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[Music]

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this afternoon I'll be talking about are

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any species that are relevant to the

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origin of life or are any wool are any

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wool tunic if you are not familiar with

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irony there are three biopolymers in

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current world let's the three major

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biopolymers one is DNA that to

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information stories and there is RNA

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that is kind of reached between DNA and

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protein linking the information or

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carrying the information coded in DNA

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into the protein and proteins are the

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functional molecules that to all

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structure looking catalysis and binding

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and so on and so forth I'll be talking

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about RNA hr1 one special class of RNA

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that may be related it is related to the

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origin of life so everyone in the room

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is interested how life originated in the

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planet or something similar to the

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prebiotic all may be happening somewhere

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around the universe so we have a very

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nice talk about these small molecules

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which are prebiotic chemicals in the

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previous lessons one widely accepted

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hypothesis about what is enough life in

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our theories RNA world hypothesis which

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tells RNA is the first molecule first

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biomolecule appeared which sort in both

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information stories and functional

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capacity so in the prebiotic world is

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small organic chemicals organic

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molecules appeared somehow and then

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these condense together form bigger

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molecules which appear similar to or

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exactly similar same to like for RNA

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monomers that we see today a dilution

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guanine cytosine and uracil these for

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RNA monomers polymerized in a phosphate

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phosphate backbone giving us primitive

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RNA oligomers these oligomers

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learned to like it with other RNA

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oligomers

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making themself a bigger and they learn

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to call

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themselves in a fateful way and then

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learn to change the sequences around

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muted like causing mutations and the

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more favorable mutations were selected

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or successful mutations were selected

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and the same biopolymers are folded in a

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special way creating a functional

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molecule and somehow this informational

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molecule and this functional molecule in

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turn are enclosed in our lipid vesicles

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creating a first system that is able to

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store information or that is able to

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support itself and store the information

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which we sometimes called a rebel cell

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arrival site and then from this point on

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the Darwinian evolution started so once

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the Darwinian evolutionist I drew the

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the system start just getting started

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getting more complex and complex so this

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is a rival site and the complexity in it

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gets increased with the evolution and

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with increasing complexity RNA itself

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cannot do could not do all the reactions

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needed so it needs it needed the help of

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other molecules that has the required

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functional groups for example 4s I'll

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transfer reactions simply carbon

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transfer reactions it needed our salt

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for functional groups or tile and the

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coenzyme a appeared in electron transfer

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reactions and oxidation reduction

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reactions it needed nad and 8 years and

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slave in Co enzymes and all these

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coenzymes appeared all common also there

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is one common thing about these

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coenzymes which is they all have this

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adenosine part attached to it which is

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which which tells us that these are

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coenzymes could be of molecular fossils

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I will be talking basically about

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coenzyme a suppose I made involves in s

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and transfer reactions because it has

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this sulfur or tile functional group at

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the end someone is talking about kick

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that database at least get some less so

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you got kicked it away certainly and if

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you go to the kick database around ten

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percent of things I am stunning in our

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show right now need this molecule for

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the function so this is an important

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molecule and if this molecule is

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believed to exist since the sins are any

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water since it has higher functional

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group there it can form a Thai

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restaurant which is thought to serve as

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a free energy source during the during

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the evolution during the reaction in

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evoking evolution so the question is how

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this coenzyme a molecule formed for the

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first time and to answer that question

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we have selected from thousands of

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trillions of RNA library some RNA

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sequences that can catalyze the

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synthesis of this molecule itself so RNA

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can catalyze the synthesis of coenzyme a

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which we call them coenzyme a synthetic

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those ribozymes so this tells us that

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RNA itself may have synthesized the

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required functional groups that it that

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the right side needed in the RNA world

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so this thing us there could be that

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could have been a complex metabolic

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system composed just of RNA so the

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question is the question now is so har

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na can catalyze the formation of

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coenzyme a in the formation of the

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thyristors are these molecules are these

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queens I'm a link to her molecules

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existing current biology if they do they

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could give us a clue about how all these

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things happened in the periodic world

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because these selections these molecules

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are from are selected from artificially

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created random

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library containing 10 power 14 to 10

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power 15 unique sequences so the

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question is to query linked to RNA exist

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in current biology and the till balloon

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from Harvard insert this question and

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the answer is yes there are we linked

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RNA species and we Tyra Stirling tyranny

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species existing in the RNA pool of

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current bacterial cell at least in

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bacterial cell we know we know that

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maybe in the eukaryotic also we don't

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know

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the sequence is down here and what they

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are doing right now if we are functional

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since we don't know the sequence we

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don't know how they evolved and what

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they what is the role in the cell so the

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goal of my research is to capture these

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RNA sequences based on this tile

