Transplastomic Plants and Molecular Farming: High-Yield Pharmaceutical Production

[Music] [Music] welcome to nptl online certification

course on pharmacognosy and metabolic engineering we have come to WS the end of this course so this lecture number 62

and 63 basically I have Focus to tell you something about molecular farming so lecture 62 is basically the uh creation

of or generation of transplastomic plants and molecular farming means you want to produce some pharmaceutically

important products in plants so normally I have mentioned all these things but here particularly Focus will be

either antibody or some hormones so in the next class we are going to talk about that but first I in this class we

will see that why transplastomic plants first of all what is transplastomic plants is basically when you transfer a

gene of Interest into plastid and if the gene is integrated with the plastid genome then uh it will be possible for

you to raise the transplastomic plants so Gene transfer to plastid and obviously what is the advantage why

should we transfer the gene into plastid then we'll see Gene construct selectable marker genes plasy transformation

vectors and then expression cassets so these are the concepts we will discuss in this course and in this Le in this

class and the next class we'll talk about the application of this knowledge okay so what we what you need

to do now is that nuclear genome and plastic genome will will compare so uh nuclear genome one important point is

that multiple insertions are common whereas in the plastic gor specific site by homologous

recombination this may have some advantage over the other and another point is that in case of transcription

it is basically each gene usually expressed individually so that means for each gene you have to put a separate

promoter and Terminator whereas the plastic genome is basically procaryotic type so it's it's

it's follows more or less opon concept so therefore a single promoter uh can control the expression

of multiple genes in the form of a operon so that is the advantage that means that multiple genes can be

expressed relatively easier way in the Plastics and uh the current limitation with the nuclear genome is that the

level of expression is unpredictable because where it will integrate it's a random integration whether you do um

Vector lless transfer or agrobacterium mediated transfer so if it integrated in transcriptional active size then the Gen

will Express if it is integrated in the heterochromatin region so it will not express so these problems will be there

and then there are other problems like gene silencing so that can also happen whereas the difficulty in the plastid uh

transformation is that obtaining homoplasmic so homoplasmic term I will explain little bit later homoplasmic

means in a brief that a plastid contains circular DNA so the number may vary from 20 to 100 so and ideally each and every

plastic circular DNA should contain your Gene of interest and if that is happen then homoplasmic will be achieved so

that is difficult to obtain and that that is the challenge okay uh and uh other there are some other advantages

with the plastic transformation which is basically maternal inheritance and all other

things now we compared the chloroplast and nuclear genetic engineering okay uh in in case of level of gene expression

the when you see about the level of gene expression so uh it's poly flid results in abundant trans gen transcripts and

high accumulation of foreign proteins why it is so because as I said that uh single plastid May contains from

2200 uh circular DNA and if each circular DNA receive the gene so then mean that means each plasted contains if

there is 100 circular DNA 100 copies of the genes and then a cell contains say around 50 plastid so 50 into 100 that

means uh 5,000 copies of genes so that means the level of expression will be so high and you cannot compare with that of

the nuclear transformation and other import interesting points I have mentioned that it is basically the genes

are arranged in operons so it is possible that polyconic RNA can be produced and multiple trans gen can be

expressed in a single event whereas in case of uh nuclear transformation it's basically monocistronic mRNA that mean

each individual genes to be controlled under individual promoter and Terminator and other important point is this that

position effect that means the plastid does usually the de combination happens through homologous recombination so and

that eliminates posit uh position effects so whereas in case of uh nuclear genome that is the problem now gene

silencing is not at all found in case of plastic transformation but whereas in case of nuclear transformation there is

a possibility of gene silencing another most important point is the maternal inheritance that means the the

plastic transformation is the transplastomic plants are maternal that means the poen a poen does not contain

the transgen so and that means that is a problem with the transgenic plants the

Poland Escape leading to the formation of super weeds so such problem can be alleviated if the transplastomic

approach can be used uh so that means in nuclear genome it is paternal inheritance other important point is

this when you transfer a gene into plastid and if the genes expressed in plastid then it will produce the target

