbial communities has early monitors
for a change for environmental change right
hope talking to talk about a concept
that's a little different from that killed concept to do here Alcala
I so functional populations these are
populations within a community pick your help the same function
so I one of the three studies utilization
of methylamine and here's our
false colored up picture a mixed population
of following you lazar's so they carry out that same function whatever news
but they use different enzymes and metabolic pathways
so it's functional redundancy
not have to file a genetic level but
have to functional module level
different enzymes different genes
but they carry out the same pathway absurd thing popped
soap how do we uncover the unknown
with you and I so functional populations
that's the focus on this project and that's this
issue that they've touched on a little
got the chance better dominates in carrying out a specific function
may not be the same as those identified in lab cultures
and this is a step beyond the idea
that the organisms might be the same this is the concept
cappuccino might not be the same okay so here's to
idea I'm very visual so
I'm here's your population your community in nature
it's all turn out the same function a minor part is that
contains the known she but the maturity
contained in unidentified teen
never been connected with that function
okay so bad hun identified gene
his invisible you there
but fortunately it's invisible invisible to prolapse
and primers an annotation an even know
hits the dominant gene for that function
you can't see it because it's apparent that
vast group I've genes of unknown function
in your mad at you know next sequence so
although the metronome and all the meta transcriptome
data in the world don't help you
because it's never been connected to that function that's the issue
okay sup add your link function
to novel genes when this thing to tap those chains
have never been ice leader study it's a very typical problem
and that's the problem we took on in this new project
and we can just take it on for kicks
okay awake first let me tell you about our sunnyside
hope so we've been looking at Lake Washington it's really handy
tried outside our back door in up
University Washington so nice to look at traffic Lake
oxygen into the pot
oxygen goes to the minimum out one centimeter and
sediment so we look at that top centimeter
whole lot happening where that oxygen goes to zero
we've actually study side for 25 years study 21 cycling
we use the Site Madison Park site that actually has been studied
per about forty years how we do culture engine light breeze CT NE libros
lots and lots of background about C$1 cycling
in this site and we have a managed only database
from stable isotope Hinrich TNA
and this is all coming from our unicef microbial
Observatory project and so we have this
high-resolution metagenomics project
mainly carried out by my colleagues Miele
just assertive a and marina counts nya
and the Joint Genome Institute
so the way this works you can't repeat samples with c13 labeled
substrates and we've used to for once
an active bacteria in corporate that c13 into their DNA
you isolate the c13 label TNA I do seek radiance
and then chichi I generated mention only
sequence okay so
hum so we have this database has been published
not going to talk about this too much but here was the issue
it has to do with the genes for methanol oxidation
K we know a lot about methanol oxidation
it's been studied for over 35 years
there's a class called methanol dehydrogenase superior low
cool econ own connected in Sun as to sub units
his cytochrome c but the problem we had
is that in this matter to know next sequence where we know
there's lots of methanol oxidation activity in that community
we see these genes very very under-represented
and in addition through this project
there were newly isolated been a pretty picture in the pilot rose
i lovers tillis and they don't have either
the activity this classical ensign or the jeans
and we know the stranger significant
and methanol oxidation in Lake Washington
K so to make a long story short marina
solve this problem had found
after all these years a study methanol utilization
there's a different methanol oxidation system
I'm it is still a PQQ lincoln's not
but it's a single sub unit and sought you have to use
different ASI conditions to see it any toes in class
file a genetically with a classical methanol T
Hydrogenics he classifies with type 3
alcohol dehydrogenases so in Mendocino sequence
his sanity did either as a No present
if not the androgynous which everybody knows
these ethanol tea and Roger nieces don't oxidize methanol
but this one does right to never seen before
so here we have are like Washington Community
we have small percentages after classical
methanol dehydrogenase and others that now I have this new one which we call
and each tune so again this one was invisible
tour proves inner primers an annotation
I'm but there's another issue
