Kind: captions
Language: en
oh yeah i'm ready yeah okay okay are you
ready so i
will introduce you uh
okay good morning all the the students
and not only all students but i think
i saw others colleges
okay just for me
okay we i'd like to start so
good morning uh everybody so for the
students and also for the colleges
faculty members from others university
and also good evening to raj
so today raj will
present the
class so i will i would like to
introduce rad first
so i would like to introduce uh
professor raj
so prosourat is uh professor emeritus
i call him raj he is now a professor
of biological sciences at the nicole
state university usa
thinks here he received a number of the
awardees and also i think the
professorships and also uh
presidential awards in dr raj also
received the nicole state university
president award for teaching
excellencies
and he has more than 30 years of
research
experience in the area of bioremediation
and bioprocessing
his senses involved by reminiscing of
hercules chemical including oil spill
explosive
vertical treatment of visual treatment
and tibiotic consistent genes
in the environment and also bio
ethanol production of biofuel production
and also currently perforat is a
physician professor at the faculty of
industrial technology in
i think he was here in the 2019
in the in itv his colleagues of mine
i know him since 1986
so it was more than 30 years
uh i would like uh
to invite professor rice to give the
lecture on the
genetic although it is a really
sciences i think so for all the students
in engineering background will
uh will take the message and then
the advantages of the genetic
in your work please write the time this
year
all right thanks very much chandra it's
uh i'm very happy to
be here even though it's evening almost
dinner time here
six o'clock sunday night and good
morning to everyone
um so as professor has um
you know mentioned that today i'm going
to talk about um
basically the talk is on genetic
engineering
but i broke my talk into three different
sections first i'm going to do some
basic information on
the gene gene structure transcription
translation those
information some of you might already
know from taking
microbiology classes and then i'm going
to introduce a genetic engineering
concept how to manipulate genes
and then what kind of various products
you can make um using that
and then the last part is the the new
science we call synthetic biology
where you can synthesize your own
genes and and produce a lot of products
so i broke
down into three different um categories
so i'm going to be
using three different slides okay i'm
going to start with my first one
so uh so i'm going to talk about
introduction to genes and
genetic engineering and synthetic
biology
um if you look at the introduction
this is very common term most of you
know from high school biology
so um genes are there as a hereditary
factor
you you have the codes and it's passed
on
as an inheritance okay so the
transmission of
all biological traits from parents to
offspring
is passed on through these genes and the
expression
and variation of these straights depend
on what kind of codes
are in those genetic material and then
if the code changes an expression
changes
as well so just to go basic
you know you have the organism level you
have different
organisms and inside at cellular level
if you break it down
at the cellular structure inside the
cells
in a in a eukaryotic cell you have this
nuclear membrane inside those nuclear
membrane chromosomes
and in the chromosomes you have dna okay
but in bacteria which is prokaryotes
which
don't have the nuclear membrane and this
chromosome is right on the cytoplasm we
call that naked dna
and this genetic code is and they and
the uh
on the chromosome with in the genes
broken down into various parts in the
chromosomes
so just some concept of
terms definition the term genome refers
to
the sum total of all genetic material of
an organisms
uh most of these genome mixes in and
they reside in chromosomes and some
appear
in non-chromosomal side because we have
in bacteria called plasmid
plasmids are a tiny extra piece of dna
and in
eukaryotic organism in some organelles
like mitochondria and chloroplasts
they have their own dna so most of the
genetic material are in chromosomes
it's not moving um so here's a picture
of
the cell um you can see the cell in
chromosome in in bacteria you have
plasmids
apart from regular chromosome and then
in eukaryotic cell
there are some dna present in
chloroplasts and mitochondria
and then viruses have its own
organization of
some of them have dna some of them have
rna that has
the genes um so the term
genomics refers to study of an
organism's
entire genome so so for
for example human genome project we know
we
already know all the genes all the
nucleotides that make a human and that
is called genomics if a term refers
genomic refers to
all the organisms entire genome
as i said chromosome before chromosome
is a discrete cellular structure
composed of neatly packaged dna molecule
so genes are the basic informational
packets
and the genes classically defined as
functional unit of hereditary
um but preferred definition nowadays is
is a segment of dna
that contains necessary code to make a
protein
or an rna so that's a
a definition of what is a gene okay so
common people know gene is a in a
hereditary function
and a molecular and biochemical genetics
says is a site on the chromosome that
provide information for certain cell
function
but at a molecular level what is a gene
gene is a piece of dna
that contains necessary code to make a
protein or an rna
so another term is called genotype
genotype is some of
all uh genes consisting of an organism
um distinct to genetic makeup and that
that spells out the genes how they are
present that is called a genotype the
phenotype
refers to the physical expression of
genotype when you have the gene
uh how you look like and what kind of
function that genes
provide that is called phenotype so
these are some
basic terms some of you already know
about it
um so let's look at the the size of the
genome
for example e coli which is a common
industrial organism we use in
manipulation of genetic gene genetic
engineering
e coli has only one chromosome and it
has
4 288 genes okay
if you pull out that chromosome out of
the e coli
it's a one millimeter long uh although e
coli is in
you know point three point four micron
in size
if you unbound this chromosome it can be
bigger than the e coli itself it's a
thousand times longer than the bacterial
cells are neatly
packaged in tightly bound inside the
cell
and it contains 4288 gene
on the other hand human cells roughly
have 30 000 genes so anywhere from 20
000 to 50
000 genes but they are packaged in 23
pairs of chromosomes have totally 46
chromosomes
so equal has one chromosome human cell
has 46 chromosomes
and if you look at e coli in one
chromosomes you have 4288 genes
um so here's a picture of the e coli
that shows um the bacterial cell
and the g the chromosome pulled out you
can see that
a thousand times bigger than um
bigger than the cell itself so that's
all the
gene i mean the one chromosome pulled
out x and showing
how big it is okay
so let's uh just look at the the
structure of
dna dna as i said is a basic
um the basic unit of a dna is a
nucleotide like another term
so each nucleotide contains a phosphate
molecule
and a sugar called deoxyribose sugar
and it has nitrogenous base so this
nitrogenous base
is the the basically carries the cores
and there are two kinds one is called
purine
another is called pyrimidine the purines
and pyrimidines
totally we have four of them um we have
adenine which
the letter refers to a which always
pairs with
another nucleotide called thymine which
is t so
we have adenine is a purine thymine is a
a pyrimidine molecule and we have
another
nucleotide called gonine which is again
a purine
as always paired with cytosine c so a
always combines with t g always comes
with
c that's how um in on the dna
you have this uh nucleotide arranged
so here's a structure of a piece of
dna molecule if you look at all the
parts we
i show i showed you we have phosphate
molecule
and you have the the sugar deoxyribose
sugar
and then the nucleotide you have the
pyrimidine and purine
g and c and and t
and a um they combine c always comes
with g
and t always combined with a ad9 thymine
coenin cytosine
and so this is how a dna is
structured and the dna is double helix
which everybody knows it's uh you have a
um two two strands that uh um
you know twist uh and tightly packaged
inside the cell
and each of those unit is is called a
base pair okay one base pair is
one computer and another pyramiding
command together is a base pair
um a little bit about some enzymes there
are a lot of enzymes involved in
replication of dna and so here is
listed on this table so each one has its
own function
and you have helicase primase dna
polymerase like a stockpile
isomerase and the important ones are the
dna polymerase which basically
add um the nucleotide during a
replication
and the base pair uh to the um
the daughter chromosome molecule and of
course the other
enzymes are also involved in
dna replication so when we talk about
um genetic engineering we talk about dna
polymerase talk about ligases
and we also talk about cutting gene
cutting enzyme we'll talk about some
other enzymes okay
so the basic function of this dna it
carries codes
and it when it is necessary when the
gene is turned on
it's going to make a messenger rna that
process is called transcription
and this method rna carries the codes
and that codes code for specific
protein okay and when there's um
code is carried to a a ribosome
and that is where the translation takes
place that's where the actual protein
synthesis takes place so
basically this um uh the
the codes that are residing in a dna is
transferred to a messenger rna
which is transcription and then the um
the transcription
go the messenger rna goes to the
ribosome where translation takes place
which is basically
making a protein um so we're going to
talk about
how this is done even though some of you
already know about it just bear with me
um so transcription is um uh
making a messenger rna and translation
is producing protein so
simple definition okay um there are a
wide variety of other rnas that are
there to regulate
uh gene function how to turn the genome
gene off and the dna that codes for
these
crucial rna molecules are commonly known
known as junk
dna but there have other functions
but people finding out every day there
are different functions of
those so-called junk dna okay you call
it basically
intron and exon exon are the genes that
express something intron are spacers in
between the gene
and and those are one time they call
junk dna now they're finding out that a
lot of regulatory functions
so this is just to show the whole
process of transcription and translation