protein and it has been found that Target protein once it produced it it it it will undergo post transational

modification uh such as D suide Bond formation and other folding leading to the formation of car bioactive proteins

so uh that is possible whereas in case of nuclear transformation you have to

Target that particular protein towards endoplasmic reticulum so that means a signal peptide sequence to be added and

you have to make sure that the protein one goes inside it should not come out so therefore uh a certain modification

needs to be made in the construct itself and uh and so so the trans Gene lines are

uniform gene expression so these are the lot of advantages of the nuclear Genome of the

chloroplast genetic engineering if you compare with the nuclear genetic engineering but there is one important

point to mention here that uh when you transfer a gene into the chloroplast so chloroplast lies within

the cell so it has a double membrane structure and then is there in the plant cell so

uh and then outside the plant cell is basically you have the cell wall the light portion is this

is this is the plasma membrane and uh this is Bayer chloroplast membrane and this is

cell wall so when you transfer a gene into plastid the gene

should be able to enter through the cell wall and then it will also pass through the membrane then

it will pass through the cytosol and eventually it will enter into the plastid so that means a lot of energy is

required and therefore the particle bombardment method is usually used to transfer the Gen of Interest into

plastid now point is this that as I said that this plastid contains uh circular DNA for the shake of

Simplicity I put only one and this circular DNA will receive the gene so here is basically the gene so it is

coming so maybe here and here it will get it but so but in this case this is basically the case

of heteroplasmy this is the case of heteroplasmy now homoplasmy will be

achieved when all the

circular DNA of the plastid contains your Gene of Interest

so then this will be homoplasmic so achieving homoplasmy will be a challenge and if homoplasmy is

achieved then the level of expression will be very high so uh so next question is that how homoplasmy is a achieved so

how hoplas me is achieved so this is basically achieved through the use of selectable marker Gene so what is the

selectable marker gen so the selectable marker Gene mostly used is a

a so a a stands for uh Amino glycosite three

prime adino adile adile sorry adile

transfer is so so this is basically the uh

[Music] adile specto

mine so normally this pomy is active but this spyine is basically

inactive that means if you grow the tissue in presence of spectrom so spectrom myin is basically will bleach

the cells so that means it musically bleach all the plastic So eventually the tissue will die

whereas uh if you uh use the plant which contains the a gene so a gene upon expression

will make a which is amino adoy 3 uh uh adile transfar so that upon expression will inactivate spectrom by

transferring into Adin and spectrom and that is how the plant Will Survive so uh so the transplastomic plant should show

resistance to this aminoglycoside type of antibiotics so how it is being done so I will show it in a uh another

schematic diagram but say for example this is the plastid which contains uh several uh

circular DNA and uh it receives the gene one or two so this is of course in a tissue and that that tissue is will be

incubated in a medium and that Medium contains uh a AA as antib IC so the selection

medium containing then what will happen here in the next case

that only these two Will Survive whereas this all will

degrade so in the next phase what will happen we'll continue to do this selection so I'll put it

here so you'll be able to achieve a condition where these two these two pled when the

cells started dividing so these two circular DNA also replicates so as a result of

that it makes two so now this survived and it contains

sorry so this contains this Gene of Interest so

this and the next round what is going to happen so you continue to give a a selection so a a selection means you

have to give the uh you have to grow with the spectrom so better we write spectrom here so

spcy spcy spectrom myin So eventually what will happen

you'll be able to achieve a plased where all this

six circular dnas will be produced by the subsequent cell division and all

this will have the Gen of Interest here so this

is so this condition first this condition is called

heteroplasmy p a s m y p l a s sorry p l a s m y

[Music] p l a s m y so this is heteroplasmy this

is partial homoplasmy okay and then this

is full homoplasmic when it is full homoplasmic

then you will have your desired genes also if you if you made the construct in such a way so the construct contains the

antibiotic resistance Gene along with the gene of Interest so all the circular DNA of a single plased contains the gene

of Interest along with the antibiotic selectable marker and in that case all the plased within the cells will get the

