gfp Gene Expression

Summary and Findings of gfp gene expression in the article “Expression of Green Flurescent Protein in Lactococcus lactis”

The study conducted by Pilar Fernández de Palencia, Concha Nieto, Paloma Acebo, Manuel Espinosa, and Paloma López demonstrated that GFP could be used as a florescent protein reporter of the bacteria Lactococcus lactis. Using polymerase chain reactions, or PCR, a scientifically engineered vector, in this case, a plasmid, was used as a vehicle to transfer foreign genetic material (gfp gene) into the Lactococcus lactis bacteria.

Lactic Acid Bacteria (LAB) interferes and prevents the growth of infectious bacteria, and because Lactococcus lactis has the capability to produce lactic acid, it has many uses in the food industry. Many different strains of the bacteria are used in the preservation of fermented food, upholding sensory properties such as taste, color, odor, and feel of perishable products such as milk, cheese, yogurt, meat, and certain vegetables. The cloning in this experiment used two different strains of Lactococcus lactis, MG1363 and CRL264, and helped the scientists more adequately understand the bacteria Lactococcus lactis, which in turn allows for the further development of new procedures within the food preservation industry.

Tracking the expression of the gfp gene is another important factor in this study because the scientists are able to create and discover means by which the GFP protein expression is regulated. The scientists are also able to track the metabolism and the growth of GFP, which is important because it is a reporter protein, and can be used to “tag” other types of bacteria in order to see if the mechanically created vector is truly implanted into the target cell.

The process of PCR in creating a fluorescent Lactococcus lactis bacterial cell is quite complex and begins with two plasmids, pLSI (the host plasmid) and pJDC9GFP (plasmid containing the target gfp gene). The gfp gene is not expressed in the original plasmid pJDC9GFP as it had not been correctly activated by the promoter. The goal is to construct a combination of these two plasmids, creating the plasmid (vector) pLS1GFP. This occurs when DNA is cut from the plasmid pJDC9GFP using restriction enzymes ClaI and EcoRI. The sticky ends from these cut strands of DNA are made blunt by the Klenow fragment of DNA polymerase I (PolIK) in order to remove the incompatible ends, thereby creating compatible ends for ligation. The new DNA fragment now contains the promoters Pm and Px of the bacteria and the genes gfp (florescent tag) and tetL, bla and erm, which encode proteins that are responsible for resistance to tetracycline, ampicillin and erythromycin. The gfp in this plasmid is under the control of the promoter Pm which promotes its expression by directing the transcription of malM. DNA from the pLSI plasmid is then cut using the restriction enzymes EcoRI and HindIII, which removed an unwanted 109-bp DNA fragment. The sticky ends were again made blunt by the Klenow fragment or PolIK in preparation for ligation. It is important to note that each plasmid (pJDC9GFP and pLSI) had different restriction sites, or locations where the specific restriction enzymes cut the DNA sequence. Both of the severed strands of purified DNA were then ligated to transform S. pneumoniae and create the new pLS1GFP vector.  The pLS1GFP vector was then transferred into the Lactococcus lactis strain by electroporation.

The Lactococcus lactis cells containing the newly inserted pLS1GFP plasmid, and the original pLS1 plasmid were then grown in either a PBS buffer, or a M17 medium and were supplemented with either glucose or maltose. Their respective fluorescence was measured by a technique known as fluorescent microscopy, and allowed for the comparison of the original pLS1 plasmid’s fluorescence with the newly created pLS1GFP plasmid’s fluorescence. In table 1, it is shown that the MG1363 strain of Lactococcus lactis, showed increased levels of fluorescence in L. lactis cells containing the created vector pLS1GFP. The fluorescence values obtained from the pLS1 plasmid were subtracted from the values obtained from the pLS1GFP plasmid in order to account for the amount of “glow” that was being generated solely by GFP, not from the cell glowing on its own. The results of the subtraction are displayed in table 1 under the column titled “corrective fluorescence”, which ultimately indicated that there was twice as much GFP fluorescence in the cells that were suspended in the PBS buffer, as compared to the cells grown in the M17 medium. This data was also shown in figure 3 graph B, which shows the relationship between fluorescence due to GFP in the Lactococcus lactis cell containing the pLS1GFP plasmid.

These results can be explained by the fact that the process of inserting the gfp gene and creating the desired vector is quite complex. First of all, Lactococcus lactis is part of a bacterial group that creates an acidic environment, which, in turn, created a difficult environment for gfp to thrive in. This is because gfp performs best at a neutral environment (at a pH of around 7). This setback was accounted for by the addition of a PBS buffer, which lowered the pH of the environment, explaining why there was more recorded GFP fluorescence in the cells that were suspended in the PBS buffer, as the environment was more suitable for gfp expression. The addition of the buffer was represented in Fig. 3 graph C, and showed that the fluorescence intensity without the buffer drastically declined as the pH became more acidic (specifically around pH’s of 4-5). It also showed that with the PBS buffer, fluorescence did level off, but the strong decrease of fluorescence never occurred because the buffer did not allow the pH to reach highly acidic levels. Another difficulty was that Lactococcus lactis is a micro-ariphile; a type of bacteria that likes environments containing small amounts of oxygen. This creates a problem for the measurement of GFP activity, because after translation, the protein must be oxidized. Oxidation requires oxygen, and thus the scientists must expose the cell to aerobic conditions. This exposure could affect the detection of GFP. The scientists indirectly accounted for this setback by using the mutated form of the gfp gene that increases GFP solubility and florescence.

The scientists then furthered the experiment by attempting to grow Lactococcus lactis bacteria in milk, a more natural environment that can be used to benefit human life. At first, it was impossible to grow Lactococcus lactis in milk because it did not carry the correct plasmid involving lactose utilization. In order to allow Lactococcus lactis growth in milk, the lactose proteinase pLAC264 was transferred into the bacterial cell containing pLS1GFP. The acidic pH levels made it difficult for GFP to thrive, however this effect could again be reversed by the addition of a PBS buffer. It was found that GFP fluorescence can be detected up to a pH of 5.2, and due to the PBS buffer, at a pH of 4.8 roughly 73% of GFP fluorescence can still be detected. The results of this study can be applied to the food industry, allowing for the monitoring of bacterial cells in milk, cheese, and yogurt production systems.

The results of the study indicated that GFP could be used as a reporter protein of certain bacteria in this host. This system of PCR and gfp expression can be used in the food industry as a process allowing for the detection of the effectiveness when preserving the sensory characteristics of food, and by means of detecting Lactococcus lactis or any other lactic acid bacteria. GFP has also been used for ecological studies involving the intestinal tracts of rats, and subsequently is used in the study of Lactococcal probiotic strains of bacteria in living animals.