there is a whole chromosome here's a
piece of a dna
the first thing when when this
organism needs a specific protein to be
expressed the gene will be turned on
a specific regulation function and
when that happens um the first thing it
does is it's going to
transcribe the code and transfer that
code into a messenger rna
and that process is called transcription
and in transcription you need these
different rnas we have a transfer rna
messenger rna ribosomal rna and
transcription
carries this code to the ribosomal rna
and transfer rna carries the amino acids
and messenger rna is the one that has
the
codes itself and then we have a lot of
this other
rna that have regulatory functions okay
so the actual translation takes place in
ribosome
when muscle rna is cut and
come to the ribosome and the ribosome we
have the transfer rna comes and carries
um the amino acids for the sequence
and put in to make a specific protein
um product okay so as a transcription
part and
translation part this is the major
function of
how a gene works so let's talk about
um um the connection between dna and
organism trait
the proteins primary structure is
determined by
how it is shaped um in a
three-dimensional view
if you look at it the shape and function
of a protein structure
protein is ultimately determined by
phenotype
and so the study of organisms complete
set of
expressed protein is called proteomics
another term
and so proteomics is all the protein
that a cell can make
and so dna is mainly a blueprint that
tells the cell
which kind of protein to make and how to
make them
and how much to make them and when to
stop them so there is a specific
regulatory function that turns the gene
on
and then turns the gene off when you
don't need that protein anymore you turn
the gene off so
it's completely controlled by a certain
regulatory function so just put all this
thing
together um if you look at the dna
every three letter i talked about that
cga the nucleotide every three lecture
on a dna
carries the code specific code okay and
that code is
on a dna it's called triplet chord
triplet
is three right tri means three and that
is
um each one is a nucleotide three
nucleotide
in on a dna is a triplet when
that is a message is passed on to make a
messenger rna
that three letter is transferred to make
three more codes transfer
and each of the three nucleotide
messenger rna is called
codon okay so we have triplets on a dna
um nucleotide and then that one is trans
uh when it is transcribed and a
messenger rna it is a codon
and each of those three letters that
nucleotide letters
um codes for specific amino acids
to make protein so here is a a codon
right there is another codon here is
another codon and so on
and each codon codes for
specific amino acids so here is amino
acid one to five
and and what order these amino acids
should be combined those are all
specifically coded in this it passed on
to measure rna
and when these amino acids are combined
together
then you make a specific protein okay so
this is the gene protein connection
so so gene carries the code
and through transcription and
translation you make protein
so each three letters on that um
and the gene is a triplet code and
measuring rna
is called codon so i'm going to go
through that very quickly and and show
you how this whole
function work uh the the major
participation in transcription and
translation there are a lot of
molecule involved they are the first one
is measuring rna that carries the
code for protein then transfer iron make
carries
amino acid regulator rna
ribosomal rna and just should be
ribosomal rna
um where the uh
actual translate translation happened
that mean actual protein production
happens
and then we have several types of
enzymes involved and also you have many
raw material that means you need
a nucleotide to make the method rna okay
all these things are involved in making
um a protein okay so just to put
together
the different rna involved so we have
method rna transfer rna ribosomal rna
and regulatory rna there are a lot of
different regulatory rna
and we have a primer and ribozyme and
splices so
so the major ones are these three rnas
okay
and then we have a regulatory function
of this and each one
has the specific uh information
what kind of codes they have how it
functions in a cell
is listed here okay the only rna that is
um that is controlled
that is that has this um thing is the
messenger rna okay
so here is a a transfer rna um
the transfer rna has this three letter
code
and that code is called anticodon in
transfer rna
for example um for
i um assign in in uh in in the code
always come into the eurocell i'm going
to talk about what
u stands for um um
so the anticodon will have exactly what
to have in codon
so here is a messenger rna codes they
mentioned like a a
u g a a comment with u a comment
u comments with um a and g combines with
c so here is your
codon here is an anticodon and when she
reads that
um then the amino acid is passed on okay
so
it transferring has an anticodon it also
has
amino acids so when transfer rna reads a
method rna
it it transfers the amino acid by
reading the codes
okay let's look at
tools in the cell assembly line there
are difference in rna structure
just i want to go through what rna
structure is
rna is a single stranded molecule and
it contains uracil instead of thiamine
so
in dna you have thiamine in rna
or uracil that's why the codon has u
instead of
t okay that's your complementary base
pair
and the sugar in rna is ribose sugar in
dna is deoxyribose sugar that's a
difference between rna and a dna
and rna is a single stranded and dna
double stranded
um so methyl rna transcripts
of structural genes in the dna and it's
synthesized
in a process similar to the synthesis of
leading strand
during a dna replication and codon
is a series of triplet code that holds a
message of the transcribed messenger rna
again to go back one more time to show
you what
trna contains it's a sequence of bases
that form
hydrogen bond with complementary section
of the same trna strand
but it carries anticodon okay it's found
at the bottom of the loop of the
clover leaf structure and and it
designates the specificity of
transfer rna and complements the
messenger rna codon
and then ribosomal rna is a long poly
nucleotide molecule
that forms complex three-dimensional
figure
and that's where all these rna come
together it's like an
assembly line and it uh
all the amino acids are put together to
make specific
protein so here is a
all of them put together in one place
okay
so the message from dna is transferred
to messenger rna
in the form of every three letters as a
code
and that comes to the ribosome
and on the ribosome um
we have the transfer rna calm and it has
anticodon
and it also carries amino acid so here
is
every three letter is a codon here is
three letters
anticodon if there is a adenine here
the corresponding anticodon should be
uracil
if it is going in here the anticodon
should be um cytosine
c right so a use um
and c g and so on right so when it reads
the correct code
it says okay this um amino acid should
be put in here
so the amino acid is cleaved and put in
and then the next
um transfer rna reads the next code
and cleaves that amino acid and put it
together
and and and this right this is like an
assembly line
and this um transfer rna move into the
exit side and
and got out and then yeah another
transfer runway goes through this
ribosome
until it reads a code called stop codon
and and the whole thing stops and the
protein is cleaved and processed uh
further okay so this is just picture as
like a assembly line
all of them get together in the ribosome
and those three
rnas ribosomal rna is a place for
protein synthesis
messenger rna carries the codon transfer
rna carries anticodon and brings the
amino acids to make the protein
um the actual uh transcription i just
just to show you how the code is passed
on
um here is a dna when
when the dna is turned on to make
specific
messenger rna the rna polymerase
enzyme come and sits and unwinds the dna
and in the cytoplasm we have this
nucleotide pool
that has all this nucleotide am
adenosine uracil
cytosine so wherever this letter
on the dna a u is put together
wherein a wherever there is a c g is
added together right
as a result you have this initiation of
um
a messenger rna made okay so rna
polymerase comes here
unwinds and from the nucleotide pool
the codes are red and messenger rna is
made
okay so every three letter carries a
code
so here is a the whole process there so
here is a
metronome transcript and every three
letter is a codon
actually transfer from a dna into
this messenger rna this is the
nucleotide pool all these
nucleotides are present in the cytoplasm
okay so
initially we start with rna polymerase
that's called initiation
and then elongation which is putting
together this messenger rna
until you see the stop codon okay and
that's how the transcription process
takes place initiation
elongation and third one is termination
um so once the messenger iron is put
together and when it comes to the stop
codon
it says this is where we stop and it's
it cut it off and the measure on it
cleaves
and this whole process is called
transcription
making the code transfer from dna to
messenger rna this method now carries
code for
specific uh protein and this goes
to ribosomes for translation
okay so the central principle of
translation is
messenger rna nucleotides reads codons
and the codon dictates which amino acid
added to the growing chain
except for a few cases this code is
universal for bacteria
archaea eukaryotic virus so almost all
organism has same codes okay
so this is just to show you the the
basic
master genetic code there are 64 codons
in messenger rna 61 code
codon codes for an amino acid specific
amino acid then we have a
one start codon and two stop codon right
so every three ladder is a uracil uracil
uracil
that codes for an amino acid called
phenylalanine
and a letter uua uracillura cell
and adenine that calls for leucine so if
there are three letters and a messenger
rna
like arranged like this then that codes
for specific amino acids so here is a
first letter second letter third letter
and you can see various amino acids
and codes and the codons listed here
okay so you have a aug which is the
initiation start codon it also codes for
amino acid in the middle of this
middle of the messenger and they have
this top code on
okay and totally we have 64
codes on the messenger rna
and there are some redundancy put in
place and said i amino acids represented
multiple codons
you can see that there are multiple
codes for same amino acids
and just to you know it's like a
redundancy purpose
and so and also it allows for insertion
of correct amino acid even if there is a
mistake is made in the dna sequence okay
so this is just to show you the whole
process again
how these whole things work um so here
is your dna
um triplet codes um thyme in
gold9 39 pesos with diamond time