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functional group sequence them and then

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find out how they evolved and what they

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are doing there so the first question is

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the first of all is capture them and

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find out what are the sequence by doing

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sequencing and the second goal of the

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project is find out how these RNA

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species are being made in the cell what

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is the mechanism there could be two

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possible pathways that these are any

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species can some can be formed when it's

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co-transcriptional which is since this

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part is Eddy notion and which kind of

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which is exactly same is ATP this part

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can initiate the transcription by

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competing with ATP under certain

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promoters so this is a cool

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transcriptional mode of formation which

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could be happening and there is another

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possible pathway which is there may be

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some enzymes linking this first poop

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endeth in part to ATP initiated RNA

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forming these colonies

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so we don't know what whether the

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colonies are being made by

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co-transcriptional mode or whose

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so the goal is to capture the cuellar

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and sequences and cuellar any species

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and sequence them and figure out how

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they are being made so to capture them

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we have to develop a RNA capture

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protocol or method and to develop our

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method we need our eat we need an easy

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access to lab synthesize quailing

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colonies so we are our lab have has

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previously developed a technique to

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label coenzyme a on the RNA phi prime in

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since coenzyme a has that any notion

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part on it it can compete with RNA under

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all right promoter under t7 promoter

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with t7 RNA polymerase in the some RNA

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transcript will automatically be labeled

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with coenzyme a but we need a default

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photo enzyme a to initiate the

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transcripts on there and this differs

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for coenzyme a itself is a problem it it

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is extremely expensive molecule well not

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let's not say extremely but it is

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expensive extremely the kind of TOEFL so

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and then we started with developing our

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one method for the to make this d4 spoke

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we we started we we use the enzymatic

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strategy nature has two different

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pathways to dedicated for the synthesis

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of coenzyme a so we use a pathway called

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coenzyme a salvage pathway which has

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three total enzymes while cloning these

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enzymes we came up with a new cloning

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strategy which is shown in this picture

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which is based on just a PCR read

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we don't need restriction digestion and

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ligation steps maybe molecular

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biologists understand understand this

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better

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we came up with a new pruning technique

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which is very simple and efficient and

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able to publish this cloning method in

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plus one two years ago if we if someone

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in the room is facing a difficult time

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in like crowning using a restriction

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dilation in like some method we can

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maybe we can talk about the new method

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which does not need digestion and

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ligation

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using the new method we call two enzymes

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of the coil salad

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into a plasmid we created using this

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plasmid we made two proteins which are

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able to make this device work away from

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the commercially available Penta teen

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this molecule and then this carried out

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this enzymatic synthesis we had some

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purified distance between Jamie and we

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were able to publish this this part in

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by organic chemistry last year so now we

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have a method to make why are any in the

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lab and we have an easy access to defer

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spoke way we can go ahead and work on

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cuellar and it captured protocol so I

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started we are any capture protocol with

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the lab made a RNA and then I was

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playing with chemistry on this tile so

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the idea is you have a RNA pool that has

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where as summit RNA sequences are

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labeled with coenzyme a and most of them

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are not so we prepared three types of

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solid beets which can specifically react

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with this tile so these are beets and

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under in the base this is a Malamute

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functional group

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with this tile and links this tile and

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whatever link to this tile to the beets

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so that we can specifically capture the

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tile containing molecules on the on the

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beets and another one another beets with

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I work with is iodoform generalized

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beets this iro also reacts with this

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tile and then linking the tile

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containing molecule permanently or

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covalently with the beads so that we can

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capture them or purify them by some

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chromatograph you check some

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chromatographic techniques later and the

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third type of beets I work with is tile

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containing beets

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this these tiles can make disulfide

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bonds with us with another tile

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containing molecules so that we can use

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them to capture these tile containing

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molecules specifically so I prepare

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these beads and then incubated days

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beats with a RNAi poop and the

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expectation is is this so if it did

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malee made will react with tile i

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also react with tile and that this tile

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column will give me a disulfide and then

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once this is captured once this RNA is

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captured we have to release it so that

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we can sequence it and the release for

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the release I used three different

280
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protocols one being cleaving a disulfide

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there in releasing the RNA part this is

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done by DT T releasing a RNA and in the

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Kyoto beats we used some some group

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called photo cleavable group which can

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be cleaved off by signing certain

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wavelength of UV light and in these type

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of beats I use light to cleave off the

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RNA sequences from the beats and in

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another type of column which is melamed

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functionalized columns I used our

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special files of enzyme called the new

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dekes hydrolysis to release this RNA

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sequences captured there so new tricks

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00:13:59,910 --> 00:13:57,200
enzyme is a special class of hydrolysis

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00:14:02,680 --> 00:13:59,920
that cleaves right there so this is a

