Bacterial transformation and gene expression - Lab Report Example

Bacterial transformation and gene expression Bacterial transformation is the process of introducing the foreign DNA into the bacteria. E.coli is a Gram-negative bacterium, which does not have the natural ability to accept the foreign DNA. Hence calcium chloride solution was used to increase the competency of the cell. The plasmid pUC18 and lux operon were used for the transformation. The LB agar medium was prepared with the antibiotic inclining and the plasmid was introduced into the E.coli using the competency and heat shock method. Calcium chloride solution neutralizes the cell membrane and creates a pore in the cell membrane for the uptake of the foreign DNA. After the plasmid was attached to the cell membrane, heat shock method was used for the insertion of the plasmid DNA inside the cell. Keeping the competent cells at 42 °C for few seconds increased the pore size of the cell membrane and enabled the insertion of plasmid. Immediate cooling in the ice bath closed the pores and stabilized the lipid membrane. It was observed that in the medium that contained no Ampicillin, there was lawn growth and in the plates that contained Ampicillin colonial growth was observed. In the plate that contained Ampicillin antibiotic and PUC plasmid, the transformation had occurred. In the other plates only limited growth was observed. The lux gene with Ampicillin plate had selective growth. There was little growth in the plasmid plates with and without Ampicillin. Bacterial transformation was complete in the plates containing Ampicillin and the plasmids pUC18 and lux operon. In the LB/Ampc plate, the transformation efficiency was found to be 1621.6 and for the LB/ Amplux plate, the transformation efficiency was found to be 1297.3. Introduction: DNA is the blueprint of our life. Life without these molecules is not possible. DNA are transcribed into RNA and translated into proteins. These proteins are important for the biochemical functions of the cell. In bacteria, apart from DNA, there is extrachromosomal DNA called as plasmids. They are double stranded DNA which forms circles with size ranging from 1 kb to 200kb (kilobase). (Jeffery et al., 1996). Plasmids are very advantageous for the genetic engineering. Plasmids code for many antibiotic regions and they have the ability to accept the gene of interest. The transformation of our gene of interest into the plasmid is called recombination and the bacteria are called recombinant bacteria. Thus plasmids can be used as cloning vehicles or vectors. These plasmids are not essential for the survival of bacteria, but in some instances, for survival in the different environments, they can provide some extra advantage. The best example is the survival of the bacterial cell in the presence of an antibiotic drug. Antibiotic resistant bacteria like Escherichia coli are used for the transformation of the gene of interest into the host cell. (Jeffery et al., 1996). Gene expression is the process of obtaining information from the gene and using it for the synthesis of a functional gene product. This requires direct manipulation of the genes, transformation and antibiotic/pesticide/herbicide resistance with a longer shelf life of the organism of interest. (Tang et al., 1994).Transformation and cloning are the two important genetic engineering tools used for the expression of the foreign gene of interest in the given bacterial cell. Three conditions required for the transformation are 1) the host into which the foreign DNA is inserted, 2) a method for the insertion of the DNA into the host cell, and 3) methods to identify the transformed cells and select them. (Jeffery et al., 1996). The most common bacterium used for transformation is Escherichia coli. E. coli is a Gram- negative bacteria and its plasmid is suitable for the insertion of the foreign DNA. Foreign DNA is delivered into the host cell using a vector. Plasmids are the extraterrestrials DNA used to amplify the gene of interest. A plasmid contains the resistance to an antibiotic and is used as a vector. All the plasmids contain a selectable marker, an origin of replication and a multiple cloning site. The multiple cloning sites contain many restriction enzyme sites, where the gene of interest is added. The plasmid is tagged with a suitable marker gene and inserted into the E.coli. If the transformation is success, then the marker gene will express inside the E.coli. The vector used in gene expression is a small plasmid pUC18, which contains 2686 nucleotide pairs. (Casali and Preston, 