prominant DNA technology has widespread applications in various Fields including medicine Agriculture and biotechnology the initial stage of recombinant Technology involves DNA preparation during this phase we select the gene of Interest ensuring it aligns with the production of our desired protein insulin for example our Gene of interest can be chemically synthesized but first we need to know its code sequence for this purpose today we utilize complimentary DNA libraries to collect the desired Gene DNA sequence but first let's delve into how these libraries are constructed the first step involves isolating the MRNA from the cell because most
mrnas have a polya tail at the three prime end a short aligo DT molecule is analed to this tail to serve as a primer for initiating DNA synthesis by the enzyme reverse transcriptase which uses the MRNA as a template to synthesize a complimentary DNA strand following that the DNA polymerase enzyme is employed to synthesize a second DNA strand the outcome is the formation of a double stranded cdna molecule subsequently sequencing methods are employed to determine the cdna sequence which is then incorporated into the libraries now the gene of Interest which codes for a specific protein
can be synthesized once we have our Gene of Interest the subsequent step in recombinant DNA technology is the preparation of a transfer Vector the vector is a DNA molecule utilized as a carrier to transport a specific DNA segment into a host cell as part of a cloning vectors can take various forms including plasmids cosmids viral vectors or artificial chromosomes the selection of a vector depends on the size of the insert DNA in the case of large DNA molecules artificial chromosomes can be used indeed plasmids are the most commonly used they are small circular DNA molecules
found in a variety of microorganisms notably bacteria following the preparation of the genetic material the subsequent step in the cloning procedure involves restriction enzyme digestion in this process DNA can be precisely cut at specific sites using specific enzymes numerous restriction enzymes have been identified from various bacterial strains restriction enzymes hydroly The Cove valent phospho bonds within the DNA some of these enzymes can cleave DNA at specific sites resulting in the creation of sticky ends While others can cleave DNA at specific sites producing blunt ends to initiate the Restriction enzyme digestion the solution containing our Gene
of interest is treated with the addition of the Restriction enzyme solution each restriction enzyme recognizes its specific sequence of nucleotides in double stranded DNA and cuts the DNA at a specific location similarly the plasmid Vector is treated with the same solution of restriction enzymes the most commonly used cloning vectors are eoli plasmas such as pu8 which Encompass three functional regions An Origin of replication that enables the plasmids to replicate independently of the bacterial cell cycle a drug resistance Gene such as the ampicilin resistance Gene and a region where DNA can be inserted such as the
laxi gene which contains multiple cloning sites also called a polylinker consisting of a short segment of DNA with numerous restriction sites each restriction enzyme recognizes its specific sequence of nucleotides within the multiple cloning sites of The Lax Z Gene after treatment with the restriction enzymes the next step in the cloning process is the ligation of DNA molecules for this purpose the solution containing our treated Gene is added into a new tube afterward the plasmid Vector solution is added to the gene solution for the ligation process DNA Lig enzymes are employed this enzy facilitates the joining
of DNA strands by catalyzing the formation of a phospho bond the formation of this Bond requires a co-actor typically ATP which is hydrolized to am for the recombinant process the LI enzyme solution is added to the sample containing the gene in the vector the gene is integrated into the plasmid and the insert DNA is physically attached to the plasmid backbone through the action of the LI enzyme The Next Step involves introducing the plasma into the host cell various transformation protocols exist and in this video we will focus on the chemical transformation method for the chemical
transformation calcium chloride can be employed in the process additionally competent cells such as ecoli serve as the host cells for the transformation a new tube is placed on Ice subsequently the calcium chloride solution is added to the tube followed by the addition of the competent cell solution finally the recombinant DNA is introduced into the tube then the reaction mixture is incubated for 10 minutes eoli is a gr negative bacterium its membrane comprises the inner membrane the periplasm and the outer membrane ecy carries a net negative surface charge this charge arises from the phosphate groups within
the phospholipid molecules additionally recombinant DNA carries a negative charge due to the presence of phosphate groups in the nucleotides because DNA carries a net negative charge and the bacterial cell surface is also negatively charged repulsive interactions occur between the plasma in the bacteria as calcium chloride is introduced into the mixture and calcium ions are positively charged they will bind to both the plasmids in the bacteria effectively neutralizing the negative charge this facilitates The Binding of DNA to the surface of the cell to perform the heat shock the tube is removed from the ice and immediately