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pen dip in part of coenzyme a and this

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00:14:07,720 --> 00:14:05,210
is the RNA part so once knew once new

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00:14:10,540 --> 00:14:07,730
dekes reacts on these are any species or

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whatever it capture there it it cuts off

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it cuts right there in between these two

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00:14:21,040 --> 00:14:15,380
phosphates and releases whatever is

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there in the RNA part so after the

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00:14:26,740 --> 00:14:25,040
releasing we ligated our adapter

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molecule on both five Prime and three

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00:14:31,840 --> 00:14:28,670
prime end of the RNA so that we can

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sequence them after ligation we did

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reverse transcription get back to TNA

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00:14:38,800 --> 00:14:35,600
and then did pshare to amplify the

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00:14:41,020 --> 00:14:38,810
signal and did next-gen sequencing so so

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far so good winning we developed our

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00:14:44,950 --> 00:14:42,680
method we got the RNA from total

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bacterial pool and we send around 20

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clones for the sequencing but when we

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saw the sequencing result I was very sad

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because I was expecting to get this coil

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00:14:58,210 --> 00:14:54,380
entirely from this protocol but what I

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00:15:01,060 --> 00:14:58,220
got was some key RNA sequences so we

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00:15:03,070 --> 00:15:01,070
know the tRNA sequences are heavily

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00:15:05,470 --> 00:15:03,080
modified the tRNAs are heavily wouldíve

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modified with tile this is this is an

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00:15:09,580 --> 00:15:07,610
example this is a type 4 high reading

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there are other multiple modified you

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readings with tiles so this protocol

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00:15:20,440 --> 00:15:16,310
captured these tire readings on tRNAs

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not the coy RNA so since Cuellar and A's

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00:15:26,680 --> 00:15:23,960
are very less like their abundance is

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very less compared to the tRNA we simply

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00:15:30,820 --> 00:15:28,210
missed Co irony

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since this protocol failed I moved on

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and then developed other two strategies

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to capture these coy RNA species so one

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00:15:43,660 --> 00:15:41,180
is based on the enzymes which we call

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the enzymatic strategy and another one

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00:15:48,820 --> 00:15:45,290
is chemical strategy based on the

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00:15:51,160 --> 00:15:48,830
chemistry so for the enzymatic strategy

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I'm looking for any specific enzyme

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called acid core I like it in the class

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00:16:00,190 --> 00:15:55,780
of a sheet coral I guess that can

339
00:16:03,100 --> 00:16:00,200
catalyze the the linkage or covalent

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00:16:05,320 --> 00:16:03,110
bond formation between some fatty acid

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00:16:08,050 --> 00:16:05,330
modified with biotin on one end and

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00:16:09,760 --> 00:16:08,060
coenzyme ellington asa so these enzymes

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00:16:13,780 --> 00:16:09,770
this is a huge class of enzymes that

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00:16:16,330 --> 00:16:13,790
catalyzes a reaction of coenzyme a and

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00:16:19,240 --> 00:16:16,340
free fatty acids but there are no exam

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00:16:21,640 --> 00:16:19,250
in nature that can work on modified

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00:16:23,890 --> 00:16:21,650
coenzyme a let's say boiling - RNA and

348
00:16:26,410 --> 00:16:23,900
modified fatty acid let's say biotin

349
00:16:29,130 --> 00:16:26,420
fatty acids so using structure guided

350
00:16:34,170 --> 00:16:29,140
engineering and some sub mutation

351
00:16:36,610 --> 00:16:34,180
mutation are guided by structure

352
00:16:39,550 --> 00:16:36,620
proteins are three dimensional strict

353
00:16:42,820 --> 00:16:39,560
structure we are creating we're trying

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00:16:45,610 --> 00:16:42,830
to create our new enzyme that can accept

355
00:16:47,950 --> 00:16:45,620
modified coenzyme a sorry modified fatty

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00:16:51,940 --> 00:16:47,960
acid and modified coenzyme a is a

357
00:16:54,430 --> 00:16:51,950
substrate and covalently links the

358
00:16:56,860 --> 00:16:54,440
carboxylic acid and the tile part

359
00:16:58,900 --> 00:16:56,870
creating a thioester bond once it is

360
00:17:01,570 --> 00:16:58,910
once the molecule is enzymatically

361
00:17:03,580 --> 00:17:01,580
labeled with biotin we can capture this

362
00:17:07,410 --> 00:17:03,590
biotin molecules on the step traveling

363
00:17:09,810 --> 00:17:07,420
beads and purified purified and then

364
00:17:12,640 --> 00:17:09,820
next starting is user

365
00:17:14,710 --> 00:17:12,650
pending in Alex which is we are trying

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00:17:19,120 --> 00:17:14,720
to modify the panteth in part with