interest with 39 going to pair with c
this is your triplet code on a dna when
this is transcribed you make your
messenger rna
so we have uh on the measure rna
that is um the diamond um uh
should be uh you know matched with 39
and in rna there is no timing
instead you have euro cell so rd9
matches with uracil cytosine match with
g so aug codes for something
cu g quotes or something and so on this
is now called codon
and then this goes into um
then if this goes into ribosome and the
transfer iron make carries the
anticodon so the anticodon is a to u
u to a g to c this is an anticodon
and this also carries amino acids right
so
aug codes for methionine amino acid
ceg codes for leucine amino acid ac
codes for three on an amino acid
acg calls by three and amino acid and
the
anticodon is the same thing for these
amino acids
so when these things are lined up and
these are the amino acids that put
together
to make a sequence of amino acid to make
a peptide
and each peptide is a protein okay so
this is the whole sequencer event
that takes place in your body
every time millions of times right
and in bacteria it happens it's almost
the same code
like we have all this um
this the dna sequence that specifically
codes for
as some specific protein
um all these elements needed to
synthesize proteins are brought together
i have masonry amino acid ribosome and
these are the three
event i talked about initiation
elongation termination to make messenger
rna
and here is the whole process to show
you one more time
the whole process of ribosome where you
have messenger rna come together
and you have um the transfer rna reads
the codon
an anticodon reads the codon and get the
amino acid cleaved
and the next amino acid put together
the one two and then three four five
more amino acids together and make a
peptide which is a protein all right
it's just to show you a long protein
molecule build up
each one is the amino acid all the way
down
and that is called peptide right and the
bond is called peptide bond between each
one okay
so the things to know is start codon
stop codon and
start code and start the process stop
codon stop okay
if the stop codon comes in the middle of
the
sequence then there is a mutation that
makes some
uh instead of putting some correct amino
acid you're stopping here that's called
mutation and there's another phenomenon
called translocation
the process of shifting the ribosome
down the master rna strand to read a new
codon
that is called translocation so you see
the one
uh transfer rna comes and another
transformer comes so
the shifting of ribosome in between is
called translocation
and after the protein is made there is
some
modification done to the protein that is
you you cleave the
um uh the the area that doesn't code for
anything that's called
splicing of introns and then you have
adding some cofactor adding some
phosphate to make phosphorylation to
make that protein functional that is
called
post-translational modification to make
that protein
function okay and this is just to show
the same picture
uh it's like a big factory in a cell
factory then you can see this
ribosome and the messenger rna
all like a ribbon sitting on a lot of
different ribosome and continuously
making
protein okay so trans transcription
translation in a cell this is the actual
picture
of bacteria making protein
and just to very quickly how the these
genes are
regulated there are certain region of
the gene
that has a regulation function to when
to produce this protein when to stop
these proteins and
they have a circle in bacteria we call
operons regions
and those are specifically controlled by
the need of the cell so you don't want
to produce this always
okay so we have an inducible operon
and that codes for a specific
enzyme to start the process um
apparently sometimes it needs to be
induced because when the substrate is
present
the substrate itself is going to turn on
the gene
and it has to go to the operon region to
turn on the gene
and then there are structural gene that
codes for a specific
function and and so those are all
regulated
uh highly in in every cell
okay and then we have repressible operon
which is
uh contain codes for you know anabolic
enzymes several genes in this series is
turned off that is
uh when you don't need them you don't
have to make this
protein so it's already in off position
so you need to
have the uh the product itself to make
it turn it on
okay so you have a regulator gene you
have a
on the regulator gene in the control
locus you have a promoter region
and operator region and then the
structural locus is where actual codes
are present
in making a particular
protein so this is given in e coli first
time described in e coli how
e coli metabolize lactose and this is
called lac
operon and that carries for
digesting lactose making a specific
enzyme
um it's just like a allosteric site
so when the lactose is present
lactose is going to go sit on this
controller gene and turn the controller
gene
structure modify the structure so it
falls off the
control region of the gene so the rna
polymerase can come and start the
transcription process okay so in the
next picture we're going to show you
okay so here is a control region of the
gene
to turn the gene on so you have a
regulator a repressive protein sitting
on the operator
region of the gene it's stopping rna
polymerase to
bind to start the transcription right so
when this repressor protein sits on this
part of the gene
that means the gene is turned off right
and muscle rna cannot be produced there
is no transcription okay
so in this case lactose um when the
lactose is available the lactose is
going to go one of the molecule of
lactose is going to go sit on the
allosteric site it's going to change the
shape of this repressor
protein and it's going to fall off and
then rna polymer is now going to sit on
the operator region and and start making
transcription
uh process and then you make measure rna
and then a method rna continues to
go through translation and make
necessary enzymes to
digest the lactose okay when all the
lactose are
processed by the bacteria there's no
more lactose left
there's one lacto that is sitting on
that repressor protein right
that represent protein that lactose is
also will be digested by this enzyme
so now this repressor protein goes back
to original
shape it goes to go back down the
operator region and lock
rna polymerase to you know go through
the transcription this
when it goes back when this last last
other lactose molecule is digested
then it goes to the off position so you
can see how the genes are turned on
are the genes are turned off by the
substrate itself
the availability of the substrate
um another molecule is a repressible
operon in this case the gene is all
always on um so
when the when the expression over
expression of the gene
uh will turn the gene off okay when the
product is made
too much and it become the product
itself a co-repressor
it will go turn the genome so there are
two ways to control the gene one
gene is an off position the substrate
needs to be
uh combined to turn their
uh off position to on position and the
other one is the
uh the repressible operon where the gene
is always on
but when the product is made too much
the product itself become a core
repressor
and turn the genome so these are the two
major
regulatory functions of how the genes
are turned on and done
so here is rna polymerase continuously
producing uh in a uh transcription to go
on
and then when when too much of this
product are
made and that one other product gonna go
sit there
and change the shape of this and repress
the protein
and which can go and sit on the operator
and stop the
rna polymerase to bind and the
transcription stops so here is the
gene is always on position the product
itself become a core repressor
and and make the genes to turn off so
there are two ways to control
and these are the two basic ways the
genes are turned on
and turned off um
the next process is called recombination
recombination is an event in which a
bacterium donates a dna to another
bacterium
the end result is a new strain of
bacteria
okay it now causes the nutrient
obtained from another bacteria okay
most of the time a plasmid to plasmid
transfer of this
gene takes place sometimes it takes
place in extra chromosomal dna
so every time a new gene is
taken from another cell now we call that
recombinant
dna a recombinant organism so recon
organisms are
organisms that have more than a
gene from more than two or more sources
okay
so that process is called horizontal
gene transfer so normally genes are
transferred vertically from parents to
offspring every time
a cell reproduces um in the horizontal
gene transfer to adult cells exchanging
genes
and and that is a horizontal gene
transfer
and this is how bacteria develop
antibiotic resistance okay
through horizontal gene transfer from
one organism to another organism
and again plasmids is a small a circular
piece of dna
that replicate independently of
bacterial chromosomes they allow
transfer dna between
cell is found in most bacteria and some
fungi it contains
a few dozen genes okay and the plasmid
genes has no
um gene that calls for any survival
function but it codes for other traits
especially
antibiotic resistant heavy metal
resistance
so the process naturally that
makes this horizontal gene transfer are
conjugation
transformation and transduction so
conjugation is
basically like bacterial sex between
one um bacteria to another bacterial
gene is transferred
uh through a appendix called pili or
pilus
and the particularly combine a bridge
and then the gene is transferred from
one bacteria to another between two live
cells
transformation is a gene transform of
dead bacteria to live bacteria when a
bacteria die
and decompose the dna is released into
the environment
that dna is called free dna and when
another
bacteria happen to come by close to this
free dna
if if it has a right receptor on its
cell membrane
it's going to capture that free dna and
you know put it inside the chromosome
and now you can have a new trait okay so
when
from a free dna from a dead cell to
livestock dna
transfer takes place it's called
transformation and transduction
is basically um accidental gene transfer
through a viral replication so
when bacterial virus replicate from one
replicate from one bacteria another
bacteria it sometimes accidentally
transfer a bacterial gene into another
bacteria
so these are the three ways uh bacteria
obtain horizontal gene transfer
conjugation between two live cells
transformation is from a dead cell to
life cell
transduction is through accidental gene
transfer through a viral replication in
bacteria okay
so just to show you the same thing i
talked about that's a conjugation
and definition okay and here is how it
happened so
here is a bacteria through a pili from a
bridge
and then dna is transferred from donor
bacteria to recipient bacteria and the
