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the theory of a biogenesis states

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that life could have originated from

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non-living organic molecules

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present on earth during the prebiotic

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time

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the first cell like structures that were

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possibly made out of these

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organic molecules are called the

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protocells

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the membrane component of this process

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might have emerged from amphiphilic

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molecules

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which could have spontaneously assembled

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into vesicles and encapsulated the

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chemical systems that are required for

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protocols if knee if it needs to be

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considered as a cell

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keeping aside all other other cellular

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aspects or characteristics

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should have the ability to grow and

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divide

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there are various proposed methods by

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which a producer could grow and

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eventually divide

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it could happen via the addition of

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fatty acid precursors

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that are released from either

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surrounding mycel or

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other vesicles and then after the growth

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of the protocell

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spontaneous division could have been

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occurring

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in the early times as the only means of

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protester cytokinesis

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and this could again happen due to

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various factors or reasons

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like internal forces which includes

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osmetic pressure or mechanical forces

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which can lead

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to pearling in stability as shown in the

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to become a modern cell the

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protocellular compartments

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must have made the switch or a

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transition

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from having fat gases as their membrane

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molecules

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to phospholipids as shown in as the

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green molecule

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with phospholipid addition to the

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vesicle the growth of these

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mixed lipid vesicles would have

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increased in comparison

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to the vesicles which are only having

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fatty acids

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and then did the increase of the

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strength of these membranes

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within with the increase of

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phospholipids the spontaneous division

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have a less chance of occurring which

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eventually leads to the formation of new

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genes

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and catalysts which would have evolved

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to make

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but which catalytic mechanism would have

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led to the division of the protester

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so there is an idea which proposed that

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maybe some

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extent enzyme that are needed for

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lipid synthesis could have led to the

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growth and the division of protolouses

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however till date no such enzyme was

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found hence a new hypothesis

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was made which states that

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maybe the lipid synthesis could have

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started in an external environment

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rather than an internal one and later

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some ancient enzyme

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or proteins may have assisted in the

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division of the cells

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after it has acquired the necessary

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before going into the catalyst spot i

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would like to mention the advantages of

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having a periodically timed division

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the cells controlling the timing of

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cytokinesis by any enzyme or early

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protein

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could have gained the advantage of

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coordinating the inf

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the replication timing of informational

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molecule

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with the early cell membrane burning

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process

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in order to successfully and efficiently

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transfer

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all of its genetic information and

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metabolic molecules equally to

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this has led to our hypothesis where we

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think

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that fjse might be the early protein

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which is needed

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for the process of division of the

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producer

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so what is fdac actually safeties is a

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gdps

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protein encoded by fdazi gene that

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assembles into a ring-like structure

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the future site of bacterial cell

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division fda is found in almost all

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bacteria

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archaea chloroplast and mitochondria

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that is

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very much essential for cell division

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process fds is also an ancient protein

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it contains very less amount of amino

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acids like arginine lysine

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phenylalanine etc which were

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hypothesized to be the last one to be

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added to the genetic code

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hence it suggests that fdac evolved as a

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functional protein way before the

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genetic code was even complete

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and fdac is the only gene controlling

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cellular division

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present in all resident minimal

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bacterial genome that are being analyzed

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the activity of ftec on various in vitra

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and in vivo system

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which mimics the protozoa structure has

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been already observed

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like for example here in the case of uni

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laminar vesicles

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the filaments of fdaz form patches

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inside the liposomes and these patches

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are reorganized to form ring-like

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structures which are parallel to the

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plane of the membrane

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some projections were observed at the

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outer portion of the vesicle like birds

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or

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tubules and the position of the

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projections is actually coinciding with

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that of the rings of the patches that

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are made by fdaz

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which shows that rings that are made by

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fdasi are able to construct the

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membranes

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of the vesicles partially which could be

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a cue for protocellular division

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and the similar things has been observed

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in other in vitro systems as well

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like slbs or supported lipid bilayers

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these model systems along with fdac

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could be able to resemble the divisional

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machinery that were present in primitive

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cells and help us understand the

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replication pathway

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therefore our project or our work is

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focused

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on the reconstitution of fdac on various

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relevant in vitro and in vivo systems

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like

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liposomes and others while observing its

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dynamics and the ability to

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fda fdz usually binds to the membrane

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not directly with the help

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of another protein which is either

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an fds a or zepa and it binds to this

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so before adding the protein on the

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invitro systems and doing the studies

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we have changed some tips and bits of

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the protein

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which could help us visualize the

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protein better on these systems

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so this is a construct or one can say a

