Panther I.D.:
“The expression of ampicillin resistance and luciferase enzyme genes by E. coli transformation using the pUC18 and lux plasmids”
Abstract
The transformation process allows the uptake of DNA from the environment, with the incorporation and expression of genes. During this process, changes in the DNA of the receptor cell occur, producing some selective advantages in some cases, such as resistance to antibiotics. pUC18 and lux plasmids were used to determine the transformation process in Escherichia coli. Cells were made competent by the addition of calcium chloride and heat shock in order to induce the incorporation of the DNA to the cell. This process causes the alteration of the pores in the membrane, allowing the DNA to pass through it. A control without plasmid was performed. Selection of the transformant was done by addition of ampicillin to the LB medium, and observation of the luminescence. Efficiency of transformation was determined. Results agreed with the expected ones, finding colonial growth on transformant cell and lawn growth on non-transformant ones. Luminescence was found in only one colony of lux transformed E. coli. Lack of luminescence detection could be associated to the lack of darkness in the room. Both plasmids, pUC18 and lux, transformed the E. coli cells, being the lux plasmid more efficient. Some factors other than plasmid size may affect the transformation process, since smaller plasmids, such as pUC18, are able to produce more copies of the plasmids. Results showed gene expression in E. coli cells transformed with pUC18 and lux plasmid.
Introduction
Transformation is the process for the uptake of DNA segment from the environment where bacteria is present, and the expression of those genes for the cell which received the genes, called the recipient cell. During this process, the recipient cell gets some new characteristics (Pommerville, 2004).
A non-resistance genotype can be changed to a resistance phenotype when exogenous DNA is introduced in the bacteria. In the case of E. coli, the presence of some plasmids coding for the resistance to a particular antibiotic, such as ampicillin, can make the species grow by expressing the resistance gene. The presence of antibiotic resistance gen and a bacterial origin of replication in a plasmid allow its selection when transformation occurs in the laboratory. E. coli is usually treated using ampicillin because this species is susceptible to being killed by this antibiotic.
Three types of growth can be observed when bacteria are inoculated onto agar: lawn, colonial and no growth. Lawn growth is related to too many microorganisms in the inoculum. In that case bacteria grow in the whole surface of the plate, with all individual colonies coming together. On the other hand, colonial growth is related to the formation of colonies which can be observed separately. Colonial growth is present when the number of cells is smaller than in the lawn growth. Another way to obtain colonial growth is by the addition of an inhibitor, which reduces the possibility of cells to grow. The other type is no growth and it can be related to the absence, or inhibition of microorganisms.
In this experiment, ampicillin will not E. coli because, it will use different plasmids that contain the resistance gene, making this organism resistant to ampicillin. In that way, when E. coli is transformed, it is able to grow on ampicillin-containing agar.
Plasmids are extrachromosomal, circular DNA found in bacteria. They can be used as vectors to transfer genes. The pUC18 plasmid derives from pBB322. It has the ampicillin resistance gene and is also a high-copy plasmid. The molecular weight of the pUC18 is 2x106, so it is a small sized plasmid (Lin-Chao et al., 1992).
The lux plasmid is a plasmid containing the lux operon which genes codes for the production of the luciferase, an enzyme that catalyzes a reaction with the emission of light, and for other enzymes that produce a substrate for light e-emitting reactions, called luciferins. Lux plasmid also contains the ampicillin resistance gen. Its molecular weight is around 4.5x106 (Engebrecht, and Silverman, 1984).
Changes in membrane permeability or surface receptors are important in competence of a cell. Particularly, during normal growth, Escherichia coli do not develop competence. In the laboratory, this species can be induced to take up DNA fragments by adding calcium chloride at 4 oC and then heating it quickly at 42 oC (heat shock) (Pommerville, 2004).
Bacteria can be grouped by a Gram staining reaction into Gram + and Gram -. This stain process is related to the cell wall. Components of a cell wall include the peptidoglycan, outer membrane, lipopolysaccharide (LPS), and the periplasmic space. In Gram + bacteria, the cell wall has a thick layer (20-80 nm) attached to the outer surface of the cell membrane. A high concentration of peptidoglycan is found in Gram +. No outer membrane and periplasmic space is present in this type of bacteria.
On the other hand, in Gram - bacteria, such as Escherichia coli, the cell wall is thinner than in Gram +, but is more complex, having less percentage of peptidoglycan. An outer membrane is present, forming the outer surface of the wall with a small periplasmic space. Another periplasmic space is present in the inner surface of the wall. This space is wider than the one present in the outer wall (Black, 2005) (figure 1).