dna is substituted
in the recipient bacteria so you can see
that whole process
okay and this f factor is called
fertility factor
and the bacteria that has pili is called
left plus the bacteria that doesn't have
a philly which is a hair like projection
is f minus okay so this is
called conjugation process um
so one of those antibiotic resistant is
commonly transferred through this
process and called resistant plasmid
resistant factor so bacteria develop
antibiotic resistance
and transformation as i said before it
is
only happens is competent cells the
competent cells has to have
right uh receptor on plasma membrane
to to to dock that dna onto that protein
to take it inside okay so if there is no
right receptor then
it doesn't do it so when the free dna is
available it captures it
and transfer inside and and it
changes the genetic reorganization so
here is an example
of a lyso a dead cell when the bacteria
die they it releases the dna into the
environment
this becomes a free dna and then free
dna
is now um when another live cell come
close to the free dna
if it has a right receptor on its cell
membrane it's gonna
dox it's gonna sit on it and then goes
in
inside the cell and it substitutes its
own dna
and take this far in dna from another
source
and that is your transformation process
and transduction as i said is a
accidental gene transfer through a virus
that virus
in bacteria is called bacteriophage and
here is a picture of it so
virus injected genome into a bacteria
for viral replication and it goes into
the bacterial genome and
bacteria is going to replicate virus for
them and sometimes it actually takes a
bacterial gene with it
and and when it reproduces go to the
next cell
it now injects the some of the bacterial
cells along with
virals um genome into that okay
bacterial genome and vital genome
combined together into another
bacteria this process is called
transduction so this is the three
processes involved in nature how a
recombinant organism can
develop through horizontal gene transfer
apart from all this there is another
piece of elements called transposable
element
and which is commonly known as jumping
genes
and this was first proposed by
a scientist barbara mclintock in iowa
state university in iowa in 1951 she won
nobel prize for this discovery
and this jumping genes have no
particular
place on a chromosome it doesn't have a
permanent place
it always moves on the cross so every
time it moves and
and and disturbs the arrangement of um
dna
uh it's gonna make mutations and
uh so that changes the expression of
gene and
uh so this is called um jumping genes
okay
so this is how the transversal elements
look
these transversal elements have no
permanent place on a chromosome
it always moves shifts and every time
it goes and find a different spot it's
going to rearrange the whole
um genome and then that's going to
change the expression of the
whole organisms that's called
transposable elements
so there is a um this is these are
called into also called insertion
element if the transverse elements are
very small it's called
insertion sequence and then
retrotransposon is another term for
type of transposable element that can
transcribe
dna into rna and then back into dna
uh into a new location that is called
retrotransposons okay
these are some more terms that you need
to learn when we
do genetic engineering process okay
and the other transversal element
contained gene that call for
antibiotic resistant toxin production
and all that
so what happens the general effect of
these transposable elements
you scramble the genetic code and you
make a mutation happen so sometimes it's
beneficial to the organism sometimes
it's adverse
depending on what kind of shift took
place on the
gene okay where it is relocated what
kind of genes are relocated
so most of the time the transversal
lemon and bacteria change the colony
structure color of the colony and
bacteria
and antigenic characteristics and
replacement of damaged dna
and also inter microbial transfer drug
resistant basically antibiotic
resistance
so that's uh concludes the basic part of
um
genetic gene structure
and transcription translation how
bacteria
transfer genes um now i'm going to
shift the genetic engineering process
if you have any questions in this um i i
i can open up the question uh
do you want me to continue um chandra
you want me to
take questions here i think sir
i think there's there's a there are a
question okay
uh the first one is from uh
ryan okay how to how to modify
dna in a micro it is this will be part
of your lecture or next one yeah next
one okay okay yeah
okay i'm going to do that and then next
slide is coming up
this one is just to show all the parts
and the component the process and terms
now i'm going to talk about genetic
engineering
yes okay okay then as you said from uh
my question
is because of it's look very complex
right
what we have been present is very
complex
how this infra information derived i
mean
how the scientists can get the
information they said
uh working like a first one this model
and the experiment and service how how
does this information derive because
basically it's a lot of work
so you you you isolate the dna
and then you read the the codes
and then you understand what this code
stands for
so it's like a 60 70 years of
a lot of people working on this field
and put all
this together the language of this
genetic codes and for example the 64
codon
that people put together to make for the
which is universal for bacteria to human
and that took uh you know almost 50 to
60 years of research
a lot of people and a lot of people want
nobel prize
also doing this work yeah yeah yeah
it's a basic research yeah yeah okay
then
okay i think it's uh uh tobago's
question will be coming up
uh i think in your uh okay
all right let me start the next section
yes which is
with this is genetic engineering how you
manipulate the gene how you make a
desired product
and then the third section is synthetic
biology okay
let me open the slide here
can you see my slides everybody
uh yes but it's you can okay
okay now okay okay all right now i'm
going to talk about how to manipulate
the gene
and make different product okay so
here is a dna toolbox so
we call that sequencing of genomes so to
to all the genes that make a particular
organism so people have been working on
different organisms and
i i talked about human genome now we
sequence
so many thousands of different bacteria
a lot of different organisms the whole
genome sequence been done so more than 7
000 species
of organisms have been sequenced um
last 20 years okay so we know exactly
all the genes for that to make a human
all the genes that make for
several bacteria so these dna sequences
depend on
a lot of modern technology there's so
many new instruments has been developed
and starting with making the recombinant
dna
okay so in recombinant dna the
nucleotide sequence from
two different sources um in put
from two different species are put
together
uh in test tube in which uh in vitro
and then you put them in in vivo into
the cell
okay you can manipulate that in um
in a small centrifuge tube and then you
put a bacteria inside
and allow the bacteria to take this gene
inside so
so you can manipulate the gene i'm going
to show you some technique okay
so methods for making recombinant dna
are central to genetic engineering
if you want to manipulate the gene you
need to cut
cut the dna in specific places
and insert another piece of dna into
that cut
dna places and then you combine them
it's like a
putting a a tape
and make a dna again hole and that dna
then you re-insert into a bacteria
and and then that bacteria going to
hopefully take that piece of dna and put
it
into the chromosome now it's going to
express whatever the gene you put into
the cell
to whatever function that you want okay
that is basically genetic engineering
yes so dna technology has revolutionized
biotechnology
now we make a lot of different products
most of them pharmaceutical industry
and agriculture we make a lot of uh
genetically modified organism
plants especially um and so an example
of dna technology
and nowadays is micro array which is uh
on a small piece of um on a slide glass
light
you can measure thousands and thousands
of gene
expression whether the gene is active or
not active you can do that
okay that's one of the revolutionized
invention microarray technology so this
is just to show you on a small
piece of slide you can see more than
3 000 genes wherever that yellow color
means that gene is expressed the red
color is gene is not expressed
so you can this is called microarray
technology
so you can put that on a small chip all
the genes
and manipulate it and make sure the gene
is
producing methyl rna and not producing
meth if it is producing machine rna that
gene is
turned on if it is not producing
methanol the gene is turned off so you
can do this
nowadays in and the prices are coming
down
and you can do that between 100 to 300
dollars
now you can do this um let's talk about
dna cloning and
how to do the genetic engineering part
okay
um so to work directly with specific
genes scientists prefer
well-defined segment of dna and make
identical copies
and that process is called dna cloning
so when you make
exactly same copy of a dna that is
called dna cloning okay
they have same information um
so most methods of cloning a piece of
dna
in the lab have general features okay
uh you need such as use of bacteria and
the plasmids so
bacteria is essential and plasmids are
as i said before it's a small circular
dna that present inside the bacteria
and the clone genes are used for useful
for making
a particular gene and producing protein
product okay
so gene cloning involves using bacteria
to make
multiple copies of a gene so once you
put the
modified gene inside the bacteria the
bacteria
every time it replicates it's going to
produce that same gene over and over
right
so you're making the clone of the gene
uh every time the bacteria replicates
and so the foreign dna insert into a
plasmid and the recombinant plasmid is
entered into the bacterial cell
reproduction of the cell make you more
of
newly made foreign dna and this makes
a production of multiple copies of a
single gene okay
so this is a just a picture to show you
how genetic engineering works so
here your bacterial cell this regular
chromosome is a plasmid
right so you you take this plasmid out
and then you take a gene of interest for
example in human this is how
we do insulin production nowadays you
take human gene
that makes insulin and and then you
you treat them with a particular enzyme
called
restriction endonucleases the
restriction endonuclease enzyme will
cut the gene at specific places it cuts
right so when you cut the plasmid
and you cut the specific human gene and
you combine them together
and you add another gene called dna
ligase
the dna ligase function is to seal the
cut