even when we add these in it really doesn't count
for all the skills we know are oxidizing methanol
so we're sure there's a third one
it's not only methanol oxidation where we have missing jeans
we have a whole series of other C-one functions
I we know we're missing sup we need a way
to find these unknown sheen's
involved a methanol oxidation and other functions
and we can't understand what's going on in these communities
with potion until we find these missing James
K so this is the problem
time to walk you through the approach trap
crashed applying this to a number functions
and c1 cycling primary focus on methanol oxidation
for simplicity so I'm gonna walk you through the steps
and then I'm couldn't go through up how it works
and what we've done I'm so you've got this
mixed population we have a situation where
a total population but safe cycling methanol
is maybe only five percent
a total bacterial population in this environment
so we need to enrich functionally for that group
can we to that using life still sorting
in a redox guy gonna tell you about
so we still sorting you can enrich for
that uncultured population the carriers out
that function like methanol oxidation
alright now we've got this in rich population
and we screen at the single-cell level for those
actually oxidize methanol not sure how we do that
now we have the single cells
that come out tonight as methanol we wanna cultivate those
and keep your cultures and once we are pure cultures
week cheetah type to identify the strains
that don't have chains we know about
and then once we find those normal strains
and this is the part where just starting to to
sheezy then we can do sequencing and further phenotyping
right so will walk you through each of these steps
but remember that the coal is
to find these novel organisms
and work with them K so the first step
is to enrich these target populations
we take samples from the top layer the sentiment
and we use this um in Peter chin die
called reduction surgery it's a lot of cell
redox dot arena to law to work with this
this work was published last year news feature no
I'm not going to go through all the controls an optimization cheated
but who just optimize conditions
um you take the samples there are mixed population
you have to read oxen's for green a specific level
any at the substrate at a specific level
and it only takes about an our for the souls to respond
and the ones that are actively metabolizing
turn green who
its water
just
ok now good some other ones
better green for already active and they're in here
and some of them were inactive and responded to
such a like methanol so it's a mixture
but its enriched in the ones that respond to methanol
and then uses so store indicate the active population
Anakin created a lotta work
with pure cultures and start cultures and I'm
lots of background work not to
optimize what that gated population should be
now you have a population enriched
who knows pack an oxidized methanol okay so
hot show you some examples when you look at the cells
under the microscope about how they respond
I'm and this is Sarita permanent a Dell system
have Michael kind of ca develop for us and yes
this it sounds fun he is a postdoc in my lap
he's not really a microbiologist he's a chemist
that is becoming a microbiologist am soap
her and he's really great in optics in at these in elsa systems
so this year retail supplier essence
from single cells under the microscope
their uncultured cells that have been
enriched with the redox encircling I'm
and so I'm gonna show you three kinds of responses
some other styles okay so this is time in this fluorescence
you had three dots inter-korean some of them
practically were sparring and their bright and then later when you add
methanol there's no change
so these are the non responding but actively responding cells
ok alright then here's what happens with responding cells
yeah reductions for Karina really damn but then when you had never know
food may come up up by the way this is just noise in the analysis
this isn't looking at multiple cells
this is just you don't want to sell you have noise in the readout
but you can see that you can't activation
um and so these are responding cells
and then solar cells not too many actually getting it
when you have the substrate pride and we're interested
in these ones the respon- so the redox inter-korean
does allow us to enrich for
on these responding cells okay so
now you have this in rich population
they want to screen
happy individual soul level for those
Peckham utilize methanol you can do this pretty some shred
so be his technology that was developed why
up up program at the University Washington
call the microscale Life Sciences Center pesar
class micro well got basis to
you can't used standard microfluidics
because those shoes by design
materials better permeable oxygen
week ready when you have souls but if you want to measure
oxygen consumption who after nap option impermeable
materials and actually class works
the best so these are classed chips