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plasmid map containing the genes

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that our design protein is having so

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instead of

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having a zip a or fdsa as a membrane

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anchorage protein

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we are using e coli mindy mts

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so fdsa had a profound effect on the

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dynamics of fdaz when both of them were

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reconstituted in phospholipid

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vesicles which overshadows its

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capabilities and functionalities

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hence it has been replaced by a

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non-interacting membrane binding protein

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which is mts

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and therefore the c-terminal region of

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ftse is not needed hence

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the plasmid contains fdac without a c

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terminal

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and mts and a fluorescent tag which is

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amnion

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in addition to this we also constructed

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two other plasmids

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and constructs that are having gtps

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mutant version of fdac

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to see how gdps mutants can actually

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affect the protocellular division

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after the construction of the constructs

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the protein is harvested

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and purified from competent strains of e

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coli

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because stringent purification processes

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the protein is usually checked

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first before any further

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downstream processes are been done on it

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we perform the sedimentation assay for

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the purpose of checking the activity

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a sedimentation assay is nothing but an

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inviter asset to observe the

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polymerization of the protein here in

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our case

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the protein if active should be able to

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form long filaments when gdp is added

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and if in the meantime the reaction is

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centrifuged under high speeds

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then filament should go down in the

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pellet section showing as the presence

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of polymerization

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and here one can see that that fdac

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is being polymerized or being active

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even our protein even our

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even after our protein purification

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process

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the amount of pellet in the presence of

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gdp as shown in this lane

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and this lane is way higher than the one

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having the control one having no gdp as

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shown in the lane seven

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and also the concentration of the ftsd

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in the pellet increases both with

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increase in fdac concentration

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the first system that we used is that of

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speroblast

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so it is a bacterial bacterial cell or

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plant cell that is bound by its plasma

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membrane

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only and without a cell wall this could

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stand as a

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model cellular system during which could

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have evolved

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during the evolution of early microbes

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where the cell wall

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and cell membranes were still evolving

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the turbo pressure

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in this case is very low compared to the

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membranes in the normal cells

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which could be mandible by fdaz as it

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could be easily mandible

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or deformable few vestculation events

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could have been observed

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in the cells after the addition of the

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protein

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so we took a dh beta cells having our

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constructs

258
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and gave an induction of arabinose and

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formed spiroblast

260
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and observed spin and observed pinching

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at various degrees

262
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and in various number as given by the

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red arrows

264
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this led to the fact that our construct

265
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could lead to the potential division of

266
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spheroblast or the models

267
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the next system we studied is an invitro

268
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one the lipid droplets the droplets were

269
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either made up of

270
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popc pope or only purebc

271
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the protein was allowed to polymerize on

272
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the top of the droplets

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the top row shows the images that are

274
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taken in wide field microscopy where the

275
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bottom one

276
00:10:25,190 --> 00:10:23,279
were taken with fits a filter showing

277
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the protein fluorescence

278
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binding of a protein was observed in the

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droplets with

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the ftse fluorescence coinciding with

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the membranes

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of the lipid droplets as shown by the

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then we did sim microscopy and observed

284
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the z section of the droplets

285
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and saw that the protein is making some

286
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kind of cellular

287
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circular like structure over the

288
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droplets

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inside the circles no fluorescence from

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fda's is

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at all observed this points to the fact

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that maybe the protein

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might be present on top of the membrane

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loosely all over the

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lip lipid droplets but in some place

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they were able to condense to make

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polymers and

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could form rings on the droplets which

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is a signature move

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that fda does before constructing a

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bacterial cell

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however no plausible constriction was

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observed

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after observing the proteins on these

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two systems we are planning to perform

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the experiments on liposomes

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and that are made of mixed fatty acids

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and see how fds is behaving on them

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to see any blebbing of escalation events

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that could

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be occurring and if it happens then fdac

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could stand out as a potential candidate

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for being the primitive protein

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needed for protocellular division and

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the same thing would be observed on

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slbs2 to find out about the mechanism

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at the end i would like to thank my

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funding sources my institute

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naiser the university under which my

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institute is homi baba and department of

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atomic energy

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india the project

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the the whole work and the ongoing

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processes will

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be the part of my undergraduate research

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project

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which is done under dr norman jim

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srinivasan and i would also like to

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acknowledge one of his

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grand student that's ajayakumar sharma

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who has helped me in every process

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each and every process of the

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experiments and also

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to the members of srinivasan and matthew

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lab

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so these are my socials i would be more

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than happy to take in any questions

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regarding the

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work and will be also happy to share

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any updates that have been happening