Cells express genes through the mechanisms of transcription and translation. Transcription is the process when DNA information is copied into an mRNA transcript . The transcript contains the instruction on how a particular protein will be made. During translation, the ribosome uses this instruction to direct the sequence of amino acid in a protein . However, not all genes can be expressed all the time. To conserve energy, genes contains regulatory sequences that helps cells control their expression . One important regulatory sequence is the promoter. A promoter is a short segment of DNA on which RNA polymerase bind to initiate transcription (BQ3) . The right combination of transcription factors must be recruited to either activate or inhibit the binding of RNA polymerase on the promoter . Hence, the promoter decides whether or not a certain gene will be expressed or not.
Aside from the main chromosome, bacteria (and yeast) contain circular, self-replicating plasmids which can hold metabolic genes, virulence genes, or resistance factors . Once a bacterium takes up plasmid DNA, it can express the genes contained in that plasmid . A plasmid can therefore act as vector - a means by which genes can be transferred from one cell to another. Scientists often exploit the ability of the plasmid to possess special genes in different studies. One method in which plasmids are useful is known as genetic recombination. Prokaryotes and some single-celled eukaryotes can undergo gene recombination through a process called transformation . Transformation occur in nature when a cell takes up a remnant DNA fragment (i.e. from dead bacteria) and consequently incorporate its genes to its own genome resulting into a recombinant DNA . If the fragment codes for functional proteins, the cell can use it to make new protein products that may or may not be beneficial to its survival.
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Transformation can be hard to accomplish even under optimal conditions in the lab. In order to ensure the uptake of plasmid, scientists can employ several methods that will render the cells susceptible to plasmid intake. One such method is the use of divalent cations (Ca2+, Mg2+, Mn2+). Cells are suspended in a cold solution of divalent cations and then a heat shock treatment follows allowing intake of the plasmid DNA . Another common technique to achieve cell competency is called electroporation. As the name implies, a controlled pulse of electricity is applied to the host cells in solution with the plasmid DNA. The electricity opens up the cell membrane pores for a brief period of time allowing the plasmid to enter the host cell . Although it may cost more than chemical transformation, it has been proven to be more effective in transforming cells . Whichever method is utilized, the most important thing is to generate a large number of transformed cells for study. The complexity of the transformation process makes it logical to perform it with unicellular organisms like E. coli cells. They grow and divide rapidly and can be easily manipulated (A1BQ1).
To determine which cells carry the plasmid, scientists often employ antibiotic selection. Before introducing the plasmid to the host cell, a gene for resistance to a specific antibiotic can be added to the plasmid. This allows it to confer an antibiotic resistance gene to the bacteria. The pGlo plasmid used in this experiment contains an antibiotic resistance gene . Once gene expression takes place, bacteria with that plasmid can grow on a plate with the antibiotic . Depending on the antibiotic, resistance can act in different ways. Ampicillin, for instance, interferes with cell wall biosynthesis important for growth and replication (BQ1) . If the plasmid contains resistance to ampicillin, the corresponding gene will produce β-lactamase that degrades ampicillin (BQ2) . Otherwise, cells that do not possess the resistance gene will not grow in the presence of ampicillin (A1BQ2). Hence, antibiotics can select the correct bacteria and eliminate the undesirable ones.
The procedure for this experiment was obtained from the lab manual .
Table 1: Observations of different culture plates
about 30 big colonies, glow negative
The colonies are made up of ampicillin resistant cells, but GFP gene was not expressed.
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about 50 big colonies, glow positive
The colonies are made up of ampicillin resistance cells. GFP gene was expressed.
The plate contains cells with no resistance to ampicillin.
about 500 tiny colonies, glow negative
The plate contains normal E. coli cells.
The E. coli colonies used to prepare the pGlo transformed cells were grayish yellow, circular clusters. There are about 100 colonies on the plate with sizes ranging from 0.5 mm to 1.0 mm. After transformation and incubation, the colonies present in the plates were circular and whitish in color. Plate 4 displayed a lawn growth, while plates 1 and 2 have clustered growth.
The genetic transformation performed in this experiment was successful. As expected, plate 2 demonstrated a positive cell transformation while plate 1 showed a negative cell transformation. The cell colonies in plate 2 glowed under UV light which means that the cells took up the pGlo plasmid. The pGlo plasmid contains the gene that codes for the green fluorescence protein (GFP) . Most notably, the availability of arabinose in the plate allowed the expression of the GFP gene. This means that the GFP gene is controlled by the arabinose operon. Arabinose activated the operon by permitting the RNA polymerase to bind to the DNA at the promoter. Then, expression of the GFP gene ensued (BQ4). In contrast, although plate 1 also received the pGlo cell suspension, it does not contain arabinose. Hence, the GFP gene was not expressed and the colonies did not glow under the UV light.
Plates 3 and 4 served as the controls for this experiment because they both contain untransformed cells. The addition of ampicillin in plate 3 makes it a negative control. It should select only for transformed, ampicillin resistant cells. Because no growth was observed in this plate, it means that no contamination was apparent. Meanwhile, plate 4 served as the positive control. The plate only contains the LB nutrients and should therefore allow growth of E. coli. As expected, a lawn growth of E. coli cells was observed in this plate which proved that the cells used were viable.
The results of the experiment showed how bacterial transformation can be very useful in the field of cell biology. Additionally, the experiment highlights one of the many uses of GFP. GFP can give off green light without the use of antibodies, co-factors, or enzyme substrates . It can also be linked to specific genes and be used to stain biological molecules and structures without being toxic to the cells . These characteristics make it a very convenient and effective detection tool. Its many uses contributed to the significant progress in the field of biology.