2003). The plasmid pUC1 contains Ampicillin (antibiotic) resistance regions. The gene of interest can be inserted into at the Ampicillin resistance regions and transformed into the E. coli. Ampicillin resistance region of E.coli enables the cells to grow in the presence of the antibiotic and the gene codes for the enzyme beta-lactamase is present in this region. Beta lactamase hydrolyses the beta lactam ring of antibiotics and inactivates them. (Brinas et al., 2002). Similarly, lux operon of the Vibrio fischeri codes for luciferase enzyme and has a molecular weight of 4.5x 10^6 and catalyzes the light- emitting reaction. (Wilson and Walker, 2010). The plasmids are introduced into the living bacterial cells by the process called as transformation. The bacteria are made to take in the plasmid DNA using the calcium chloride solution. Calcium chloride increases the cell’s ability to pick up the plasmid. After transformation, the bacterium obtains the ability to grow in the antibiotic medium and the plasmid can be easily isolated from the bacterial culture. These competent cells will now have altered cell walls and they can uptake DNA very efficiently. The cell competency is increased by creating pores in the bacterial cell suspension in the high calcium concentration solution. (Old and Primrose, 1981). From the research, it was evident that under the Ampicillin gene resistance, the transformed E. coli will be able to grow in the plates and the non-transformed E. coli will not grow in the plates. Method: Competent cell preparation: Calcium chloride solution and E.coli tube were kept in the ice bath and 590µL of CaCl2 solution were transferred into 50 µL of bacteria. The tube was tapped gently and incubated in the ice for 10 minutes. The competent cells were thus prepared and they have the ability to take up DNA from any medium. DNA uptake by competent cells: The tube containing the plasmid was kept in the ice bath and 5 µL of control plasmid was added to the new tube and 70 µL of the competent cells were added to the tube and tapped for proper mixing. Similarly, in another tube 35 µL of the competent cells were added to a separate tube and no plasmid was added to it. 275 µL and 150 µL of nutrient broth was added to the control and plasmid containing tubes and kept in the water bath at 37°C for 45 minutes. Selection of antibiotic resistant cells: 3 agar plates were labeled as control tube(C) , did not contain plasmid (NP)and lux plate (Lux) and to each plate, 130 µL of mixed bacterial suspension were added and incubated at 37°C. Transformation efficiency: The plates were examined for the transformation and transformation efficiency was calculated. The transformation efficiency is calculated using the formula: To determine the number of colonies in the given plate, the following formulae are used. The total amount of plasmid DNA = concentration (µg/µL) of DNA x volume of DNA (µL) Total volume of cell suspension = amount (µL)+ amount (µL) of LB+ amount(µL) of cell suspension. Fraction of DNA spread = volume (µL) spread on LB/Ampc plate Total sample volume (µL) in control DNA tube Total amount of (µg)of DNA = µg of DNA x fraction of DNA spread. Transformation efficiency = total number of colonies on the LB/Ampc plate Total amount of DNA spread on LB/Ampc plate Results: Table 1: treatment results of LB plates with and without Ampicillin in the control and normal plates of pUC18 and lux plasmids. C= control plasmid, lux = lux plasmid, NP = no plasmid, LB = Luria Betrani broth nutrient and Amp = Ampicillin. Treatment Observed growth type Bioluminescence Reasoning for observed results LBc Lawn No Absence of Ampicillin LB/Ampc Colonial No Bacterial transformation in Ampicillin resistance LBNP Lawn No Absence of Ampicillin and no transformation LB/AmpNP Colonial No Presence of Ampicillin and absence of transformation LB/Amplux Colonial growth Yes Transformation and cell growth LBlux Lawn No Absence of Ampicillin Fig. 1: The transformation results for plates containing LB control and LB/Ampicillin control, LB do not contain plasmid, LB/Amp control plasmid, LB lux plasmid with no Ampicillin and LB lux plasmid with Ampicillin. In the given plates, it was observed that there were lawn growth in the LB control plate, LB NP plate and colonial growth in Ampicillin resistant plates with the plasmid pUC18 and lux operon gene. Trasnformation efficiency: The total amount of plasmid pUC18 DNA and plasmid lux DNA: 1) Concentration of (µg/µl) DNA x volume of DNA( µl) = 0.005 x 5 = 0.025 µg pUC DNA. 2) Concentration of (µg/µl) DNA x volume of DNA( µl) = 