DRAFT: This module has unpublished changes.


Recombinant DNA technology

      Have you ever wondered how scientists create genetically modified organisms or splice genes or create antibiotic resistant bacteria? If so, this is the class for you. In this class, we created bacteria colonies that fluoresce under UV light and used a polymerase chain reaction thermocycler. Some of my lab reports are posted below. 


Henock Tsegaye
Dr. Bettica
         The experiment was designed to ligate BamHI/Hindlll digested pAMP and pKAN fragments using T4 DNA ligase and form a recombinant pAK plasmid that encodes genes for both ampcillin and kanamycin resistance. Subsequently, the ligated plasmid, including pAMP and pKAN controls, were transformed into MM294 Escherichia coli bacterial cells. The transformed colonies were then streaked onto plates labeled LB/AMP, LB/KAN and LB/AMP+KAN. After replica plating these master plates, it was identified that 4 colonies had dual resistance and therefore were able to thrive on both LB/AMP and LB/KAN plates. The dual antibiotic resistant colonies are phenotypic indicators suggesting that a ligated plasmid was in fact produced and successfully transformed.

       PAMP and pKAN are short plasmids which encode for ampicillin and kanamycin resistance genes, respectively. Like other antibiotic resistance plasmid, pAMP and pKAN plasmids have numerous applications in many biological techniques. For instance, they can serve as phenotypic markers for identifying bacterial colonies that have successfully taken up a certain gene of interest. However, sometimes it is necessary to utilize a plasmid which encodes for two or more antibiotic resistance genes. In those situations, a recombinant plasmid can be synthesized.
             DNA is structurally the same in all living organism, thus, DNA sequences from multiple sources can be enzymetically ligated to form a recombinant or chimeric DNA. Similarly, a recombinant or chimeric plasmid, pAK, can be synthesized by ligating pAMP and pKAN fragments, hence creating a plasmid with dual antibiotic resistance genes with a single origin of replication. When digesting pAMP and pKAN plasmids with restriction enzymes, BamH1 and HindIII, two fragments of each plasmids will be formed since each plasmid has a single recognition site for each enzyme. The restriction cleavage of pAMP will result in one short fragment with 784 base pairs and a larger fragment of 2332 base pairs containing the origin of replication and the ampicillin resistance gene. Correspondingly, the cleavage of pKAN yields fragments of 1861bp and 2332bp; the kanamycin resistance gene is located on the 1861bp fragment. Using T4 DNA ligase, it is then ligated with the 3755 bp AMP fragment to form a 5616 bp recombinant plasmid.
             Classical transformation procedures can then be applied to insert the pAK ligated plasmid into MM294 Escherichia coli cells. Classical transformation is the preferred technique over rapid colony protocol because it has enhanced efficiency in transforming ligated DNA, which produces 5-100 times fewer transformants. Replica plating the transformed cells on LB/AMP, LB/KAN and LB/AMP+KAN media can be used to identify the bacterial colonies that possess dual resistance.