part of the gene together it's like a
tape it tapes them together
right so you take the plasmid you take
the gene of interest from human self in
this case say insulin
production and you treat them with
endonuclease to cut it
and cut it here and put them together
in the test tube and you put a dna
ligase and it makes
the cut dna uh make a
cut dna and and to seal them
and then you insert that back into the
bacteria all right
and then the bacteria every time it
reproduces now this is a recombinant dna
a plasmid from bacteria human gene here
right so this is now the recombinant dna
every time the bacteria replicates now
you make clones of this gene
identical gene right you can do one way
you can harvest the gene
and do whatever you want you can put the
gene back into human
and the people that have diabetes and
insulin
you know deficiency you can harvest a
bunch of this gene and put them back
into there and hopefully
it will take the gene and put the
corrective genes
in the in the place of defective gene
like that gene therapy
okay are you allow this bacteria to
express this
gene and make the protein for you and
you can use that protein purify this
protein and make insulin out of it so
this is how insulin is made nowadays
all right and insulin is purified this
is where the chemical engineer comes and
and and they do the scaling up of in a
fermentation process and a reactor
process and make this
recombinant e coli to multiply and
purify the protein all that comes into
play so here's some
example of you can make the clone and
you can
express the protein itself in bacteria
so
harvest the gene so you got the genes
and
insert the gene into a plant for example
if you want to plan to modify
a resistant to a pesticide or
insecticide
you can put that in that's a genetically
modified organism
you can put a specific gene in a
bacteria to clean up contaminated site
okay and then you can you know put in
the
um product itself
and as a protein for example human
growth hormone
you can make this growth hormone inside
a bacterial stuff
and now you purify that protein and and
give it to
people and they can you know use that
here's another example of protein
dissolved blood clot in
heart attack therapy and that protein is
now produced in
in bacterial cells so this is basically
genetic engineering you're manipulating
um bacterial gene and human gene put
them together
the two major enzymes are the enzyme
called restriction endonucleases cut
specific places dna like a seal
the genes back together so you
cut pieces of dna from two sources and
put dna ligase
in a test tube and you make this
recombinant dna and insert the dna
back in the bacterial cell every time it
replicates you make a clone
okay so i'm going to show you some
specific information how they do it
so here's a as i said before you take
the plasmid
you take the gene of interest from human
cells
and then you um put as
this thing together and plasma put in
the bacterial cell
and every time it replicates it's going
to make a clone okay
um and then you can um express them as a
harvested gene or make a protein
whatever you
whatever need that you need you have you
can use
them okay so the enzyme i talked about
is called
restriction enzyme this restriction and
ventricular restriction endonucleases
these enzymes these enzymes cut the
molecule at specific
places that place is called palindrome
sequence so palindrome means um
in if you read if you read a letter
forward and backward it spells the same
all right for for example mom mom mom
you can read it backward right that is a
palindrome dad d.a.d
dad that's a palindrome on a dna
sequence
if the sequence can be read forward and
backward exactly the same way
that is a palindrome sequence and these
enzymes
cut the sequence on the dna
when the sequence can be read forward or
backward exactly same
okay and the wrestling can eventually
make many cuts and yield restriction
fragments that cut small pieces of the
card restriction fragment
and then you put this cut dna
with other genes and that at the end
that's sticking out where the genes are
removed is called sticky ends
and then you put them with a here's an
example of
palindrome sequence and you can read the
sequence forward and backward
forward and backward exactly the same
and when you put this restriction enzyme
there are a lot of different restriction
enzyme cut in different places so
it's going to make the cut exactly like
this in this part
and this part so this gene is cut and
now
this is called sticky end it sticks out
in this part of the dna it's a sticky
end
right so now all you need to put is
another gene that
for example human gene if you want to
cut that human gene exactly like this
and put it back and seal it together
like
sticky tape put them on a on the jean
and seal it together
exactly like that so once you put
the cut gene together and treat them
with
dna ligase and other enzyme it seals and
bonds
this restriction fragment together now
you repair that
gene you put a foreign dna into that
place and and
cut it together and you make your
recombinant dna now okay
so just to show you how it works so you
have the restriction site which is a
palindrome sequence read forward and
backward
you treat them with restriction enzymes
you have a sticky hand
and then the the genes are cut now this
this dna is exposed here it's
waiting for exact opposite of
um dna to combine and then you
um dna the gene that from another source
you can add
and then you it's going to pair with
exactly
what is exposed it's going to pair with
another gene from different source it
and then you treat with them dna ligase
to seal them back
and so you put dna ligase dna ligase
will bind now this become a regular
piece of dna right if you look at it
this you cut it you treat it with a
restriction fragment with another gene
treated with ligase now it's sticky tape
you put them together and seal the
new gene so this is a recombinant dna
now you have
plasma genes and you have a another gene
from
say humans pro human gene inserted
now this is a recombinant dna okay
so these two enzymes are important
restriction enzymes
dna ligase presence and cut dna
ligase see the enzyme back
um so you can do that in industrial
cloning you can make it in
you know this is where engineers comes
when the biologists
make this and give it to the engineer
they can make it in a big scale
uh in big industry they make it in in
you know in thousands and thousands or
liter reactor they produce this okay
so the gene cloning the original plasmid
is called cloning vector
the plasma in the trigger where you put
that
piece of a human gene is called cloning
vector
cloning vector is a dna molecule that
carry the foreign dna
into the host and replicate that okay
so here's another example to show you uh
how
a gene from a hummingbird and e coli are
combined together with the same example
okay um so they
used the same principle of using these
two enzymes
and took a piece of a bacterial gene a
piece of a hummingbird gene and podium
together
so here's a bacterial plasmid that
cut with the restriction enzyme now it's
all open and cut you have sticky ends
sticking out
and then you have the hummingbird jeans
that
you know meant for metabolizing sugar
um and then you um cut them gene and it
has a sticky end too
and then you put the um ligase and
combine them together you can the
recombinant plasmid put them back in e
coli
and plate it out to show you which one
is um
carrying the gene which one is not
carrying the gene that expressed with a
different color
okay so here is the colony carrying
recombinant
um dna here is a colony carrying
not carrying the recovery it expressed
differently okay
and just to show you the same thing in
more details
okay how this process works so this is
basically genetic engineering
you cut and seal you cut the
plasmid this is a cloning vector
and you put the piece of dna that you
wanted to
express and you seal them together and
insert in a bacteria and bacteria will
replicate it for you
and this is where engineers again come
to
at an industrial scale to make them
and just to show you in closer view how
it is done okay
and uh in order to know that uh this is
this
research has been done for a lot of
different things and
and nowadays you can do this genomic
library
that is basically using bacteria the
collection of recombinant vector clones
produced by cloning dna fragments from
entire genome
you don't have to do this anymore now
you can refer genomic library
and it shows you which enzyme to use and
which enzyme
will cut and which enzyme will seal and
which part of the dna will go that is
called genomic library
the genome will already made using
bacteriophage and stored
as a collection of fudge clones so you
can buy this now
and it's this technology getting cheaper
and cheaper nowadays
and so now this this library how they do
it is they use the artificial chromosome
that called bacterial artificial
chromosomes because the chromosome from
plasmid is so small
right so you can only have small piece
of
information so they they use this
bacterial
artificial chromosome called bach and
you can make this library bigger
and you can store the genomic library
and um so this is how it has become an
industrial scale
and these the companies the biotech
company now sell them you can
if you'll check their catalog it shows
whichever
library whichever clone you need they
will supply to you
okay uh just to showing how the
genomic libraries are made in using the
bacterial artificial chromosomes
is a large plasmid it can have more
information there okay this is another
type of vector basically okay
so now i'm going to introduce another
concept called complementary dna
which is called cdna library so i
so far we talked about information from
dna to rna
right so you can make um
dna from an rna so if if you have a
piece of messenger rna
you can make a dna because whatever
codes on a messenger rna if you put a
nucleotide pools
in a test tube it's going to pair
with whatever rna has but i for example
if rna has a
cytosine c it's going to combine with a
guanine g right c pass with g
you pass with um adenine always pairs
with
um you know uracil so if methyl rna has
a code if you put a nucleotide pool in a
test tube
you can make a dna right so that is
called
complementary dna which is basically
making a dna from a messenger rna
right nowadays if a cell expresses a
method and if you want to make the dna
you can make that dna from a messenger
rna that is called complementary dna
and this is already been done and there
is a library
available for c dna library
with a lot of genome information that
shows the transcription of method rna in
the original cells
so this is in order to do that you need
a specific enzyme called
reverse transcriptase the reversal of
transcription that's why it's called
reverse transcriptase so here is a
example of how c dna is made
um so here is a messenger rna that is
expressed in a
