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Genetic transformation is the technique of introducing a recombinant DNA into a living cell. In this experiment, we introduced pGLO plasmid into E. Coli bacteria through the heat-shock method. CaCl2 solution was used to make the E. coli cells competent.
Genetic transformation is the technique involving introduction and expression of foreign(exogenous) DNA in a living host cells. Scientists cut out interested gene from human, plants or animals DNA, insert it into a vector to produce a recombinant DNA and introduce the recombinant DNA into host cells which express the exogenous genes and produce interested proteins under appropriate condition. Demonstrated by Frederick Griffith in 1928, transformation has been applied in various areas of biotechonology. For example, genes coding for human insulin can be genetically transformed into bacterial cells. In this way, genetic transformation allows the production of protein products on a large scale.
The introduction of a foreign DNA into a host cell requires the use of a vector. Vectors are small DNA molecules that can be used to combined with foreign genes and transfer them into the host cells. In a laboratory research, plasmid is one of the most commonly used vectors to transform foreign DNA. The recombinant plasmid used in this experiment is pGLO plasmids. pGLO plasmids contain two genes that are useful: the gene coding for beta-lactamase and the gene coding for the green ¬‚uorescent protein(GFP). The expression of beta-lactamase gene provides resistance to the antibiotic ampicillin.
GFP is extracted from Aequorea victoria (bioluminescent jelly fish). It is a polypeptide consisting of 238 amino acid(Cubitt et al., 1995). For its non-invasive characteristic and capacity of resisting interferences, GFP has been widely used as a visual marker for gene expression for more than 20 years(Gilbert et al., 2000). Even under normal light, GFP presents yellow-green color that can be easily observed.
The objects of this experiment were to perform a transformation of E. coli with a plasmid containing the GFP DNA gene and force the GPF to be expressed in a particular environment. In this experiment, we induced E. coli to take in pGLO into their cytoplasm and grew the E. coli cells in different plates. CaCl2 solution is utilized as transformation solution to increase cell membrane permeability, thus plasmid vectors are able to enter the cell. The ampicillin-resistance gene in the plasmid was utilized as the selectable marker, which means E. coli cells transformed with plasmid are able to grow in an environment with ampicilin. Because the GPF expression is driven by the arabinose-stimulated PBAD promoter, it is expected that the E. coli cells grown in the plate with sugar arabinose would express GPF DNA and present a yellow-green color in the UV light.
Materials and Methods
Plasmid could be easily taken in by E. coli cells if the cells had been treated with calcium salt. First of all, two micro test tubes were labeled with either +pGLO or -pGLO. Each tube was added with 250μL of CaCl2 solution and then placed on ice. A single colony of E. coli bacteria was added into each tube. The cells were dissolved into the solution by gently spinning the tubes. Then both tubes were placed on ice again.
After the entire colony was dispersed, the tubes were examined with UV light to make sure that there were no visible green clumps of cells in the solution. Then 10μL of pGLO plasmid was added into the +pGLO tube and gently mixed, while no plasmid was added into -pGLO tube. Both tubes were placed on ice for 10 minutes.
Following the 10-min incubation, the tubes were heat shocked. Both tubes were transferred into 42°C water bath for exactly 50 seconds, and then immediately placed back on ice for 10 minutes. The heat shock process must be rapid. After heat shock, 250μL of Luria broth (LB) were added to each tube and the tubes were placed at room temperature for 10 minutes.
Selection of GFP Transformation
To grow and select the cells with GFP DNA, four agar plates were obtained: 1 LB, 2 LB/AMP and 1 LB/AMP/arabinose. Among them, 1 LB/AMP plate and 1 LB/AMP/arabinose plate were labeled with +pGLO, while 1 LB plate and 1 LB/AMP plate were labeled with -pGLO. 100μL of cells from +pGLO tube was added to each of the plates labeled with +pGLO, while 100μL of cells from -pGLO was added to each of the plates labeled with -pGLO. New sterile loops was used for each plate. The whole process was conducted near fire.
Then four plates were stached upside down at 37°C for 24 hours. A photo of the plates were taken in the UV light the next day.
Figure 1 on the next page shows a photo of four plates. Table 1on the next page illustrates the observation results of each plate.
From the photo, we can observe the growth condition of colonies on each of the four plate.
Plate 1: the untransformed E. coli colonies grew normally and form a lawn on the plate;
Plate 2: no colonies grew;
Plate 3: transformed E. coli colonies grew and fluorescenced yellow-green in UV light;
Plate 4: E. coli colonies grew and presented white color in UV light.
Fig Photo was taken after the plates had been incubate at 37°C for 24 hours.
Table Observation of the colonies in each of the four plate
E. coli -pGLO plasmid
E. coli -pGLO plasmid
E. coli +pGLO plasmid
E. coli +pGLO plasmid
In the plate containing LB and E.coli -pGLO (Plate 1), bacterial cells formed a lawn, because there was no antibiotic in the medium. The cells grew normally as in natural condition. Plate 1 is a negative control which excludes possible contaminants.
In the plate containing LB/amp and E.coli -pGLO(Plate 2), no colonies grew, because the ampicillin in the medium killed the cells by inhibiting the cell wall from producing.
In the plate containing LB/amp/ara and E.coli +pGLO(Plate 3), colonies were grew, because these bacterial cells contains plasmid carrying ampR gene which perform a resistance against ampicillin. Under UV light, the colonies emit green-yellow fluorescence, because the GFP gene on plasmid was expressed in an environment with arabinose.
In the plate containing LB/amp and E.coli +pGLO(Plate 4), white colonies were grew because of the presence of ampR gene and absence of arabinose.
The presence of ampicillin in the medium is to identify if the E.coli cells have taken in plasmid and thus acquired ampicillin-resistance gene on the plasmid. The results of Plate 4 compared with Plate 2 indicate that E.coli cells have taken in plasmid successfully.
The presence of arabinose is to identify if the E.coli cells containing recombinant plasmid successfully inserted with GFP gene. The results of Plate 3 compared with Plate 4 indicate that the plasmid was successfully recombined, thus the cells express GFP under the induction of arabinose.
In this experiment, GFP transformation in E. coli was perform in order to examine how the recombinant plasmid can be introduced into bacterial cells, incorporated into bacterial genome and express recombinant protein. The experiment shows the expected results, which successfully support the hypothesis.