0.005 x 3 = 0.015 µg lux DNA. Total volume of cell suspension prepared : Total volume (µl)= amount of plasmid + amount of LB+ amount of cell suspension Total volume for pUC18 DNA = 3+275+70 = 348 µl Total volume for lux DNA = 3+275+70 = 348 µl Fraction of DNA spread for pUC18 and lux plasmid DNA : Fraction of DNA spread = volume (µl) spread on LB/Ampc plate Total sample volume(µl) in control DNA tube Fraction DNA spread for pUC18 = 130/ 348 = 0.37 Fraction DNA spread for lux DNA = 130/348 = 0.37 Total Amount of DNA present in the plate: Total amount of DNA (µg)pUC 18 = µg of DNA x fraction of DNA spread = 0.025 x 0.37 = 0.00925 µg. Total amount of DNA (µg) lux = µg of DNA x fraction of DNA spread = 0.025 x 0.37 = 0.00925 µg. Transformation efficiency = total number of colonies on the LB/Ampc plate Total number of DNA spread on LB/Ampc plate Transformation efficiency for pUC18 = 15/ 0.00925 = 1621.6 Transformation efficiency for lux DNA = 12/0.00925 = 1297.3 Discussion: E.coli cannot uptake the foreign DNA naturally as DNA is very hydrophilic in nature. So to make the bacteria take up the plasmid, they must be made competent. So competency must be induced by chemical methods using divalent cations such as calcium, magnesium or manganese. The permeability of the membrane was altered to increase the DNA uptake. This was done by creating small pores in the bacterial cells by suspending them in the high concentration of calcium chloride. Calcium chloride solution neutralizes the unfavorable interactions between the DNA and the polyanions. The DNA and the competent cells are further incubated on ice for thirty minutes to stabilize the lipid membrane. Heat shock was given for a short period at 42°C. Heat shock method increases the size of the pores in the membrane permitting the DNA to enter the E.coli. Six plates were prepared. The first plate contained only LB agar medium. This plate served as the control. The second plate contained Ampicillin resistance gene. The third plate contained no plasmid and only E.coli cell negative control. The fourth plate contains Ampicillin antibiotic and no plasmid. The fifth plate contains lux gene along with Ampicillin resistance. The sixth plate contained lux gene along with LB plate. After incubation period, lawn growth was seen in the LB control plate, no plasmid LB plate and lux gene LB plate. Colonial growth was seen in the Ampicillin containing LB plates with pUC18, NP and lux plasmids. Transformation was observed only on the pUC18 plasmid containing Ampicillin resistance gene. Since the medium contained the antibiotic Ampicillin, transformation was complete. In the presence of ampicillin, the cells that contained the plasmids with the antibiotic resistance were able to grow in the mediuma and they formed distinct colonies in the medium. Bacterial transformation occurs when the bacterial cell takes up foreign DNA. Transformation occurs in the plasmids. E.coli treated with pUC18 solution developed a resistance to Ampicillin and they were able to grow well on the Ampicillin containing plates. The plates that were not treated with the pUC18 Ampicillin resistance could not grow on the LB medium. The plate without Ampicillin showed lawn gorwth and the cells were non-selective. The competent cells were able to take up the foreign DNA. Thus it was concluded that the plates that contained Ampicillin and plasmids pUC18 and lux gene had bacterial transformation and other plates did not have transformation. The transformation efficiency of pUC 18 plasmid DNA was found to be 1621.6 and that of lux plasmid DNA was 1297.3. References: Brinas, L., Zarazaga, M., Saenz, Y., Ruiz-larrea, F., and Torres, C. (2002). β- Lactamases in Ampicillin- resistance Escherichia Coli isolates from Foods, Humans and Healthy Animals. Antimicrobial Agents and Chemotherapy. 46(10): 3156-3163. Casali, N and Preston, A. (2003). E.coli Plasmid Vectors: Methods and Application. New York: Springer Science and Business media. Old, R. W. and Primrose, S. B. (1981). Principles of Gene manipulation. Oakland: University of California press. Jeffery, M. B., Cladwell, G. A and Zachgo, E. A. (1996). Biotechnology: a Laboratory Course, London: Academic press. Tang, X., Nakata, Y., Li, H-O., Zhang, M., Gao, H., Fujita, A., Sakatume, O., Ohta, T and Yokoyama,K. (1994). The Optimization of Preparations of Competent Cells for Transformation of E.coli. Nucleic Acids Research. 22(14): 2857 -2858. Wilson, K and Walker, J. M. (2010). Principles and techniques of Biochemistry and Molecular Biology. London: Cambridge University press. Read More