The Processes of Bacterial Transformation - Lab Report Example

Lab Report The basis of this report is to show the processes of bacterial transformation. This follows systematic steps starting with the introduction of the plasmid lux and control plasmid (pUC18) into the Escherichia coli (E.coli). This takes four basic procedures beginning with the placement of calcium chloride solution and E.coli tube in the ice bath. Transfer of 630 ul cacium chloride solution to the tube containing 50ul of bacteria using a sterile pipet and mixing the solution. This is followed by incubating the cells for at least ten minutes on ice. The above procedure is appropriate for this experiment since the E.coli provides a host into which the foreign DNA can be inserted, means of delivering the DNA into the host cells and way of identifying and selecting the transformed cells. The experiment exhibited a colonial growth in the E.coli seen in two large patches each patch holding hundred individual colonies. Introduction The main aim of the prevailing lab experiment is to insert the genes in order to make E.coli resistant to the corresponding ampicillin. Genetic transformation is the active uptake of the free DNA by the existing bacteria cell coupled with the incorporation of the underlying genetic information. The blueprint of life is normally found within DNA. Nevertheless, life cannot exist without supporting molecules DNA is transcribed into the RNA making it easier for the blueprint within the DNA to be read by the translational machinery through conversion of the blueprint message into proteins, the language of the cell. The protein is expressed in a cell that determines the physical and biochemical, structural properties and operation of the cell. The underlying molecular machinery plays fundamental rolein transfering information from DNA to RNA in order to enable the protein to fundamental perform the function of life in the cell. The cellular pathway is normally the initial step for the synthesis of RNA molecule from corresponding DNA templat via transcription. With the nucleus, a mature RNA can be translated into the polypeptide sequence through translation. Moreover, the pathway is commonly developed with certain modification within the eukaryotes and prokaryotes. The process is regulated by the cell, but it is also susceptible to manipulation by researchers particularly prokaryotes. Besides nuclear DNA, there exists the supplementary form of DNA such as plasmids. Plasmids are small, circular DNA molecules that exist apart from the chromosomes within most bacterial species of the nucleoid region. Nevertheless, Plasmids are not significance for the survival of the host bacteria. Plasmids normally give extra advantage to the survival of the bacteria they are placed in the particular environment. Moreover, plasmids are capable of carrying genes that when expressed aids in the survival of bacteria. A bacteria cell containing plasmid can live and multiply in the presence of the antibiotic drug. Thus, antibiotic-resistant Escherichia is segregated into numerous sections containing genetic information products, which is capable of interfering with the action of several diverse antibiotics. The laboratory experiment aids in introducing a plasmid containing an ampicillin resistance gene into E.coli. Plasmids were introduced into the living bacteria cells through the transformation process. The bacteria acquire the ability to take in plasmid DNA molecules when placed in a solution of the calcium chloride thereby escalating the cell competency. Cell competency is the capability of the cell to pick up a plasmid. The procedure offers a way of preparing massive plasmid DNA molecule. Due to the development of the bacteria in the existence of the antibiotic, the underlying plasmid DNA is readily segregated from the corresponding bacterial culture. Hypothesis The hypothesis of the lab experiment is that E.coli having plasmid will survive on the dish full of ampicillin and no ampicillin. The second hypothesis is that the E.coli that does not possesses plasmid will not survive within a petre dish devoid of the ampicillin and will also succumb in the dish full of ampicillin. Materials and method Materials: Starter plate containing starting bacteria, 3 micro test tubes, ice bath, foam tube rack, 630μL CaCl2,sterile pipets and loops, stopwatch, 275μL LB nutrient broth, rack on the ice, 3 poured agar plates and preheated water bath at 37oC. Method: Aseptic technique General procedure Summary In the laboratory experiment, plasmid lux and a control plasmid(pUCl8) was introduced into the E.Ccoli by transformation. The process of introducing E.colie took four fundamental steps to the procedure. The bacteria cells were treated with the CaCl2 solution in the bid to enhance the uptake of the plasmid DN. Competent cells were incubated with plasmid DNA. The cells, which have taken plasmid DNA by growth on an ampicillin-containing medium, were selected and were examined in the dark. Procedure A: Preparation of competent cells A vial of CaCl2 solution and tube of E.coli were place in the ice bath and 630μL CaCl2 solution was transferred to the tube containing 50μL of the bacteria utilizing a sterile pipet. The tube was tapped with finger index to ensure proper mixing. The cell was incubated for 10 minutes on ice to make then competent of up taking DNA from the medium. Procedure B: Uptake of DNA by competent cells One small Eppendorf tibe was labeled C for control plasmid DNA and the other tube was labeled lux for the plasmid lux DNA. The two tubes were placed within an ice bath. 3μL of control plasmid was added into tube C and 5μL plasmid into the tubed labeled lux using sterile micropipette. The two tubes were left in the ice bath. 70μL of competent cells was added into each of the tow tubes using sterile transfer pipet. The solutions were tapped with finger index to ensure proper mixing and then stored on the ice bath for 15 minutes then 35μL of the competent cells was added into the third tube and labeled NP(No plasmid). The tubes were then transferred to the preheated bath of 37 degrees for 5 minutes. 