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00:17:20,770 --> 00:17:19,130
biotin and some linkers some some label

368
00:17:23,079 --> 00:17:20,780
layer so that these molecules will be

369
00:17:25,270 --> 00:17:23,089
sent to the bacterial cell bacteria will

370
00:17:26,549 --> 00:17:25,280
level their query RNA with the with the

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00:17:28,409 --> 00:17:26,559
new pending

372
00:17:30,090 --> 00:17:28,419
since Pete and Alex are modified with

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00:17:31,669 --> 00:17:30,100
biotene we can capture them money step

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00:17:36,509 --> 00:17:31,679
evident Peaks so these are two

375
00:17:39,330 --> 00:17:36,519
strategies and I'm working - working to

376
00:17:42,049 --> 00:17:39,340
ask them trying to get the enzyme like I

377
00:17:47,039 --> 00:17:42,059
purified multiple enzymes from bacteria

378
00:17:49,379 --> 00:17:47,049
from e.coli trying to like expect

379
00:17:52,289 --> 00:17:49,389
expecting the enzymes when modified we

380
00:17:54,419 --> 00:17:52,299
can do this reaction so that enzyme

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00:17:56,820 --> 00:17:54,429
reaction will be specific and then I'm

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00:17:59,460 --> 00:17:56,830
modifying the pendant in part with by

383
00:18:02,009 --> 00:17:59,470
putting some putting a biotin and

384
00:18:04,649 --> 00:18:02,019
multiple lights and residues on it so it

385
00:18:07,200 --> 00:18:04,659
has a pendant in part that will be used

386
00:18:08,909 --> 00:18:07,210
to make way RNA by the cell and it has

387
00:18:10,430 --> 00:18:08,919
multiple license residues that will help

388
00:18:13,499 --> 00:18:10,440
this molecule to get inside the

389
00:18:15,450 --> 00:18:13,509
bacterial cell membrane I'm in two key

390
00:18:19,049 --> 00:18:15,460
to get across the cell membrane and then

391
00:18:21,479 --> 00:18:19,059
I'm doing the transfer D study of

392
00:18:24,570 --> 00:18:21,489
studies of these molecules and luckily

393
00:18:26,669 --> 00:18:24,580
these molecules are being acted like are

394
00:18:28,349 --> 00:18:26,679
accepted by the enzymes of COI synthetic

395
00:18:30,690 --> 00:18:28,359
pathway so there is a good chance that

396
00:18:34,710 --> 00:18:30,700
these molecules will end up in the RNA

397
00:18:36,409 --> 00:18:34,720
of the bacteria and then I'm also

398
00:18:40,169 --> 00:18:36,419
working with Cuellar and a metabolism

399
00:18:42,389 --> 00:18:40,179
trying to answer how these core RNAs are

400
00:18:46,169 --> 00:18:42,399
being turned up like how the toner what

401
00:18:48,779 --> 00:18:46,179
happens I'm not talking a lot about this

402
00:18:52,769 --> 00:18:48,789
area since the time is up and then do it

403
00:18:54,779 --> 00:18:52,779
this hopefully I convinced you these

404
00:18:57,690 --> 00:18:54,789
qualities are very important to

405
00:19:01,080 --> 00:18:57,700
understand how RNA the world started and

406
00:19:03,899 --> 00:19:01,090
they can give us a good clue on how our

407
00:19:09,660 --> 00:19:03,909
and will start it and I'm doing working

408
00:19:13,950 --> 00:19:11,269
[Applause]

409
00:19:17,789 --> 00:19:13,960
your question yep

410
00:19:18,450 --> 00:19:17,799
I know you yeah okay so um quick

411
00:19:20,999 --> 00:19:18,460
question

412
00:19:23,129 --> 00:19:21,009
you want to use the biotinylated Panther

413
00:19:24,749 --> 00:19:23,139
theme as a way to basically labeling

414
00:19:26,249 --> 00:19:24,759
darnay's yeah but are you concerned at

415
00:19:27,599 --> 00:19:26,259
all that if there is some kind of

416
00:19:29,970 --> 00:19:27,609
post-transcriptional modification

417
00:19:31,859 --> 00:19:29,980
happening that those enzymes would not

418
00:19:33,180 --> 00:19:31,869
be able to add the modified panda thing

419
00:19:36,060 --> 00:19:33,190
and therefore you're not actually saying

420
00:19:41,140 --> 00:19:36,070
- yep Bejo yeah cuz you might not see a

421
00:19:49,460 --> 00:19:47,450
yeah all right one more quick one all

422
00:19:51,110 --> 00:19:49,470
right if not we're gonna move on in the