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[Music]

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I'm June my Raj I'm from I just started

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my ph.d program at Georgia Tech this

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work is my master's thesis work that I

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carried out at NASA Ames before I

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started at Georgia Tech this is a

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real-time autonomous instrumentation for

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lab based microbe experimental evolution

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so basically extremophiles have somehow

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adapted to learn in to learn to live in

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uncomfortable environments it might be

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extreme temperatures it might be extreme

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amounts of salts it might be extreme

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acids acidic environments extremely

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basic environments and lack of oxygen no

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matter what you think they they have

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learned to live in those environments

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through natural selection natural

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selection is a process of adaptation of

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an organism to its environment by

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selectively passing on the changes in

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its genetic Constitution experimental

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evolution that we are interested in is

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the process of mimicking natural

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selection artificially by the process of

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exposing microbial community to

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intentional stressors to improve the

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resistance through artificial mutation

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basically experimental evolution is

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forced natural selection using lab based

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procedures how do you carry out

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experimental procedures basically it

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just grew up microbes you subject them

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to a stressor that you're interested in

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remove unfit microbes and you retrain

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the process and at some nth iteration

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you have a microbial colony that has

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adapted to your stress despite having no

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information as to how to do it initially

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in the initial stages so the proof of

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experimental evolution is this data

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basically they use we use

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ecoli microbes for by exposing them to

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40 seconds and 60 seconds of UVC

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exposure which is supposed to kill them

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but they actually learned to grow

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despite the UV stresses and in the

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seventh iteration they had almost ran to

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the power 6

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you know factors of increase

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the growth rate so this has proof that

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experimental evolution actually works

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and this is proof that lab in the lab a

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natural environment can be created and

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this can be made to work this is this

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work is a black box approach I am an

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instrumentation engineer and I in learnt

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biology for the further part of this

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project so basically it's a black box

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approach we have a microbe that's

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radiation by the way we're exposing

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we're exposing them to radiation and

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it's learning to grow stronger and

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robust despite the radiation we are not

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focusing on what is happening to the DNA

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when this process is happening and this

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is a black box approach we are not

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interested in what's happening here we

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just care that this is happening and we

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just care about producing an

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instrumentation technique that can allow

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the problem with manual experimental

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evolution processes is that it's tedious

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it's extremely time-consuming it's

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highly prone to human errors it's it has

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high reproducibility and repeatability

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issues and that affects your moods and

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also it kind of hurts that a one-celled

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organism dictates a multicellular

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magnificent organism such as ourselves

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in the lab it's not supposed to dictate

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our schedule so what do we do we ought

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to be the process so this was the first

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generation device that was designed at

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NASA Ames it had four sub components it

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had sensors environmental controls

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fluidic systems and data storage using

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Arduino so that's how it looked and yes

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I repaired it when I started at NASA

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Ames it had temperature sensing in

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optical density which was done

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automatically eco light likes to grow in

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37 degree searches so it had that kind

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of sensing UVC system was the stressor

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that was used and temperature also could

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be used because it was also a sensing

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parameter the fluidic system included a

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one chamber growth this is the one

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growth one chamber growth chamber it had

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parasitic pumps for in letting fluids

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letting the nutrients continuously and

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it had education because e coli tends to

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stick to the walls and that messes with

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the optical density and we don't like

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that so agitated magnetically so the

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cells are in in the limbo state and in

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between media and also Arduino control

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so everything was automated and nothing

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I included minimal human interaction

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actually that had its own problems it

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had only sent to two types of sensors

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and it only allowed one stressor to be

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applied and also one grow chamber was

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not really that fancy because if you do

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multiple chambers you can do multi

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parameter growth and you can compare

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much populations and that that was not

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allowed in this so that's why my work

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was to enable parallel growth cultures

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so have multiple chambers and there was

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absolutely new biochemical information

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as to what is happening inside the

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chamber during the evolution process and

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that's why I added a set of wet

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chemistry sensors and also enable

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dynamic application of stressors in

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response to sensor parameters and all