Gene and all the tissue will get all the plastids present in all the cells will get the gene so you you think that the

level of expression will be very high so this is how the uh homop plasm is achieved now here what is what I'm

showing here that's basically telling that is a maternal inheritance and and uh so therefore the the trait will be

restricted with the seeds so poen will uh will not contain any uh Gene of interest because the

disintegrated paternal plastids so only the maternal plasters will contain the gene of interest and uh and here what is

showing here that the multiple gene expression it is possible using the transplastomic plants and that leading

to the formation of biopharmaceuticals apart from the transgenic plant stress resistance so

now uh what we are going to see that uh this is the method of Gene delivery so as I said that it is biolistic Gene

transfer so the it requires high energy so that the the DNA that which contains the gene of Interest along with the

antibiotic resistance marker gen should be able to uh go through the cell wall cell membrane and ultimately it enters

into the chloroplast by piercing the chloroplast double membrane and then so it is again it will be coated with

either gold or stung Sten and that will enter into the plastid and eventually uh depends on the homologous regions which

is there in the construct and the similar homologous region there in the plastid so there will be homologous

recombination so now uh let us consider here this point that is the single Gene so single Gene normally what is to be

done that promoter is there and then you have the marker Mar marker means it is the Atri distance marker and the

Terminator then you have again the promoter and X is of gen of interest and then the Terminator so this is how the

single genes are transferred but multiple Gene can be transferred under the control of a single promoter

uh but then and the genes can stack together but in between you have to put a ribosome binding site and then the

construct is to be made in this way and uh then the whole construct can be transferred successfully into the plased

genome and the single promoter here can take care of the expression of the entire

operon okay uh now uh uh what I have mentioned the aada a is basically antibiotic so but there is a so it's a

negative selection marker but there is a positive selection marker where Bain alide dehydrogen is can be used so where

the if the betan alide dehydrogen is used so that will basically uh degrade the toxic betan alide normally ban alide

is toxic to the plant tissue but if bettin alide dehydrogen is is there so that will convert ban alide dehydrogen

will convert toxic ban alide to to a non-toxic

product which is glycin Bain and that is utilized by the plant so point is this so here this is a

positive selection marker so the glycin Bain is utilized by the plant for its own nutrition and balide diogenous can

be used as a positive selection marker whereas a is basically a negative selection marker now what is showing

here is uh this one is the heteroplasmic deployed Okay so so uh what you see that uh when

the genes are transferred if you notice out of these there are two different colors so this this this this so

This four or five so these Plastics are different from the other that means this has received the gene so now what has

happened that through this uh selection of antibiotic resistance marker eventually when you notice here all

these contains these genes so this is a homoplasmic achieved so how it is achieved I have explained in the

previous slide and next is this is uh this is another pictorial diagram so this is how the uh

foreign the tungsten or gold particle containing the gene of interest is inserted and here you see that it is

only one plastid one DNA of one plastid receive the gene so that is primary plased

transformation event then through round of selection round of antibiotic selection what you

see that this is a heteroplasmy why heteroplasmy if you notice carefully that out of four circular DNA only two

have the here out of four three have the things here out of four four achieved but here out of four only one here out

of four only two so it is definitely not a homoplasmy but heteroplasmy then further antibiotic selection eventually

if you see all these contents uh G so this is full homoplasmy

achieved okay so this is the beauty of plastic transformation and now we'll talk about

the vectors so normally what happens that I said is a homologous recombination so there is a region

homology here uh uh so this is the plastic DNA which is this uh left and right targeting regions so these are

sometimes this inverted repeats are also there so and when the vector has to be constructed one has to take care that

there should be a similar sequence to be put here in the vector and another similar sequence of the other to be put

here so the left targeting region and right targeting region the similar sequence to be put in the vector where

you have the marker and the gene of Interest so that means where you have the marker and the gene of Interest so

which is clearly showing here and then uh so this so this is basically the DNA which is coated with t strin or gold

particle and this will be bombarded into the cell and eventually it enters into the pl it and then a situation will come

these two will meet and as a result of that there will be homologous Rec combination so as a result of homologous

recombination what you will see that these two will fuse and form a single circular DNA where Gene of interest and

marker both are there okay so uh and then uh transform plastic DNA Mark Gene can be removed by uh subsequent

selection there are different techniques for removal so that I am not going to discuss okay now so this is what I have

said is the process so first you bombard and then

selections selection finally regeneration then additional round of selection make sure you have it achieved

homoplasmy and then you check for the traits or the products so this is basically a plastic transformation

