<h4 id="successbiotechnology---principle-and-processes-success"><Success>Biotechnology - principle and Processes </Success></h4> <h3 id="successpart-3success"><Success>Part 3</Success></h3> <div style='float: left; width:100%;'> </div> <div style='float: right; width:0%;'> <p><img alt="image" src="https://temp-public-img-folder.s3.ap-south-1.amazonaws.com/sathee.prutor.images/subject-images/iitpal/image/biology-class-12-unit-11-chapter-03-biotechnology-principles-and-processes-lecture-3_6-5q3uluerdik-1.jpg"></p> </div>

<h3 id="successgenetic-engineering-success"><Success>Genetic engineering </Success></h3> <div style='float: left; width:50%;font-size:25px;text-align:left'> <ul> <li> <p>Tools</p> <ul> <li>Polymerases</li> <li>Restriction Enzymes</li> <li>Ligase</li> <li>Vectors</li> </ul> </li> <li> <p>Procedures</p> <ul> <li>Isolation of Genomic DNA</li> <li>Polymerase chain reactions</li> <li>Restriction Digestion</li> <li>Ligation</li> <li>Competent cells preparation</li> <li>Transformation</li> <li>Screening of Clones</li> </ul> </li> </ul> </div> <div style='float: right; width:50%;'> <!----> <img src="https://imagedelivery.net/YfdZ0yYuJi8R0IouXWrMsA/f9b00594-f5a4-4e53-5778-6f2e06671500/public" width="60%"> </div>

<h3 id="successligasesuccess"><Success>Ligase</Success></h3> <div style='float: left; width:100%; font-size:30px; text-align:left;'> <p><strong>Ligase</strong> Joining two DNA to generate the chimeric DNA is the basis of cloning It is an essential steps to generate clone containing foreign DNA in a vector When cohesive end generated by the action of restriction endonuclease on DNA associate with each other, a nick remained to seal and give complete circular DNA</p> </div> <div style='float: right; width:0%;'> <p><img alt="image" src="https://temp-public-img-folder.s3.ap-south-1.amazonaws.com/sathee.prutor.images/subject-images/iitpal/image/biology-class-12-unit-11-chapter-03-biotechnology-principles-and-processes-lecture-3_6-5q3uluerdik-3.jpg"></p> </div>

<h3 id="successligasesuccess-1"><Success>Ligase</Success></h3> <div style='float: left; width:100%; font-size:30px; text-align:left;'> <p><strong>What DNA ligase is doing?</strong></p> <p>It is an enzyme requires ATP or NAD+ as a cofactor to catalyze ligation reaction. Ligase is processing ATP to generate AMP, and then AMP is making adduct with enzyme to form ligase-AMP complex This complex is binding to the 3’ and 5’ of DNA bearing nick and bringing them together AMP is released and phosphodieaster linkage is formed between 3’ and 5’ end to seal the nick</p> </div> <div style='float: right; width:0%;'> <p><img alt="image" src="https://temp-public-img-folder.s3.ap-south-1.amazonaws.com/sathee.prutor.images/subject-images/iitpal/image/biology-class-12-unit-11-chapter-03-biotechnology-principles-and-processes-lecture-3_6-5q3uluerdik-3.jpg"></p> </div>

<h3 id="successligasesuccess-2"><Success>Ligase</Success></h3> <div style='float: left; width:0%; font-size:30px text-align:left;'> </div> <div style='float: right; width:100%;'> <!----> <img src="https://imagedelivery.net/YfdZ0yYuJi8R0IouXWrMsA/7fb1a0a4-9a92-4a7c-46b5-53bcf05e2f00/public" width="60%"> </div>

<h3 id="successvectorssuccess"><Success>Vectors</Success></h3> <div style='float: left; width:100%;font-size:30px; text-align:left;'> <p><strong>The vector DNA has a two main responsibility</strong></p> <ul> <li> <p>Ability to carry foreign DNA</p> </li> <li> <p>Ability to replicate in the host Hence to fulfil these responsibility, a number of properties are desirable</p> </li> </ul> <p><strong>Cloning Vector</strong> For cloning of DNA</p> <p><strong>Expression Vector</strong> For production of protein from gene</p> </div> <div style='float: right; width:0%;margin-right:270px;'> <p><img alt="image" src="https://temp-public-img-folder.s3.ap-south-1.amazonaws.com/sathee.prutor.images/subject-images/iitpal/image/biology-class-12-unit-11-chapter-03-biotechnology-principles-and-processes-lecture-3_6-5q3uluerdik-6.jpg"></p> </div>