Figure 1. Schematic representation of cell wall in Gram- and Gram+ bacterial. (Brown et al., 2015).
The hypothesis was that if E. coli is transformed with the pUC18 and lux plasmid, the result will express the genes present in the plasmid, such as ampicillin resistance for pUC18, ampicillin resistance and presence of light in the lux operon. The objective of this study was to look the expression of the gene after E. coli was transformed with the pUC18 and lux plasmid.
Methods
Transformation of E. coli was performed using a plasmid lux and a control plasmid (pUC18). The transformation process was done in four steps (Alberte et al., 2012).
Preparation of E. coli competent cells. The competent cells were prepared before the experiment by adding 590 µL of CaCl2 solution to a tube containing 50 µL of E. coli culture. Solutions were mixed by tapping the tube and incubated for 10 minutes on ice.
Uptake of DNA by competent cells. Five microliters of plasmid were added to an Eppendorf tube, placed on ice. Concentration in both plasmids was 0.005 µg/µL. Seventy microliters of competent cells were added. The tube containing the mix was stored on ice for 15 minutes. A control without plasmid, having 35 µL of competent cells was prepared in an Eppendorf tube (“NP”). After 15 minutes, tubes were transferred to a water bath at 37 oC for a heat shock, during 5 minutes. Nutrient broth was added to the control and lux tubes, 275 µL, and the no plasmid tube, 150 µL. All tubes were incubated at 37 oC for 45 minutes. It is important to gently tap the Eppendorf tube containing solution every time it is used.
Selection of transformed cells. To select the transformed cells, 130 µL of the mixed bacterial suspension from control tube “C” was transferred onto a plate containing ampicillin medium. Cells were spread using a spreader. The spreaders were flamed using ethanol, and cooled for 30 seconds. The same procedure was done for the lux tube, “lux”, and the no plasmid, “NP”, containing tube. In all cases, 130 µL of the mixed bacterial suspension was added on the plate and spread. Plates were kept for about 10 minutes, or until the liquid was dry, and then incubated at 37 oC inverted.
Visualization of the culture. After the incubation period, plates were observed for E. coli growth. The type of growth (lawn, colonial or no growth) was reported. If growth was present, colonies were visualized in the dark and with light. Transformation efficiency was calculated through the relation between the total number of colonies in the plates LB/Ampc and LB/Amplux.
Results
The E. coli cells were transformed using pUC18 and lux plasmid. A control without plasmid was performed. Table 1 shows predicted and obtained results for both plasmids. Predicted and observed results are basically the same. For LBc, lawn growth was found in all plates. Plates for transformed cells (LB/Ampc, LB/Amplux) show colonial growth. In the case of no-plasmid control, no growth was found in the observed plate for lux plasmid. Figure 1, shows plates with growth for the control plasmid, pUC18, where lawn and colonial growth is evident.
LB: luria broth, c: control (pUC18), Amp: ampicillin, NP: no plasmid, lux: lux operon.
Figure 1. Growth of E. coli on LB media. Lawn and colonies growth is evident.
The total amount of DNA spread on each plate requires the calculation of the total volume prepared in the tube and the fraction of DNA spread on the plate from this total volume. The total amount (µg) of plasmid DNA was the same for both plasmids, control (pUC18) and lux. The calculations were done by the multiplication of concentration of DNA by volume of DNA.
µg DNA=(0.005 µg/ µL)(5 µL)= 0.025 µg of plasmid DNA.
The total volume of cell suspension prepared in the tube and its fraction spread onto the plate, were also the same for both plasmids, since the volumes used were the same. The total volume is calculated by the addition of the amount of plasmid, LB and cell suspension, all expressed in microliters. It is calculated as:
Total volume (µL) = (5 µL)+ (275 µL) + (70 µL) = 350 µL.
The fraction of DNA spread onto the plate is the relation between the volume (µL) spread onto the plate and total sample volume (µL) in control DNA tube.
Fraction of DNA spread= 130 µL/350 µL= 0.371
The total amount (µg) of DNA present on the plate is the multiplication of µL of DNA by fraction of DNA sample, as follow.
Total amount (µg) of DNA = (0.025 µg) x (0.371) = 0.0093 µg
Transformation efficiency was calculated, for each plasmid, according to the number of colonies produced on the plate.
