The Production and Isolation of Green Fluorescent Protein - Lab Report Example

The specific purpose of the study is to explore the production and isolation of Green Fluorescent Protein (GFP). This process will include several related laboratory techniques, including bacterial transformation, selection of transformed cells, Growth of recombinant bacteria and isolation of the GFP. The lab presents the results obtained for each laboratory technique and compares them with the already existing knowledge. Four plates were streaked with bacteria and from this the transformant cells was identified. In which case, plate 2 containing both arabinose and ampicillin, in the presence pGLO, contained the transformant cells since they acted as vector cells. The presence of the plasmid produced the resistant effect on ampicillin while arabinose regulated the production of GFP, which gave out the fluorescent green glow. Introduction The study was carried out to explore the production and isolation of Green Flourescent Proteins. The study of GFP was particularly of importance based on its use in biotechnology during the creation of genetically modified organism. GFP is one of the notable reporter genes found in pGLO, normally used as a vector aiding in production of GMO. In which case, pGLO is a placid containing three genes: Bla that aids in transformation of bacteria resistance, araC that controls the expression of GFP and GFP, per se,exists as a fluorescent protein, which gives the characteristic green glowing color. The phenomenal green color of GFP acted as a core material in the production of Alba, the green fluorescent rabbit. GFP is a major compound confirmed to generate fluorescence for mammals, especially the marine organisms. This confirms the wide routine use of GFP in various researches. In which case, a given gene is cloned in the presence of GFP then expressed to allow it link well with the latter (GFP). From this, the protein will start glowing green thereby allowing for easy tracking of its location among other cells in an organism. The presence of AmpR, the ampicillin resistance gene allows for selection. The expression of GFP in this plasmid serves to evaluate whether the cells have gone a complete transformation. The specific purpose of the study is to explore the production and isolation of Green Fluorescent Protein (GFP) (Chess, 2009). This process will include several related laboratory techniques, including bacterial transformation, selection of transformed cells, Growth of recombinant bacteria and isolation of the GFP. The lab presents the results obtained for each laboratory technique and compares them with the already existing knowledge. Methods Two test tubes were obtained and labeled +pGLO and another –pGLO. 250 µl of transformation solution (CaC12) were transferred using a sterile transfer pipette to the test tubes. All the tubes were placed on ice. Single colony bacteria from the starter was obtained from the starter plate followed by picking up +pGLO tube and immerse the loop into the transformation solution at the bottom of the tube. The UV lamp provided was used to examine the pGLO plasmid DNA solution. A new sterile loop was immersed into the DNA solution. The ice containing the content was immersed on ice for 10 minutes. The four agar plates were labelled on the bottom (not the lid) as follows: Label one LB/amp plate: +pGLO; Label the LB/amp/ara plate: +pGLO; Label the other LB/amp plate: -pGLO; Label the LB plate: -pGLO. Both the (+) pGLO and (-) pGLO tubes were transfered into the water bath, set at 42 °C, for exactly 50 seconds. The rack containing the tubes were removed from ice and placed on the bench top. Sterile pipette was used to add 250 µl of LB nutrient broth to the tube. The closed tubes were then tapped to allow mixing of the content. A new sterile pipett was used add100 µl of the transformation and control suspensions onto the appropriate plate. The suspensions were evenly spread around the surface of the agar by quickly skating the flat surface of a new sterile loop back and forth across the plate surface. Finally, the plates were stacked and taped together and then placed upside down in the 37°C incubator until the next day. Results In the absence of GFP gene, the colonies appeared to be white, especially in the absence of GFP regulator. The bacteria appeared to have wild-type (natural) phenotype as evident by white colonies with no visible fluorescence. The transformed bacteria were able to survive in the ampicillin plate, indifferent to the untransformed that died in the process. For the four different plates, in which the bacteria was streaked, the following is a flow chart insinuating the final conclusion made” 1st plate (LB/amp): containing only ampicillin and pGLO, in the absence of arabinose, there was formation of white colonies 2nd plate (LB/amp/ara): containing both ampicillin and arabinose and pGLO, there was formation of fluorescent green colonies in the presence of UV light 3rd plate (lb/amp): there was only ampicillin and no pGLO. The cells died as shown by the absence of growth. 4th plate (lb, – pGLO): There was no observed changed in the content of the plate. Discussion During the process of transformation, arabinose was used to regulate the production of Green Flourescent Protein (GFP) as shown by the colonies appearing to be white. It is worth noting that in the presence of arabinose, the RNA polymerase binding carries on with aid from araC protein. The arabinose achieves a direct interaction with araC usually in the presence of a binding DNA. Consequently, the binding is what leads to the production of GFP. The white color observed can be attributed to the absence of the phenomenol green glow of GFP. However, when there is no arabinose, the binding process facilitated by araC does not proceed and therefore also inhibiting the transcription of the GFP (Pommerville & Alcamo, 2007). Intuitively, the expression of GFP relies on an appropriate source of arabinose, which must be present during the lab. The presence of the arabinose turns on the production of GFP. The following is a simple char showing the production and regulation of GFP in the lab: The ampicillin used was core to determining the presence of plasmid, pGLO, in the bacteria used. In which case, the bacteria were placed in a plate containing ampicillin to test whether they could survive or not. As shown by the results, the transformed bacteria containing plasmid were able to survive in the ampicillin plate while the other colonies died. This occurrence can be explained by the fact that transformed bacteria containing the plasmid can express a gene which is resistant to ampicillin effect. The gene expressed is beta-lactamase and is produced by the transformed bacteria (Lackie, 2013). The 10 minute incubation period, for the transformed cells, in the presence of LB nutrient broth allowed for individual cells growth and the expression of ampicillin resistance protein .The untransformed bacteria, which do not have the plasmid and beta-lactamase ends up dying in the ampicillin plate. The four different plate in which the bacteria were streaked, were used to evaluate the effect of arabinose and ampicillin on the color of the bacteria colonies; both in the absence and presence of pGLO. In plate 1 there was ampicillin, with pGLO, but in the absence of arabinose. The observed white colonies were as a result of the absence of arabinose, which is responsible for turning off and on of the GFP. The GFP was required to produce the fluorescent green colonies as witnessed in plate 2 where there was both arabinose and ampicillin. In both cases, the presence of pGLO prevented the cells from dying from the ampicillin effect. For plate 3, the cells died because of the absence of the plasmid pGLO to produce the resistant effect on the present ampicillin. Consequently, the ampicillin was responsible for the death of the cells, which in this case had not undergone transformation. For plate 4, there was no witnessed change because of the absence of ampicillin and arabinose. In conclusion, the transformant cells can be identified from the four plates. In which case, plate 2 containing both arabinose and ampicillin, in the presence pGLO, contained the transformant cells since they acted as vector cells. The presence of the plasmid produced the resistant effect on ampicillin while arabinose regulated the production of GFP, which gave out the fluorescent green glow. Work cited Pommerville, J. C., & Alcamo, I. Edward. (2007). Alcamos laboratory fundamentals of microbiology. Sudbury, Mass: Jones and Bartlett. Lackie, J. M. (2013). The dictionary of cell and molecular biology. Amsterdam: Elsevier/AP. Chess, B. (2009). Laboratory applications in microbiology: A case study approach. Boston, Mass: McGraw-Hill Higher Education. Read More