incubated at 42° C for 90 seconds the sudden temperature shift induces thermal stress on the bacterial cells leading to the D stabilization of the cell membrane and the creation of temporary pores or gaps now the plasma DNA is able to Traverse the weakened membrane and enter the bacterial cell next the tube is placed back on ice for 2 minutes the cooling helps the cells recover and secures the plasmid inside the cell once the transformation steps are completed the tube is removed and a recovery medium enriched with amino acids vitamins and sugars is added to the
treated bacterial cells then the tube is incubated at 37° C for 1 hour the S so nutrient-rich environment is designed to create a conducive setting for cell recovery after bacterial transformation the next step in the cloning process is to cultivate the bacterial cells for bacterial cultivation an appropriate volume is transferred into a culture medium containing all the Essential Elements for optimal back bacterial growth then the liquid is evenly spread on the culture medium using a cell spreader following that the culture medium is incubated at 37° C overnight following the cloning process three potential outcomes emerge
firstly the bacteria may not incorporate the plasmid resulting in non-transformed bacterial cells the second outcome involves transformed bacterial cells with an unaltered Vector lastly the third possibility entails transformed bacterial cells with the recombinant vector the non-transformed bacterial cells will not survive in the culture medium this is because the medium is supplemented with ampicilin an antibiotic lethal to bacteria lacking the ampicilin resistance Gene consequently only transformed bacterial cells can survive because their plasmid carries the ampicilin resistance Gene which often codes for an enzyme called betalactamase this enzyme is capable of breaking down betal laum antibiotics including
ellin after the incubation and multiplication of bacterial cells The Next Step involves the selection and isolation of our transformed bacterial cells carrying the recombinant plasmid the unaltered vector signifies that The Lax Z Gene which typically codes for the beta Galactus at a enzyme can function normally in the recombinant vector where our Gene is inserted into the Z Gene the normal function of this Gene is disrupted leading to the inability to produce the beta Galactus at a enzyme in the culture medium xgal substrate is included for visual indication the beta galact Tod Ace enzyme initiates a
cleavage reaction on the xgal substrate releasing colorless Galactus and five bromo 4 chloro 3 indoxyl the indoxyl undergoes oxidation and dimerization forming an insoluble product that leads to the development of an intense blue precipitate so the transformed bacterial cells with unaltered vectors will manifest as blue colonies on the culture medium on the other hand the transformed bacterial cells with the recombinant vector which are not capable of producing beta Galactus at Ace will appear as white colonies next the white colonies representing transformed bacterial cells with the recombinant vector are isolated and transferred to an enriched culture
medium using an inoculation Loop following that the next step involves the growth of the recombinant DNA this can be done on a small scale typically in a laboratory setting the culture medium containing our transformed bacterial cells is used for generating additional cells that have the capability to produce or desired protein alternatively production on a large scale can be achieved using large vessels known as bioreactors these vessels are specifically designed for processing substantial volumes of cultures for optimal bacterial cell multiplication a bioreactor provides the optimal conditions necessary to achieve the desired product efficiently after producing the
desired protein the next step is its purification if the protein is produced inside the bacterial cells a step called cell disruption may be necessary initially the bacteria are harvested often achieved through a method likee centrifugation the centrifugal force causes the bacterial cells to pellet at the bottom of the tube effectively separating them from the liquid culture medium following this the supernatent is removed and cell disruption can be achieved through an enzymatic method using a lysis buffer cell disruption involves breaking open the bacterial cells to release the intracellular contents including our desired protein once the proteins
are exposed chromatography methods such as Affinity chromatography can be employed for the separation process a stationary phase pH is employed consisting of a specific support medium to which the protein will bind coal the solution of the protein mixture is directed through the stationary phase our desired protein will bind to the stationary phase While others will pass through next a wash step is implemented using a wash buffer the wash buffer is employed to remove any non specifically bound or impurity molecules leaving behind only the specifically bound Target protein after the wash step the specifically bound Target
protein is eluted from the stationary phase for this purpose a new tube is used to collect a solution then an ution solution is applied the uion buffer disrupts the specific binding interactions releasing the purified protein finally our desired protein is ready for use