bacteria expressed in a human cell you
can take that method rna um
and then and take the methodology out of
the cell
and then you put them in a test tube and
treat them with reverse transcriptase
and add some nucleotide pool
you can buy the nucleotide full from
sigma
and then this nucleotide pool wherever
that methyl rna has a a
it's gonna come in with t and and and
and it makes a
a complementary copy of a piece of dna
okay and once you made the dna then you
add a dna polymerase
and put more nucleotide full then you
can make a double-stranded copy of a dna
okay so this case this enzyme is
important this reverse transcriptase
and this was possible because of this
enzyme is common in viruses
and in nature so people start doing
research on this enzyme now
they're making industrial scale now you
can make
from a messenger rna a double stranded
dna you can make them
and so here is a cdna completely made
from a messenger
rna you reverse it you make a double
stranded dna
and this kind of dna you make from
measure rna is called
complementary dna called cdna and this
is also now
available in cdna library all around the
world you can buy from biotech companies
okay
um so now i'm going to talk about
a little bit about uh
screening our library for clones
carrying gene of interest
you can when you make an industrial
scale you make the clone right like the
genetically identified copy and those
clones
carry different functions for a
different protein
and then you can express them with
nucleic acid probe and see whether
it expresses in a cell that process is
called nucleic acid hybridization
so you can you can synthesize a probe
and if you think your cell is working or
not a cell is
functioning or not you put the piece of
probe
into the cell if it has a matching pair
it's gonna hybridize it's gonna match
with it wherever c
g gonna combine wherever t a gonna
combine it's gonna hybridize
right if it hybridized that means your
cell has particular piece of
gene express that is called um
nucleic acid hybridization you can do
that in a test tube or a plate
you can do that nowadays the dna probe
can be used to screen
large number of clones simultaneously
to see whether what kind of gene you uh
you wanted to
make in bioprocess industry okay
so once you identify the clone carrying
the gene of interest you can culture
them
you can put them in e coli and reproduce
them enlarge there
so here's an example of how it works so
you
you you want a piece of
gene that you want to see and you put
them in this
nylon membrane and you tag them with a
radioactive label
tag and you you wanted to uh
get from a library all the
piece of nucleic acid and pair them with
on top of them and then you
if there is a matching pair it's going
to hybridize
right these are all single stranded
probe
and you put the probe of interest and it
kind of probe and it's going to
combine if it combined it's going to
light up because we they put the
radioactive label probe
okay and this is how you can see what
kind of genes are expressed in a
in a in a cell and if it is not
expressed then you have to insert the
gene to express them
okay and so
if you wanted to insert a foreign gene
into a cell
you need to do that by a technique
called electrophoration
is basically applying a electrical shock
uh a pulse electrical sock and you open
up the cell membrane
so the gene that you want to put it into
a cell
after you make the recombinant dna in a
test tube
and you give a shock to the bacteria the
plasma membrane opens up then the
dna you put into the test tube will go
into the bacteria and the vector will
now put it inside in its gene and you
make that recombinant dna so that
process
of giving mild shock is called
electroporation
so electroporation opens up the plasma
membrane the dna will be incorporated
into the bacterial genome
um so it's remarkable like uh
these bacteria can take all kinds of
genes from human
from plants from because
the codes are inverse of course only the
four ladder
right so if the letters are there it
matches
it's going to take it up so so for
example you can see the
bacteria can take eukaryotic protein up
any gene you put in the bacteria and
take it
and and then if you look at the
vertebrate invertebrate
that expresses the eye um it's the same
gene
you can put a vertebrate gene into
invertebrate and invertebrate into
vertebrate
you can substitute because the gene
hybridized and it can pair because it's
a function is the same
so this is basically is what um
makes genetic engineering possible
because of this the code is universal
and the bacteria takes up any code you
put in it takes it and substitutes and
make
um its own genome expression changes
okay um another
uh thing you already familiar with is
called
pcr nowadays you did the kobe test pcr
test
uh what is pcr it's a polymerase chain
reaction
and basically you are making
a piece of dna amplifying it several
thousand million times in a test tube
so all you needed is you heat cool and
replicate
so when you heat the dna the dna falls
apart
and break the double stranded opens up
and
and then you cool it down and then you
add the nucleotide pool with
the polymerase enzyme and it builds back
that
new strand of the dna that falls apart
you you do this
over and over and you become a chain
reaction that is called pcr
so i'm going to show you how it works so
if you wanted to
take this piece of genome that you
wanted to make millions of copies of
them
you first isolate them with restriction
endonuclease enzyme you break it open
and then you take that piece in a test
tube
and you heat it up when you heat it up
the double strand break and become
single strand
right now this one strand open that all
in it
is whatever chord it is there if there
is a you need a t
if it is a c you need a g if it is a g
you need a c right
so you put the nucleotide in a test tube
the
nucleotide pool and um and then you
add this polymerase enzyme dna
polymerase enzyme okay
the three steps involve denaturation
which is heating
double strand becomes single strand
aniline
which is adding this first piece of
primer
to start the process uh wherever there
is a t is combined
and then extension put all the
nucleotides
it makes a new piece of dna right
so that's called denaturation annoying
and extension
repeat this process over and over and
over in a small test tube
and you make millions and millions of
copies of genes that you want
and this is polymeric chain reaction so
cut the
piece of dna that you want to amplify
and go through these steps
and you can buy these nucleotide tools
from
any scientific companies um
and you need the primer to start the
process and
by heating and cooling and adding dna
polymerase you can
make a new strand then you heat it again
and this is going to fall apart and make
single span
and then you continue this process and
you get
millions and copies of the gene that you
wanted to
amplify so this is the same process just
to show you how it works
so here's a new strand put together
right here
after you break open with the new
nucleotides
and now repeat the cycle make
more genes right so every cycle you know
you multiply
uh the new product that you make new new
genes you make
okay uh that is basically tcr okay
uh let's talk about um um
there's the sequence an expression of a
gene how a gene is um
expressed now and so dna clothing
allow researchers to compare genes and
allele between individuals
and locate gene expression in a body it
also determines the role of gene in an
organism so there are several techniques
involved i'm not i cut it off
because i don't want to go through there
are northern blood southern blood
the basic technique is electrophoresis
i'm going to show that
one technique and there are several
technique i removed it from my
powerpoint okay
so gel electrophoresis what it does is
when you
it shows what piece of dna you have
is a very basic um tool
uh so you you put the dna
in the test tube and you uh treat
through the restriction under nucleation
it's going to cut into several pieces
and then you put in a gel and apply um a
current electrical current um so when
the
dna cut yeah when you apply electric
current
the smaller piece move faster the larger
piece moves
um slower slower right so when they move
across the gel
it's going to form a pattern of dna the
larger pieces are at the bottom of the
gel
smaller pieces at the top of the gel
because smaller pieces move faster
and so it's going to form a pattern of
genes that is called gel electrophoresis
technique it's going to form
band basically what kind of bands are
expressed is what you're going to see
so here is the the technique of
electrophoresis
so you have the dna mixture in a test
tube and
you wanna that you want to see what kind
of dna are there
and he had this electrical current
cathode and anode in a gel
and you put it in this well you apply
electrical current
as i said um the shorter piece moves
faster
the longer piece moves slower and it's
going to have this kind of pattern of
expression
and that is basically cultural
electrophoresis
and this is possible because of a
phenomenon called
rflp called restriction fragment length
polymorphism
and everybody's gene is different
and so that is basically dna
fingerprinting
so when your gene and my gene are put
together in a test tube
and treated with restriction
endonucleases your genes cut in
in several pieces my gene cut in several
pieces not in
exactly the same location because your
sequence is different than my sequence
right so as a result when you put it on
the gel electrophoresis your
banding pattern should be different than
my banding pattern
so if you this is how they solve the
crime nowadays right
the dna fingerprinting so if you if you
find a
sample in a crime scene like a blood
uh some dna you can amplify
doing a pcr reaction and then if you
suspect a suspect
you take the dna from the suspect and
put that in the
electrophoresis and if the banding
pattern
matches the dna collected from a crime
scene then
you are the one you know did the crime
right so
this is basically dna fingerprinting and
this technique will cut
basic technique called gel
electrophoresis so you can
take a sample and do a pcr amplify the
dna
treat them as restriction endonucleases
and then
allow this through the gel
electrophoresis okay
so that's how it it shows uh you have
different banding pattern that's because
of rflp
restriction fragment length polymorphism
which is commonly called dna
fingerprinting
so this is our i show you here
restriction fragment analysis
is done using electrophoresis okay
this is called rflp restriction fragment
length polymorphism
uh you can use the same technique to
treat
um find a patient that has
carry a gene that is defective genes
okay so here is an example of
sickle cell patient and a normal patient
and you can do this cutting of genes and
putting the electrophoresis
they can see that normal alleles of the
gene look like this
sickle cell gene looks like this so you