Transformation efficiency LB/Ampc = 80 colonies/0.0093 µg of DNA= 8.625 x 103 transformants / µg of DNA.
Transformation efficiency LB/Amplux = 200 colonies/0.0093 µg of DNA= 2.156 X 104 transformants / µg of DNA.
Discussion
Transformation is now used as an important genetic recombination method that occurs in less than one percent of bacterial population. This process can occur in the environment in both Gram- and Gram+ bacteria. DNA present in the environment can come from lysed cells which break into fragments of about 10 to 20 genes. These fragments can be taken up for live bacteria that are genetically similar and competent to receive the DNA (Pommerville, 2004).
Competent cells are those cells that have the ability to take up DNA from the environment. In this process, a protein, competence factor, is released into the medium (Black, 2005). Also, the presence of calcium chloride and heat shock allows DNA, which is very hydrophilic, to pass through the membrane by creating small holes in the cell. Competence is a requirement for cells to be transformed.
The presence of growth on LB without containing ampicillin (LBc and LBNP) demonstrated the ability of the E. coli culture to grow in this media. LB is a medium of choice for some enteric bacteria including E. coli and is widely use in molecular studies (Sezononv, 2007). Transformed cells were evident for both pUC18 and lux plasmid by the growth of some colonies in LB media containing ampicillin.
The presence of ampicillin in LB plates acts as a selective substance that allows bacteria that have taken up the plasmid to flourish. Both pUC18 and lux plasmid contain ampicillin resistant genes showing that phenotype when they grow on this media. Lux plasmid also contains the luminescence genes, coding for the luciferase enzyme, but only one colony on LB/Amplux showed luminescence or glow. This failure to glow in the dark can be related to the room not being dark enough. Cells transformed with pUC18 do not produce this enzyme.
Determining the presence of colonies on plates containing ampicillin was the first approach to determine if the transformation occurred successfully. On the other hand, the absence of growth on LB/AmpNP, when compared to LB/Ampc, demonstrated the importance of plasmid presence on the bacteria to grow on media containing ampicillin.
Difference in growth, both lawn and colonial, is related to the selectivity of the ampicillin for the transformed cell. In that way, LB/Ampc and LBc, had colonial and lawn growth, respectively, which was the same result as LB/Amplux when compared to LBc. These are associated to the lack of ampicillin in LBc, allowing the growth of all bacterial cells. This is contrary to the presence of ampicillin in the media, where only bacteria having the ampicillin resistance plasmid, the transformed cells in other words, can grow.
The absence of plasmid inhibited completely the growth on LB containing ampicillin. This is reflected on LB/AmpNP results when contrasted with LB/Amplux, where transformed colonies having the lux operon growth formed different colonies. The absence of plasmid restricted the growth on ampicillin containing media, but not in LB without ampicillin, represented as LBNP, and where lawn growth was observed. Both LB/Ampc and LB/Amplux presented colonial growth corresponding to the transformed cells with pUC18 and lux plasmid. They grow in the presence of ampicillin because they have the ampicillin resistance genes in the plasmid. All results agree with the predictions, confirming the hypothesis.
The uptake DNA from the environment can be advantageous to the host organisms, since it confers new genetic characteristics, such as possibility to grow on some media or, as in this case, resistance to some antibiotics. In the transformation process, an organism’s characteristics change. Pathogenicity increases due the ability of the cell to take some DNA and incorporate it in its expression. These pathogenicity characteristics are advantageous for host organisms, but in some case can be dangerous for the surrounding ecosystem, including humans, because they can cause infections and it is difficult to treat.
Transformation efficiency was higher for the lux plasmid (2.156x104 transformant/ μg) than the control plasmid pUC18 (8.625 transformant/ μg). This was contrary to the expectations, since smaller plasmids replicate better in bacteria and produce larger numbers in the cell. The best results of transformation in E. coli, in a small plasmid are around 2-4x1010 transformant/ μg. Also, transformation efficiencies of 5x106-2-x107 transformant/μg of DNA have been reported, using CaCl2 method (Chan et al., 2013). For larger plasmids, this value is considerable lower.
Conditions other than plasmid size affect transformation efficiency, such as forms of DNA, genotype of cells, growth of cells, methods of transformation, and damage of DNA (Hanahan, 1983).
Improving the transformation process can be done by using a higher concentration of DNA and also, in this case, a very dark room for a better visualization of the bioluminescence, can help in the determination of the lux plasmid presence in the cell.
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