A. Restriction digest and ligation of plasmids pAMP and pKAN
             2 micro liters of BamHl/Hindlll restriction enzymes were obtained and added to tubes containing pAMP and pKAN plasmid DNA together with 7.5microliters of 2x restriction buffer. The reagents were then mixed by pulsing in microfuge and placed in 370C water bath for 30 minutes. The digested pAMP and pKAN were incubated in a 650C water bath for 10 minutes. 3ml of digested pAMP, 3ml of digested pKAN, 10 ml of 2x ligation buffer/ATP, 3 ml water and 1ml T4 DNA Ligase were transferred using fresh tips each time and mixed inside a tube labled LIG for ligation. The mixture was agitated and left for 2 hours for the reaction to take place.( Here three possible ligation products might materialize. The combination of 784bp and 3755 bp to regenerates pAMP, the combination of 1861 bp and 2332bp regenerates pKAN or the ligation of 1861bp and 3755 bp will produce a simple recombinant plasmid.)
B. Transformation of E.Coli with recombinant DNA
              Two 15 ml tubes each with 10 ml of mid-log cells were obtained. Once the tubes were centrifuged for 10 minutes, the supernatant was poured off. 5-10 ml of ice-cold CaCl2 was then added into each culture tube. The cells were immediately suspended by tapping. After complete cell suspension was attained, the tube were returned to ice for 20 minutes. Once again, the tubes were centrifuged and the supernatant was poured off without disturbing the cell pellet. Following that, three sterile 15 ml culture tubes were labeled +pLIG, + pAMP and +pKAN. + pAMP and +pKAN were used as control.
200ml of competent cells were then added into each tube which were placed on ice. Using a fresh tip each time, 10ml of 0.005mg /ml of pAMP, pKAN and pAMP/pKAN solution were pipetted into their respective tubes. Following a 20 minute incubation on ice, the cells were exposed to a heat-shock (420C for 90sec then returned to 00C). 800ml of LB broth was pipetted into each tube and the cells were allowed to recover by incubating all three at 370C water bath. Three plates labeled LB/AMP, LB/KAN and LB/AMP+KAN were prepared. Using a micropipettor 100ml of cell suspension from the tube labeled pAMP was added onto the three plates. The same procedure was repeated for the remaining tubes. The cell suspensions were then spread on the plates using a spreader, sterilizing it each time. Finally, the plates were stored in a 370C incubator for 15-20 hours.
C. Replica plating to identify mixed E.coli populations
        To prepare the replica plates, the replica plating gride was attached to the bottom of an LB/AMP and LB/KAN plate. Following that, a sample of cells from one colony on the LB/Amp plate were replica plated onto the fresh LB/AMP and LB/KAN plate by first using a sterile toothpick to scrape up a cell mass from a colony on the original LB/amp plate. Immediately, the same toothpick was used to make a short streak on square 1 of the LB/amp plate and the square 1 of the LB/KAN plate. Fresh tips were used to repeat this procedure for the remaining squares and the replica plates were incubated at 370C for 15-20 hours.
D. Gel Electrophorasis
        5ml of pAMP, PKAN and pAK plasmids that were set aside for genotopic analysis using gel electrophoresis were added into 1.5 ml tubes and mixed with 1ml of loading dye. Using a micropipettor, the entire contents of the tubes were transferred into the wells of a pre-prepared gel. The electrophoresis was then carried out at 100-150 volts for approximately 2 hours, and only stopped when adequate separation of the bromophenol blue and the cresol red were observed. The gel was then placed under a UV transillluminator and photographed using a digital camera. The picture was then examined for bands.
          In this experiment, close examination of the electrophorasis gels showed the predicted digested pAMP and pKAN fragments. In addition to that, the replica plates showed that 4 bacterial colonies had dual resistance and therefore were able to grow on the LB/AMP+KAN plate. The colony that flourished on a particular square of the replica pAMP plate was compared with the same square on the corresponding pKAN plate. The colonies that managed to grow on both plates in the same squares were selected because they had dual resistance as opposed to colonies that only showed growth on one or the other plate ( the plasmids that transformed did not manage to form a ligation probably because the sticky ends of the relaxed or nicked plasmid have a tendency to reattach). Therefore, it can be said that the ligation of the ampicillin and kanamycin resistance genes was successful and a few E.coli bacterial cells were able to take up the recombinant plasmid. Furthermore, growth was also observed in the LB/AMP and LB/KAN control plates, indicating that the pAMP and pKAN plasmids are in fact functional.
             In conclusion, the recombination of BamHl/ Hindlll digested pAMP and pKAN using DNA ligase produced ligated pAK plasmid which was transformed into MM294 E. coli bacterial cells. The replica plating of these transformed bacterial colonies revealed dual resistance in some colonies which indicates that some of the fragments managed to form a recombinant pAK plasmid which was later successfully transformed and expressed in those few colonies that thrived in both ampicillin and kananmycin media .




DRAFT: This module has unpublished changes.