can see whether the person carrying a
genetic defect
um you can using this technique and you
can identify that
that that is basically color
electrophoresis and this is possible for
basically for a restriction fragment
length polymorphism
and just to show how that works okay
same thing all right now
dna sequencing have it's been now
very very cheap now dna sequencing has
become industrialized commercialized and
all you need is uh as i said any piece
of
dna that you want to sequence you uh you
know
you put that fragment and send it to the
company they have this a lot of
different new instruments nowadays and
they can sequence exactly
what kind of gene sequence you have
in the sample that is basically dna gene
sequencing okay
and just to show you how they do it here
but i'm not going to
elaborate because it's going to confuse
you guys so
again it's the same technique before
nucleotide code
and all you need you're reading it over
and over on an industrial scale
you use a once you make the sequence you
use a laser and laser to read
the sequence and then the laser will
show you
the the nucleotide that are longest the
nucleus that are shortest
and it makes this graph and from this
graph you can
read and see how exactly the genes are
aligned
and what sequence they have a c t g
how it is arranged and here's an example
of short piece dna and long piece dna
and nowadays you can read through this
instrument
using laser okay
um let's talk about how you can analyze
gene expression
which is using nucleic acid probe to
hybridize
with messenger rna and see whether the
gene is expressed that gene
gene is always present but it's not
expressed right how do you find out
whether gene is
you know making method rna making
protein so
in order to do that if you take the cell
and you have this messenger rna from the
library
and put it on the gene if it matches
that means it's going to make if it
doesn't match it that means it's not
making it's not doing expression okay so
that's
basically is what gene expression is
um we do that by doing institute
hybridization method
um you just you take a piece of dna you
put the messenger rna and you
you know hybridize you put on top of
that
g basically it's an agar plate and you
push it down
and now we use this um um
the labeling so if it matches and a uv
light it's going to light up
if it doesn't match it it's not going to
light up it's called institute
hybridization technique
okay so this is just to show you a piece
of dna
yeah and wherever it matches
it lights up and wherever it does it
matches it if not
just to show you what the cell is
expressing a particular
a piece of gene are not expressing
that means that the gene is turned on
the gene is turned
off you can do that by doing insectic
hybridization
um and just to show you how
microarray works you can do this
expression of
whether the gene um turned on and turned
off in larger scale
you can do that in a small piece of
chips which is called biochips or dna
chips
so you put all the genes and put measure
on it and
and see whether it lights up or not
lights up you can do that
at 300 you can see 3000 genes in one
small slide
so this is how it works so you take
isolate and measure rna
and make a complementary dna that means
you put reverse transcriptase enzyme you
make a
double standard dna um you apply cdna
mixture into a microarray
plate and then you put the dna fragments
representing specific gene on top
and if this gene matches with the
messenger rna
it's going to light up if yellow color
means the gene is expressed
and blue chloro gene is not expressed
and here is a plate
that's showing 2400 human gene
expression in one small chip
it's called microchip or dna chip
biochip
there are a lot of same say same name
for this
this technique is called micro array
technique
and here is a just a picture to show you
how that
works okay um
how you determine the gene is
functioning or not
and you do that using in vitro
mutogenesis
as mutation is a change in
gene code when the meteor region is
returned to the cell the normal gene
function might be determined
by examining the mutant phenotype how
the phenotype change are not changed
you can tell whether gene is functioning
or not
um the gene expression is silence using
i showed you there is a regulatory rna
that rna
is called rna interference rna and you
can
use that rna to you know silence a gene
expression
this rna will stop synthetic double
stranded rna molecule
matches this sequence and then it blocks
the measure rna
and method rna cannot you know
transcribe anymore
that is called rna interference
um and then another thing to know is
called this
genetic markers called single nucleotide
polymorphism
these are 100 300 base pair
and called snips per shot the snips you
can detect them in
by pcr and this is how you can
differentiate different disease causing
gene
using this polymorphism everybody
sequence
there's a single nucleotide that repeats
over and over
in different places and that is called
single nucleotide polymorphism
and using that you can detect people
have particular
disease or not okay
um i'm just gonna i don't know
i'm gonna go through quickly how an
organism is cloned and then i'm going to
come to bio
processing okay so you already know the
the first organism clone is this sheep
and scotland
dolly right i'm going to show you the
technique how it is cloned
um it is possible because the cells
in anybody that have cells these are
called
totally potent cells the 2d potential
can generate
a complete new organism because it can
differentiate into
different um type of cells your heart
cell your kidney cell your brain cell
those are called toti patent cells if
you get that cell
and you can make a whole organism okay
from that one small cell
and so here is an example of a carrot
you cut the piece of carrot and put in
the small fragment
and allow the carrot to grow and attest
to you because this carrot cells are
totally cotton
cells it can grow into a root cell a
stem cell
um small one single cell and it's going
to grow
and make whole carrot and become other
plant right that's because
these cells are totally potent it can
express different
part of organisms and
in cloning what they do they take a cell
and they replace the the the
dna of embryo
and insert this cell from a
different part of the organism in in in
the sheep they took the other cell from
milk producing other cells
they took a cell and take another sheep
ovary and remove the nucleic acid remove
the chromosome completely
and insert this uh other cell into it
and put it back into another sheep and
that she produced
exactly a clone of the
parent cell where they took the other
cell from so
carbon copy of the of the gene of the
goat it produced okay
so because it's called 2d potent cells
and and the 2d potentials and just to
show
you the difference if you if you take
that cell
and it can make the whole organism but
if you take
a cell that is not totipotent um
it can if you take a cell that only
produce one type of
organ for in this case it this cell
taken from
tadpole from lungs it only produce lung
producing cells not the whole
organism so you need to have a 30
potential to make the whole organism
um so this is the story of how the
dolly was made in in scotland um
in 1997 researchers started
and produced this sheep so here's how it
it is done they take the other cell the
milk producing cell of a
um goat and um it took out the cell
isolate that one particular 20 potent
cells
and then you take a embryo from a donor
sheep and you remove the dna
of this sheep and you put the
other cell back into this embryo
from the donor cell and then he inserted
in
another surrogate mother and surrogate
mother now
without sperm from this 2d potent cell
produced a lab which is carbon copy
exact carbon copy of this land
genetically identical right so this is
how um genetic identical
clones are made okay this was a
in in done in 97 first organism to be
cloned now we have
clone almost a lot of different
organisms on the clone okay
so just to show the how they remove the
nucleic
acids and insert this back into the
south
and put it back into the sheep
and then the surrogate mother developed
that into
a normal sheep
okay and as i say this is the carbon
copy of
the way that other cells come from
now we clone the cats cloned dogs
we clone lot of organism cows all those
are cloned and it has some disadvantage
and but
people are every year they're overcoming
problems that had this for example dolly
died prematurely
after a few years um that's
because of some epigenetics there is
some factor that
killed that um animal but now they can
overcome that okay um so
stem cells so you can use the same
technology
to treat patients okay so you can you
can take
um if you have a defective gene you get
a stem cell from
um an aborted fetus or maybe uh
even uh you know embryonic stage
like two force um cells embryonic states
you can use the gene from that cell
to make a correct gene to to
make a patient with defective genie put
the correct gene in there that is
stem cells um in human and animal
research so
here's an example so you take embryonic
stem cells
embryonic stem cells are totally potent
cells so you can produce a lot of
different kind of cells you can make
liver cells nerve cells blood cells
okay if you have adult stem cells it can
only produce
where you took it from in this case you
took it from bone marrow you only
produce bone cells it doesn't produce
no sulfur it doesn't leave herself so
you take embryonic cells you culture
them on a plate
and then put them in different culture
conditions it turn into a liver cell and
also a blood cell
or if you take it from your own
body and adult cells wherever you take
liver cells
become liver uh you can make it a liver
cell
um if you get it from
bone marrow it become a blood cell so
you can you can take this gene that if
it is
um defective you take from another
person that has a normal gene you
introduce that gene in here
and then you put it back into the person
to correct defective
genes those are called gene therapy okay
so researchers are now transform skin
cells into embryonic cells so you don't
have to have embryo anymore
so you take your own skin cell and use
viruses
to manipulate the regulatory gene in the
stem cell
and then the skin cells become induced
pluripotent cell
called ips and exactly you can produce
whatever cell you want to produce
so this technology is now mainla
mainstream now
we treated some you know disease that um
that are normally not treated using the
patient's
own skin cells you can change into a
pluripotent cells
okay so here's an example you take a
skin cell
from a patient and you reprogram the
cells
and become ips which is induced chlorine
patent cell
and this treat treated ips cell is now
differentiated to whatever cell you want
whether you want a blood
type blood cell on a liver cell or
whatever cell
return the cell to the patient hopefully