275μL nutrient broth was added to the control and the lux tubes and corresponding 150μL of the nutrient to the no plasmid tube using the sterile pipet. The solutions were incubated at 37 degrees for 45 minutes and the results recorder in table 9.1 Procedure C: Selection of the cells that haven taken up plasmid by growth on an ampicillin containing medium 130μL mixed bacterial suspension from tube C was removed from the preheated bath and the content were dispense on to the agar and spread evenly onto the agar surface using cell spreader. The cell spreader was then dipped in ethanol, passed across the ethanol lamp, and maintained for 30 seconds after the ethanol has burned off to enable the spreader to cool. After cooling the spreader was used to evenly distribute the cell suspension over the whole surface of the plate. The cell spreader was then returned to ethanol without flaming.The procedure was repeated severally. 130μL bacterial suspension from the lux tube was transferred to the lux and spread onto the agar surface and the above step repeated. Procedure D: Examination of the cultures in Dark Each plate was opened one by one to determine if E.coli growth occurred and the growth type were noted and recorder in table 9.2. The plates were viewed in the dark and then in the light and the results recorded in the table. Transformation Efficiency was computed. Results Table 9.1 Predictions Treatment Observed Growth Type Bioluminescence ( Yes or no) Reasoning for observed results LBC Yes No LB/Ampc Yes No LBNP Yes No LB/AmpNP No No LB/Amplux No Yes LBlux no Yes Table 9.2 Results Treatment Observed Growth Type Bioluminescence ( Yes or no) Reasoning for observed results LBC Yes No No transformation of cells hence no bacteria growth LB/Ampc Yes No No transformation of cells hence no bacteria growth LBNP Yes No No transformation of cells hence no bacteria growth LB/AmpNP No No No transformation of cells hence no bacteria growth LB/Amplux No Yes The cells were transformed leading to bacteria growth LBlux no Yes Questions Question 1 a. Calcium Chloride . Calcium Chloride ( CalCl2) aids in neutralizing the negative charge of the prevailing DNA b. Heat Shock The heat shock escalates the permeability of the underlying cell membrane to DNA. It also permits plasmids into the underlying cells. Moreover, it is fundamental has it optimized the underlying type employed and the corresponding transformation situations utilized c. Agar d. LB Broth LB broth permits creation of suspension of the bacterial cells from the corresponding original colony developing on the agar plate. It also offers essential nutrients and appropriate environments for the optimal replication. Moreover, the bacteria can be plated onto the agar plates that is within the suspension thus developing a streak to cultivate the colonies from the underlying single cell or be utilized in the purification of the reaction in the extraction of the underlying transformed plasmid. e. Ampicillin Ampicillin serves the purpose of exterminating the bacteria in the experiment                       f. Plasmid Plasmid is able to autonomously replicate underlying bacteria genome hence it offers genes for the antibiotic resistance g. Aseptic Techniques Aseptic techniques prevent contamination via use of appropriate sterile technique. It encompasses maintaining agar plates closed and avoiding taking over the open plates. Moreover, it prevented touching sterilized objects with underlying non-sterile objects and working swifter with the sterile objects. Question 2 The underlying pointer on the prevailing genotype of the E.coli bacteria is the required plasmid. Moreover, plasmid is normally resistant to the ampicillin thus in case the bacteria develops on the existing plate in presence of agar and ampicillin then there is conclusion that plasmid was up taken by the bacteria cell. The prevailing Luria broth made the experiment more apparent through addition of a glow to the existing cells. Question 3 The phenotype of the transformation of the prevailing suggest the successful occurrence of the experiment Question 4 The transformed cells will be on the underlying LB/amp and corresponding LB/ amp/ ara plates. Moreover, the genetically transformed cells possess have absorbed the pGLO plasmid that depicted the presence of the ampicillin resistance gene. The cells are capable of surviving on the plates containing the ampicillin. Error analysis The lab experiment possessed numerous steps thus resulting to the occurrence of error. The entire measurements ought to be precise and accurate. Moreover, the heat shock timing was a cumbersome process. Error in the lab might have resulted from the underlying concentration of Calcium Chloride since most of it was frozen. Discussion and conclusion The prevailing bacteria that were treated with the existing pAMP solution showed resistance to the ampicillin hence developed on the underlying ampicillin plate. Conversely, the ones that were not subjected to the treatment of the pAMP were unable to develop on the medium thus making it to act as a control experiment. Transformation was not full efficient. Thus, the prevailing ampicillin + plates depicted development rather than the corresponding plate. References Doelle, H. W. (1994). Microbial process development. Singapore: World Scientific. Mishra, S. R. (2003). Bacterial plant diseases. New Delhi: Discovery Pub. House. Gnanamanickam, S. S. (2007). Plant-associated bacteria. Dordrecht: Springer. Read More