that Michael wells in them that have been etched
and we used to measure substrate dependent
single-cell oxygen utilization pair
peri-peri the Chamber's less than
one Pike leader for I am in the Avery causal lead
so you can have the substrate close the lid
temporarily he would serve oxygen decreased
so that substrate depended oxygen this consumption
just like a macro oxygen consumption chamber
but 13e and then you can raise Politiken
washin and repeat with a different substrate so
this allows you to identify cells
beckon you liza tested substrate hi
full just briefly show you this technology
a kid its Emily see technology
that was developed program million cells and million seller
really pick so we headed to quite a few modifications
to get this to work with bacteria so
the bacteria were in so pica leader volumes
these arrays have wells on
they have an oxygen sensor pattern into them and Greeks
things that's the retreat good
I'm we put cells in these by passable owning
tried a lot a loading techniques but
the most gentle and the most reproducible
his 22 slowed under optimal conditions
and about a third of these who end up with one cell
so that's what we do this trip goes under the microscope
us to to the holder but as a big president or
of the medium for the fluid on then we have a piston
that's all computer-controlled and that piston
has a glass lid on it we can bring that piston down
it will very cheaply steel
all of these wells the still since I don't consume oxygen
you watch the oxygen decrease by the readout of the sensor
and then you can resell it again pic so
I'm here's the oxygen sensor so very standard
well-known oxygen sensor to platinum poor from
one the phosphorescence increases
has the option key creases and we don't measure intensity
we measure lifetime because it's not a sensitive to
pH and things like that this shows a calibration
to pursue an option %uh persist lifetime
formula we use it's a very good oxygen sensor
for this kinda approach
soak have allotted to you with pure cultures
up here's just up 172 showing you
here's a picture the chip I can really see pictures
recruiting around here Justice Center who
other creatures background out these are actually
reductions for green label sales so you can see him
better we can see in the microscope hope
under phase-contrast a had your cell
on %uh the rest these have cells in this particular case
this shows that that oxygen a concentration
over time missus heat notice all these other
wells don't have any respiration going on
so there's a very small decrease and here's the one
that as the Respironics sell the single-cell and we can
he easily see the difference between the background
and the single-cell and Sarah McQuaid
his are very talented microfabrication tech
makes all these chips and Sam Levin
is a very very talented undergrad who takes the day at
up and marina has been involved in all this so
what we can do then his screen what a lot of these
its are pretty time consuming but we have multiple arrays
on a chip and took us about three weeks
to get a hundred single cells
hey so construe that Tatum in it
but we also wanted to with these genotypes
so once we identify they're responding cells
the ones that responder methanol in this case
%uh then we need to move amount of these wells their way too small to work with
want to move in into 96-well plates
and there's a robotic commercial system that works on two microscopes
called are still system and it'll have Sprint cells from the well
in the capital at I'm and then into a 96-well plate
this is very tedious I'm to bring a long time
this system's design for mammalian cells and their way
easy pick your top what we found a course
it's our undergrads who spent all this time
doing video games you can do this 38
really well but so before undergrad students
hump but you can be done I'm hand
I we can get these arrays where we can't these
I'm into 96-well plates okay
million ordinary discontinuing
I'm his you chose his standard kinda
I micro sample techniques
to life these carry out whole genome amplification
this is pristina now single-cell
whole genome amplification and that she's been
using rt-pcr per 16s
and for specific functional genes we're looking at
I'm and she's done lots and lotsa controls
she gets really nice rt-pcr Kors
for this alright so we have our first date is set
very exciting to us the first 200 cells
about 10 to 20 percent are responding to methanol
so are in Richmond is up from baby 125 percent
attend 20 percent makes it feasible
start to scream these hand you can see
we have a wide range genera
being represented none of these
are bacteria we have in culture from the site
and none of them are highly similar to any culture bacteria
so that's really exciting because it means
we're getting out bacteria that have never been isolated
before some movies arc routes that are no
I'll mmm sympathies are known
to have a file a terrific group in them
and some of them are not up coming up
okay so
so what we're doing right now as we speak
his we picked out to %uh the most interesting for us