Vector so where these are the regions of homology A and B so are the flanking regions and here it

contains a is the marker gen along with a promoter and the Terminator so uh untranslated region uh so it contains a

Genus to the plasmid promoter and sign dardo sequence this is sign dardo sequence and uh and a

plased unrated region UT which is red okay uh and then homologous recombination and this is the plastic

DNA and this is the vector DNA so upon homologous recombination so this aadaa will will be inserted or integrated

within the plastic DNA and so basically it happens by physical linking to the ad gen by by the flanking region and then

eventually you will be able to have this a in all the circular DNA of the single plastid and eventually in all the uh

plastic DNA present in the cell so the transgenic casset which need to be constructed should consist of a promoter

sign alard sequence one then you should have the coding region one then again sign

algo sequence two and then you have the coding region to UT like that way the

Gen to be constructed so here what is what I'm showing this is typically a vector so here the r

[Music] [Music] so a PL casset includs a

promoter uh five Prime unrated region five Prime UT and or B a five Prime translation

control Region 5 Prime TC which means P Prime UT plus a segment of the coding region so that is the difference so

there are basically way uh the constructs can be made so that you can get a better expression so that is just

for your information and then these are the different ways transformation can be done okay one is that suppose if you

want to uh do a insertional mutagenesis so that can be done where will what will happen the existing Gene will be

disrupted okay in this way and the other one is that if you want to put the gene of

interest in the plastid a new Gene by by replacing the existing Gene that is also possible uh or yeah okay and so or a

point mutation can be introduced or simply you put the gene which is not there in the plastid so

that can also be uh integrated and so that the plast pled Gene DNA will receive this so this will be more clear

here so here what you see that left flanking region right flanking region and this is the plastic DNA this is the

vector so Gene of interest is here under the control of a promoter Terminator and a is here and

then that is inserted and eventually you'll get the whole thing here so and then what happen now the

difference between the left and right part is that here uh the one single plased is here and then one single

Terminator is here but both the genes are joined with the sign dardo sequence and interpal element so that this

plastid will do the this promoter will basically transcribe both the genes so that leads to polyconic mRNA uh and then

eventually there will be cage and that makes two separate mRNA and this is uh at the last that if you

want to have optimize if you want to optimize plastic transgen expression in non-rain tissue so that is possible by

doing certain Fusion so one is that that if the RNA extracted from the uh green tissue as well as from the

ripen tissue and then what will happen that this transcriptor miix leads to the giving the transcriptional

signals so which promoters and on which promoters which is active in the chromoplast so that promoter to be

inserted here and along with that the polysome can be isolated and then that can be translated so translatomics can

be added from there translation P Prim utria signals can be isolated and that can be fused here so as a result of that

chimic expression element one coming from the unripened tissue one coming from the ripen tissue and that to be

fused and and that will be put in the Upstream of the gene of interest and that leads to eventually more efficient

expression of the genes into the plastid because of the shortage of time and because uh I am not supposed to discuss

about these techniques in this class because our image basically look on the pharmacognosy and met and the molecular

farming so with briefly uh by introducing this I will end this class so by this uh you have develop some

understanding that how genes are transferred into the plastid and what are the advantages if genes are

transferred into the plastid so that means that there will be huge level of expression and there are lot of

advantages that is maternal inheritance know no chances of poen escape the level of expression will be high and there

will be no gene silencing you can and we have the predicted expression so this this all speaks positive uh or or

advantageous over the nuclear transformation techniques so in the next class which will be the last class of

this course we will talk about molecular farming where uh human somatotropin gene will be expressed in tobacco using this

transplastomic techniques with this I end this class thank you