<h3 id="successcriterion-of-good-vectorssuccess"><Success>Criterion of good vectors</Success></h3> <div style='float: left; width:100%; font-size:30px; text-align:left;'> <p><strong>1. Low molecular weight</strong> The low molecular weight or size confers a number of advantages (1) small size vector is robust towards shear stress and easier to handle In addition, after ligating foreign DNA into the vector, the size of the resulting recombinant DNA will be small and it will be easier to deliver the recombinant DNA into the host cell</p> <p><strong>2. Post entry into the host should give phenyotypic changes</strong> Another important feature is that vector DNA should give additional phenotypic changes in the host cell so that recognition of transformed cells will be easier</p> </div> <div style='float: right; width:0%;'> <p><img alt="image" src="https://temp-public-img-folder.s3.ap-south-1.amazonaws.com/sathee.prutor.images/subject-images/iitpal/image/biology-class-12-unit-11-chapter-03-biotechnology-principles-and-processes-lecture-3_6-5q3uluerdik-7.jpg"></p> </div>

<div> <h3 id="successcriterion-of-good-vectorssuccess-1"><Success>Criterion of good vectors</Success></h3> </div> <div style='float: left; width:100%;font-size:30px;text-align:left;'> <p><strong>3. Muliple cloning site with unique restriction site</strong> A short stretch of DNA on vector DNA containing restriction site for possible site for insertion of foreign DNA is desirable</p> <p><strong>4. High copy number</strong> A high copy number is desirable as it gives high amount of DNA after growing host cells</p> </div>

<h3 id="successcomponents-of-vector-success"><Success>Components of vector </Success></h3> <div style='float: left; width:100%;font-size:30px; text-align:left;'> <p><strong>There are minimum molecular components to assemble bacterial plasmid to perform the function of vector are as follows</strong></p> <p><strong>1. Origin of replication</strong> Like any other replicating DNA, plasmid DNA needs its own independent origin of replication to provide replication start site to make more copies It decides the range of bacterial host strain can be use with the particular plasmid vector The plasmids containing ori region from Col El can be able to grow in limited bacterial species such as E coli etc In contrast, plasmid containing ori from RP4 or RSF1010 can be able to grow in gram (-) bacteria and gram (+) bacteria</p> <p><strong>2. Selection marker</strong> Selection marker in the form of either antibiotic resistance gene or enzymatic gene is essential to give phenotypic changes in host after entry of the plasmid</p> </div> <div style='float: right; width:0%;'> <p><img alt="image" src="https://temp-public-img-folder.s3.ap-south-1.amazonaws.com/sathee.prutor.images/subject-images/iitpal/image/biology-class-12-unit-11-chapter-03-biotechnology-principles-and-processes-lecture-3_6-5q3uluerdik-9.jpg"></p> </div>

<div> <h3 id="successcomponents-of-vectorsuccess"><Success>Components of vector</Success></h3> </div> <div style='float: left; width:100%;font-size:30px;text-align:left;'> <p><strong>3. Promoter</strong> Plasmid replication in host is performed by the host provided proteins such as DNA gyrase, helicase, polymerase and DNA ligase But proteins required for conferring antibiotic resistance or enzyme use for selecting transformed host cells is present on plasmid and a promoter adjacent is required to expressSetivate Windows genes present on plasmid DNA In addition, promoter is also needed to express gene present on foreign to Semmings to anthate Windows</p> </div>

<h3 id="successdifferent-vectors-success"><Success>Different Vectors </Success></h3> <div style='float: left; width:100%;font-size:30px; text-align:left;'> <p><strong>Different vectors</strong> As vector needs to replicate in different host strain, vector needs special additional structural features to make it suitable for a particular host strain Why one vector doesn’t replicate in different host strains? Replication of vector DNA is controlled by the origin of replication and it need to be recognized by the host factor especially DNA polymerase to perform replication Consequently, there are different types of vector DNA to suits the cloning of a foreign DNA in a particular host strain</p> </div> <div style='float: right; width:0%;'> <p><img alt="image" src="https://temp-public-img-folder.s3.ap-south-1.amazonaws.com/sathee.prutor.images/subject-images/iitpal/image/biology-class-12-unit-11-chapter-03-biotechnology-principles-and-processes-lecture-3_6-5q3uluerdik-9.jpg"></p> </div>

<div> <h3 id="successdifferent-vectorssuccess"><Success>Different Vectors</Success></h3> </div> <div style='float: left; width:100%;font-size:30px;text-align:left;'> <ul> <li>Bacterial plasmid</li> <li>Phage based vectors</li> <li>Yeast vectors</li> <li>Mammalian vectors</li> </ul> </div>

<h3 id="successpbr-322success"><Success>PBR 322</Success></h3> <div style='float: left; width:100%;'> <img src="https://imagedelivery.net/YfdZ0yYuJi8R0IouXWrMsA/79e5757d-1705-4ce8-7995-70142abfd000/public" width="60%"> </div> <div style='float: right; width:0%;'> <!----> <!----> </div>

<h3 id="successthank-yousuccess"><Success>Thank you</Success></h3> <div style='float: left; width:50%;'> </div> <div style='float: right; width:50%;margin-right:270px;'> <!----> </div>