because her most diverse and
or to an all too nope sequencing of the individual cells
so we'll kid partial channel sequences
because your individual cells who will give more insight
you know these are and what they're doing okay so it's great through
exciting
but there's a problem if you I
yeah only have one to sell in your kill it
he's got right so we don't really wanna kill it
hitter baby so I'm
what we really wanted to this cultivate that sell
alright so what marina's working on now
with Kochi a lot over in our lab
his when we keep peace in a 96-well plates
for who campaigning knows under Institute conditions
but in the crease Tiger really slow
I'm hand with like Washington water filtered water
um and I monitoring cross
microscopically and actually
we don't need a turban culture we only need a few decisions
really start to get out of this concept I'm kinda
close call microbiology any into
you know eight or 16 or 32 cells is enough
even if these things are crowing very well
if we can get him to divide a few times
then we can split these cultures and
key part of them and their lives
and then kill part alright so that's the idea
about the phenotyping inch you type in part of this
they were just doing now where
we split culture we hold part of it
and we to this genotype screen and you know
one seventeens we know we don't care about
it's the ones where we can't get the jeans that we know about
those were the interesting ones had no sir the ones
we target for further phenotyping
we work really really hard to get those cultured
I'm and for the whole genome sequencing
because it's still pretty expensive to do whole-genome sequencing
and you get a lot better coverage
if you have a lease five single cells
pearl genome sequencing then if you to what
so it's really the best so that's the idea
who and you know
this is unfortunately the most exciting part of this
and so far we don't have data yet so stay tuned
I'm we're gonna keep working on this
I'm now we thank your lucky and some other most
interesting cultures peco just fine we know
we've spent a lot of time culturing from this environment
you know using the techniques where we need to lose out to
extinction in just 96 well plates
and try lot of things we think these ones
that we know are out there we have in common
probably can be difficult to culture alright
so this whole thing right
so when we get there what we learned a bell
this variation and I so functional populations
it's an aspect of functional redundancy
it's an aspect that kills what we learned about this
okay we can ask what percent of the cells
kind of sad eyes one carbon compounds have known pathways
alright maybe in many environments
known pathways are the dominant ones but no one knows
right now so you can ask those questions
I come and then in those unknown once
we can go through the standard
hard work biochemistry and Jean isolations
I'm and ask what teams are responsible
in those cells to hunt down one's
responsible for consuming C-one compounds
just like we did with him th2
you can work with micro cultures and
speed a lot of his top but there's no substitute
for gene function work once we know this
no we can then go back to manage an ohmic
amid a transcriptomic and met a proteomic data base
his star chick get more information on functions
okay so I'm going to summarize a kid
remind you when are we trying to do
repeat in trying to couple genomes
a function gone on fashion
killer Trina by pre-screening for the function of interest
doing this physiological profiling
and selecting these individual self-interest
ok we're hoping we can cultivate
you know at least a few decisions actually though
the reality is even if we can't cultivate
I'm we know where to find more
are the ones we killed we can go back
it's kinda perforce we look back and find them
I'm and then you submit existing
techniques for small numbers have cells you know
thank you looking at transcript began looking at genomes
to 1005 cells and other characteristics of interest
who we love Nana since
at so we're working right now with Victoria
orphaned at Caltech who does nuisance
hope to go back into the environment
with sexiness probes oMG using
incubations UVC 13 labeled C-one substrates
and start actually connect all of this
up I'm a functional probes are not working very well yet
but we have a few chains in c1 metabolism
third fairly highly expressed way he'd be even
people to look at those with innocence right
so I'm this i think is a very
exciting interesting approach are right now
this single cell protein profiling really only available
in our lap but with a little bit more work on
developing it to a more robust age
actually you just need a good confocal microscope
and someone to make chips for you and it's pretty easy to do
I'm so I'm hoping that we can
see this technique disseminated to lots of other
environments and approaches and a course I wanna think the Department of Energy
for funding and thank you for putting up with
having listened to my voice pic do