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these sensors were added along with cell

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density which was already previously

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done I added dissolved oxygen electrical

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conductivities pH and ORP sensors to the

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system adding sensors is not that easy

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because you need to select the self

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sensors and also have a set of my the

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fluidics card needs to be completely

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changed so that it can accommodate the

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sensors

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so this fluidics card like you see for

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chambers for growth chambers the

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circular one is a growth chamber and the

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square ones and there are the sensor

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chamber slots where a sensors can be

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sensor probes could be inserted and it's

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a six layer design that was integrated

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and it looked like this once it was put

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together and this these are the sensor

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circuits we bought off the shelf

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these are basically called a class

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sensor stamps they're really stamp size

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they're really that small and its really

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cute that's why they have a slide in my

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presentation and this is how it looks on

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the breadboard design

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it's very compact it's very efficient

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and I'll show you the data you'll be

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impressed in the process of adding

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sensors we found pretty good set micro

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sensors but the electrical conductivity

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sensor that we looked at was not really

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compact enough so I designed a sensor

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electrocuted electrical conductivity

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sensor called the meek on microbio

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electrical conductivity probe it's

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basically this is a 1 ml syringe to be

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honest and this is a point eight

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millimeter nichrome wire so these

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together make the electrical

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conductivity probes and these were much

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less invasive as to what we could buy

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off-the-shelf and this is a circuit that

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controls it and this is the data that

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shows that the commercial probe EC probe

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was the me con probe was functioning

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exactly as well as the commercial probe

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in the in the range that we were

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interested in that's impressive data FYI

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so in the second generation device all

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these parts in yellow added added more

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color to it so for the sensing instead

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of two parameters it added four more so

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the total of six sensing parameters was

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enabled and if you have a we'd a sense

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it you have a way to environmentally

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control it so environmental control

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system increased from two to seven two

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to six sorry

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two to six now and fluid mechanics was

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like inner chamber flow was allowed pump

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was still there

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there was an exposure chamber and a

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sensor chamber added to accommodate the

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sensors I like Raspberry Pi along with

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Arduino Arduino space a microprocessor

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raspberry PI's an entire computer it can

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control the entire system without

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failing really badly and that

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constituted the second generation device

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that I'd have loved and this is

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important data because two reasons one

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it showed that I was awake for 24 hours

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and also this was the data that proved

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to my adviser that I'm capable of

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graduating

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so the optical density data was

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collected over 24 hours every R I had to

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collect samples and put it through an

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optical density sensor and record what

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was the optical density dissolved oxygen

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is the most interesting part here so the

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dissolved oxygen started at some point

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and then it went to zero at

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approximately six hours so after six

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hours everything that happened was

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anaerobic niccola is not supposed to

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grow in an anaerobic environment but it

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did grant it it did not have a really

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good growth rate as it had previously

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when it was Arabic but it did have an

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increase in the growth rate even after

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six hours when oxygen was completely

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depleted of it well also what's

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interesting is that add that switch

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between aerobic and anaerobic the pH

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slope changed drastically and there is

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one more slope change that I do not know

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there is a biochemist chair please help

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me

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there is also electrical contacts would

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be change at six RS there was a dip in

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the small dip in the electrical

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conductivity that that gray line that

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shows that there was a short-term

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stagnation in the exchange of ions

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between the media and the cells but

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again it regained that it's somehow

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learned to do that and despite having no

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oxygen and then there was again an

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increase in the electrical conductivity

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that means the ions were still produced

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and excreted out of the system so that

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means this system can be used to

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eliminate the black box that I had spoke

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about initially so basically you have a

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system that can enable application of

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radiation stressor temperature stressor

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increase amount of salts or decrease

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amount of sauce

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I said you can control the pH and you

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can control the amount of oxygen and all

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these stressors can be applied at the

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same time in combination or individually

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and you will have a microbial population

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that has learned to grow despite these

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stressors that means the intermediate

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black box the changes in the protein

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structure the metabolic pathways it took

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to and after that change

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and the genetic sequences that were

