Hi friends, in this tutorial we are going to discuss about PPR322, the first widely
used vector in genetic engineering. The name PPR322 was named after its discoverer Toliver and Hortvig in the 1970s
And this is the plasmid PPR322 and we will be discussing all the elements of this particular
vector in detail. And this is the first widely used genetically engineered plasmid entry component in a technology
And these are the PPR322 elements. There is an origin of replication, that is from E. coli origin
And unique restriction files, there are at least 12 unique restriction files in PPR322
examples like BAMX1, SAL1, PDU1, PSU1, etc. And the third thing is the selectable marker
There are two selectable markers in the artificial interface and region and there are also a
selectable marker in the sub-interface and region containing the genes that will impart
resistance towards this antibiotics. And this vector is 4361 base pairs in length and is the most widely used vector in genetic engineering
Let's recall ORI. ORI is derived from E. coli plasmid, E. coli ORI
The function of ORI is whenever we have introduced this vector into a suitable host, ORI is a
sequence that is essentially required for replication of this particular vector inside
the host. Secondly, restriction on forms. There are at least 12 unique restriction files in PPR322, BAMX1, SAL1, PSU1, PDU1, and all
to think the selectable marker region. And this is the function. So we are, in order to integrate our desired gene into the vector, actually we are using
these restriction files. We are actually making a cut in this BAMX1 file or any other restriction file and introducing
our gene of interest in that file. So because of these restriction and signs, in order to integrate our gene of interest
into the vector, we need to make a cliff in the DNA segments and this is why the restriction
and signs are restriction files. The third major element that is the selectable marker, there are two marker regions
This is the ampicillin-resistant region that is in barbing resistance, that contains genes
that provide resistance to the host while growing in an ampicillin medium, and this
is tetraoxygen resistance, where also this will provide resistance against tetraoxygen in a tetraoxygen-containing medium
And this will help us in selecting transformed colonies from the rest
Once the transformation experiment is complete, we'll be getting three types of colonies
Firstly, non-transformed without a vector, and secondly, transformed with unaltered vector, vector has introduced into the bacterium, but without our gene of interest, third, transformed
with recombinant vector, the colonies that we need to select. And this is a tedious process as the number of colonies that we transformed with recombinant
vector will be always very low, so we need to select isolated piece from the rest
So these are the three types of colonies. Here comes the use of selectable markers
So this is the marker plate containing all the colonies. From this, you need to find out which of the colonies contains our gene of interest
A non-transformed cannot grow on ampicillin or tetraoxygen medium as that vector is not
introduced because the bacterium doesn't have the innate capability to grow in ampicillin
or tetraoxygen-containing medium, so it cannot grow in a medium where there is tetraoxygen
or ampicillin. And the second type, transformed with non-transformed vector, here this particular vector has both
ampicillin and tetraoxygen-resistant genes so that this particular vector containing colonies can grow on both ampicillin and tetraoxygen-containing medium
The third one that we need to select, that is transformed with recombinant vector
In the case of transformed with recombinant vector, it can grow in ampicillin medium based
on where we inserted the gene. In this case, in this example, you have inserted our gene of interest in tetraoxygen-resistant
region using pan-exon of the restriction site. So once the gene has indicated into that site that gene is no more functioning, this particular
gene cannot grow in tetraoxygen medium, it can grow only in ampicillin medium
The inactivity of the gene once our desired gene is inserted is also called as infertile inactivation
So that our transformed recombinant can grow only in ampicillin medium and cannot grow
in tetraoxygen medium due to infertile inactivation. So the selectable markers are actually helping us in picking out our desired colony of gene
of interest. Hope things are clear. You are with biologicsamsury.com. Thank you for watching