the patient
will take that cell back most of the
time it does but sometimes there is a
rejection
and patient with the damaged heart
tissue now
you know the heart tissue repair itself
and recover from
it okay so this is called the new
technology called ips
which is induced pluripotent style from
your own skin cells
okay so you don't have to have another
patient it's taken from your own
cell and reprogram the cell
so this is all basically genetic
engineering so you're manipulating the
gene
in a test tube put it back into the in
vivo into the cell
and express the function of the gene
okay
so let's talk about many benefits of dna
technology in bioprocessing and a
different field
and there's a medical application as i
said you can do gene therapy
so you can find human gene that are
mutated
you can put you know genetic diseases
and put corrected gene in patients
um you can do uh you know
sequence the genome using pcr and the sn
snp which is i told you single
nucleotide
polymorphism and and to look for what
kind of disease your human has
and then you can look for the presence
of particular type of cancer
whether you're going to express in the
future you can do it
20 30 years before you develop a cancer
um so gene therapy is basically what i
just mentioned
you can alter the gene that uh
take the defective gene out or put a
current gene back using manipulation in
a test tube and put it back into your
bone marrow and um and you hopefully
that bone marrow will take up the gene
and
you're going to express the gene again
in your body and just to show how this
works in order to do that you we use
viruses to insert the gene back
and then that virus is inserted into the
bone marrow
every time the wire is replicated the
human gene is replicated
inside your body uh and then that is
taken up in the human cell
okay pharmaceutical product a
lot of um different um
cancer of protein monoclonal antibodies
are now
produced in various uh goat milk and cow
milk
we put the human gene into the
cows and the goats they express the milk
when you drink that milk and supposed to
cure some of the cancer this very common
treatment for
colon cancer people use that um
and then you have a lot of moly
synthetic molecule like i said the
the production of insulin is basically
genetic engineering technology
and then they use it that and the drug
imatin
name is a small molecule that inhibits
over expression of specific leukemia
causing receptor and this pharmaceutical
product are
uh basically protein can be synthesized
in a large scale
this is where chemical engineers come in
so the bio bio
biologists and molecular biologists
manipulate the gene and
give the gene back and you need to make
them in large scale
okay an industrial scale um
and protein production cell culture has
been done now
whole cell culture can be engineered to
secrete
protein and and simplify the tasks of
purifying it
right okay you can make the engineered
protein
um exactly what instead of
making a complex protein a regular cell
if you put exactly the same gene you
want you don't even have to purify the
protein
the bacteria are going to make the exact
protein that you want right
and all you need to do is scale up and
that's what
nowadays they do to make insulin is
become very very common
human growth hormone very common
and vaccine is now we are making in
large scale
that's because of chemical engineers and
scaling up this process
okay um
and and then the the way we express
human protein in animals is called
um transgenic animals are called farm
animals and here's an example of
um goat producing milk and expressing
human anticlonal
anti-cancer anticlonal monobody um
monoclonal antibody to to stop some
cancer
okay especially colon cancer
um the forensic
is now regularly a regular um
way to identify who who committed the
crime all you need a
piece of sample from a crime scene
that is because of the dna
fingerprinting i told you how it works
restriction fragment and polymorphism
um then we use the electrophoresis to
look at the short tandem repeat to
identify some
disease we can identify and then we can
do the environmental cleanup we put
correct
gene for making specific enzymes to
clean a particular contaminated site um
but um you cannot in the u.s you cannot
release the
uh genetically modified bacteria in the
environment you can do it in a control
reactor you can have it in a large scale
and treat the contaminated site
once it's treated you kill the bacteria
um so you don't release the genetically
modified bacteria into the environment
so this is uh in some cases people are
doing this
using genetically modified organisms
that's again
chemical engineering work and
environmental engineering work
in agriculture la almost all
crops in u.s are genetically modified
you know genetically modified cotton uh
vegetables everything is genetically
modified okay
in europe people are against most of
them
in u.s there's a lot of acceptance of
genetically modified product
okay and just to show you how it works
um you
you just same exactly same technique
for example this called bacteria called
bacillus turingiensis
it's called bt for short bacillus
transgensis is a
natural bacteria that make a toxic
protein that kill insect
okay so all you need is take that gene
from the bacteria from bacillus
and put it in a plant and the plant
going to express the toxin
and so the insect the pests that eat
that plant will die
so you don't have to apply any pesticide
and that is called
bt product basically three answers so
you have bt cotton
bt corn btv bd tomato so every crop
has bt genes in them now so you apply
less pesticide so basically you put that
gene
into the plant exactly same technique
endonucleases
ligases and put it back into the plant
and plant gonna have a nutrient
so the one of the common genetically
modified is the
bacterial gene called bacillus
thuringiensis gene um
so some ethical questions now raised by
dna
technology that's always there
government regulations you don't want to
create you know frankenstein monster so
you want to
have some control over this uh how we
are using
not going to be become harmful in the
future okay
uh so the genetically modified organism
is a big debate of using it for
food as i said there is uh
mostly restricted in european union but
in us
for some reason public acceptance is
there okay
uh let me talk about um some industrial
scale so
these are the product nowadays we make
with genetically modified
bacteria um antibiotics hormones vitamin
acids solvents enzymes and here is some
list
industrial product microorganism in
pharmaceutical
food industry and you know miscellaneous
product
by modifying genes like this is
commercially produced nowadays
uh here is the industrial products of
microorganism
enzymes you can produce specific enzymes
for specific functions so this is
application for amylase for what type of
function
cellulase for what type of function
proteases streptokinases
so this is now nowadays you know
commercial scale people are using it
um here's another uh using microscale
and numerous complex stages um this is
where the chemical engineers come and
do the scaling of bioprocessing and then
biofuels another
product we use genetically modified
organism
to produce you know biofuels from
lignocellulosic materials okay and just
to
finish up this part this this is my
research i'm going to talk about
later on in the class uh in
lecture four i just want to show you how
we did this so
so to make biofuel from um
uh sugarcane leaf liquor what we did was
we took an e coli and we put genes from
two different yeast
one is that can make ethanol from
glucose another east can make ethanol
from xylose
and put it in the e coli and then the
the e coli also makes have gene that
make um
other product like acetic acid and
other solvents we knock off the gene
like
knockoff experiment so we put the genes
into this e coli
and so just to show you what genes we
put in there
and then we eliminated a knockoff to
remove the mixed acid we wanted to only
produce ethanol
and just to show you what we did so
these are the genes we knocked off
so remove the genes from the e coli and
these are the genes we put in
and it expresses 95 percent of ethanol
it doesn't produce these products we
don't want
and it produces this so we have done
that and we uh
pilot scale we tested it out just to
show you results and
i'm going to talk about in uh fourth
lecture
uh so it's it's now degrading xylose
and it's degrading glucose and making
ethanol
and making more biomass so this
can make a theoretical yield of um
ethanol from using e-coli genetic
engineering
method okay and just a pilot plant run
we did that
just to show the reason so that is my
second part and then the last part will
be synthetic biology now i'll entertain
some questions
raj yeah uh things it might be the third
part
we may not have time oh okay
i will continue with before anaerobic
digestion yeah yeah yeah i think so
very short it's only 30 slides so yeah i
think so
there will be it might be uh in the
third
in the third class yeah class i will
start with this and then go to anaerobic
digestion
okay okay i'm sorry about this i had too
many slides i think yeah yeah
yeah okay i think so it might be
we don't have time for the question and
answer because
um probably the student will have to go
to the
next class okay classes so okay
i think so please you can turn off your
uh presentation file please
ah i think she's already did it yeah so
i think sir uh
i would like uh i'm for decision we
we don't have yet we don't have time for
the uh
question and answer right but i think
sir
uh this lecture is part
uh there will be uh another lecture and
then in
in the next next monday will be
dr himanshu raji yeah
talking about the introduction to
bioinformatics and then
the 5th of march 15th of sorry 15th of
march
uh there will be fundamental of
anaerobic but it might be
starting with the uh
yeah and then uh 22nd
of march you'll be celebrating ethanol
and microbiology especially you have
been mentioned
briefly uh right so that
that that one is so this one is
all the lectures and monday morning lake
here is part also
we now is celebrate the 80 years of the
chemical engineering
education indonesia so that the chemical
engineering
uh this year now we are celebrating
80 years 80 years wow great
so i think i would like uh to thank to
raj so the sony uh
next monday will be uh himanshuraji
i can be there with him too yeah okay in
case
i'd like to thank raj and also to all
the
the student the audience and then
friends oh that's that's my colleges is
uh susanna
luis was the the the alumni of
uh of chemical engineering so the
chemical engineering she is she's a
campaign as well
and also uh i think spariki is also
in the youtube so i would like to thank
to
to you all probably uh could you take
the
photo station before we are leaving you
thank you
yes okay please you mean
you could put your put on your video
please