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adapted can be studied with devices like

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this because it allows intermediate

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changes between population that has no

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resistance to a population that has

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excellent resistance to all the stresses

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that you apply in stay in small stages

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also this kind of study can be used to

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help and enhance the ecosystem for

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cycling food water air and waste

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so basically no organism can survive on

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its own period it needs other organisms

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to support it so that that includes

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humans so if we want to have extended

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presence in space if we want to travel

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really long distances and through space

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and time then we need to have a set of

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microbes that will support us by

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providing a cycling procedure or water

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air food water and waste so that kind of

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study will envel envelopes apply

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microgravity and high radiation levels

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and then those microbes can be actually

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sent with the astronauts to survive in

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space flight mission control environment

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for future work this device is intended

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to carry out the Iggy regimes that I

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just mentioned with individual or

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combinational stressors six stressors or

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employable stressors like with those

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sensors can be like free ion

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concentration osmotic ion stress acidity

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metal ion presence very nutrient

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availability like oxygen thermal

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stressors dissolved oxygen again as more

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stressors that can be added that means

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more sensors there is no such thing as

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too many sensors in the system so

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reactive oxygen species can be included

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and very usually availability can be

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added to the system also this was a

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proof-of-concept so we did it for one

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chamber system so a multi chamber system

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for intercultural comparisons and much

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population studies can be expanded so

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the single chamber can be expanded to

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four or six as per you like it and have

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those studies carried out

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extended sister for growth and testing

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of other bacterial species so this was

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done for e.coli so any any system any

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ill organism that doesn't require like

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beyond 200 to 300 degrees temperatures

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can be used that will not melt the

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fluidic system and you can still use

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other bacterial species to carry out

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this processes questions okay I really

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love the potential for this hardware but

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I have a ton of questions which I'll

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probably so how do you guys deal with

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sterility it's like how does the

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hardware and all sterilization so this

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was this was a microfluidic system so

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basically what we did is we assembled it

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in the presence of a flame in a Bunsen

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burner flame and before that it was

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autoclaved this system can be autoclaved

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in my presence itself i autoclave this

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at least seven to eight times and it

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still remain intact actually and so with

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the with the experimental evolution

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portion of it if you wanted to do like

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very long extended you know thousand

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generation plus experiments

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the fluidics pumps I'm assuming or you

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know moving media out and then putting

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it back okay so it's not like a chemo

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static system No

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well we avoided that kind of a system

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because of the limitations in the

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fluidic paths that it allowed so this

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system was basically you can have a

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secondary chamber which will store the

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first generation of a first generation

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culture and then it can be made to

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reflow into the fluid it's into the

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floating system and it has the ports for

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it okay very cool I'll have to talk to

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you if you have a larger system the way

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I would see it as like the sterilization

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will be harder and also if the system is

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smaller you can see the growth in a very

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short amount of time and if the system

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is really large it would actually

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simplify the application of sensors but

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that also means that you need to have a

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really robust agitation system to keep

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the media homogeneous throughout the

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chamber which is why we switch to

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smaller and smaller chambers so that

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there's not much of magnetic magnetic

359
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hey um I wonder if you think you can use

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this system to study Khan soldier of

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different species living together either

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by differentiating them with different

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chromophoric probes for example while

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they're growing or any way on any other

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means which necessitates some another

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form of detection like microscopy or

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different wavelengths so you want to add

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some kind of an imaging system into this

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is that what you asked yeah because you

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need if you want to grow different

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species together you need to track all

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these parameters for every other species

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so I was wondering if you think this

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could be done in a very efficient way so

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the center of parameters data that I

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showed was actually real-time so during

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the growth of e.coli those parameters

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were recorded so if you want individual

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parameters over a duration of time for

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multiple species there has to be some

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kind of chemical barrier between those

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systems so you can actually isolate the

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sensor parameters but it's not living

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together in a solution it's like you

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need to separate them and if you put

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them together in a solution you will get

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the resultant yeah media okay count

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chemistry and the other individual