November 25, 2012 The Effect of the pGLO Plasmid on Genetic Transformation of E. coli by Heat Shock Introduction Genetic transformation is the genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material l(exogenous DNA) from its surroundings and taken up through the cell membranes. This was first demonstrated in 1928 by Bacteriologist Frederick Griffith (Lederberg 2000). A plasmid is a small circular piece of DNA that contains important genetic information for the growth of bacteria. In a recent study, students tested the response of the arabinose operon promoter from E.
coli to two different carbon sources. They directly observe the effects of these sugars on gene expression by monitoring the ability of the transformations to fluoresce under long-wave UV light. This experiment helped students experience transforming E. coli with plasmid DNA and using antibiotics for the positive selection of transformation (Mosher 2002). The present study was carried out in order to see if plasmids could be employed to transfer genes from one organism to another using a vector. The hypothesis was that the pGLO plasmid would introduce new genetic material to E.
coli bacteria thereby changing the genetic make-up of the E. coli. Such a result would suggest that bacteria usually transfer these plasmids back and forth allowing them to share these beneficial genes. That is say, it is indispensable and important for pGLO plasmids to transfer GFP gene because a gene can be spliced into the DNA of a plasmid and when it is transferred from one bacterial cell to another, the gene will be transferred along with the plasmid DNA. Furthermore, we will also predict to find that the bacteria in non-transformation plate,which contains ampicillin, cannot survive.
All bacteria which survive in the transformation plate with ampicillin must contain the ampicillin resistance gene and would fluoresce and glow in the dark. This result can prove that we cannot destroy the marker genes such as ampicillin because it could screen for successfully transformed cells. If the gene haven’t transformed, the offspring will have no antibiotic ampicillin resistance and that means it could not live in the plate containing ampicillin. Last but not least, the two transformation plates which contains LB(Luria Bertani broth) and ampicillin and which contains LB, ampicillin and arabinose have different results.
This result helps prove that the sugar arabinose is used to bind to the regulatory gene responsible for turning on the GFP biosynthesis pathway. Without the presence of arabinose, bacteria cannot express the GFP gene successfully. Materials and Methods First, we transferred the same amount of transformation solution, which contains calcium chloride, into two tubes, and in order to increase the permeability of the cell membranes. Then we placed both tubes on ice to reduce activity of Calcium chloride solution.
Both tubes were added a single colony bacteria by two new sterile loops, and return both tubes to the ice bath. We added a sterile loop of pGLO plasmid only to one of tubes, and did nothing on the other, then continued put tubes back on ice for 10 minutes. Second, we placed both tubes in a 42°C water bath for exactly 50 seconds. When this process was done, immediately placed both tubes back on ice bath and held on for 2 minutes. After 2 minutes, we added the same amount of LB(Luria Bertani broth) to each tube and incubated them at room temperature for 10 minutes. Third, we transferred 100?
l solution in the tube that contained pGLO plasmid to the LB/amp plate and the LB/amp/ara plate, and another two plates which contained LB and LB/amp were each added 100? l solution from the tube which did not have pGLO plasmid. We stacked up four plates and taped them together, and placed the stack of plates upside down in the 37°C environment for 24 hours (Weedman 2012). We will observe the result of each tube under normal room light first and then turn off the lights and observe them under UV light. We can determine if the transformation was successful by the color of the bacterial colonies show under normal room lighting and UV light.
Results The effect of the pGLO plasmid depended on the existence of fluoresced bacteria (Table 1). It is clear to know that bacteria in the transformation plate, which contains LB, ampicillin and arabinose can reproduce new bacteria which can also fluoresce and glow under UV light (Figure 1). Another transformation plate, which has LB and ampicillin can also reproduce new bacteria, but they are incapable of fluorescing and glowing under UV light. In both control plates, no fluoresced bacteria were reproduced. The difference between LB and ampicillin –pGLO plate and LB –pGLO plate is that
a plenty of bacteria can survive in the plate which contains LB and ampicillin while bacteria in the other plate can not. Observations| Transformation Plates| +pGLO LB/amp| | – bacteria grow- bacteria cannot fluoresce and glow under UV light| | +pGLO LB/amp/ara| | – bacteria grow- bacteria can fluoresce and glow under UV light| Control Plates| -pGLO LB/amp| | – bacteria cannot survive| | -pGLO LB| | – bacteria grow-bacteria cannot fluoresce and glow under UV light| Table 1. The observations in survival and fluorescence of E. coli in four different plates.
Figure 1. The image of E. coli fluorescing in +pGLO with LB/amp/ara plate. Discussion The pGLO plasmid has the clear effect of transforming genes (figure1 & Table1). These results support our original hypothesis that pGLO plasmid would introduce new material to E. coli bacteria thereby changing the genetic make-up of the E. coli. That is to say, the pGLO plasmid primarily effects genetic transformation because a gene can be spliced into the DNA of a plasmid and will transfer DNA when it is inserted from one bacterial cell to another.
The results support the hypothesis in that the bacteria can grow and fluoresce in the plate which has +pGLO, LB, ampicillin and arabinose. The reason why bacteria can grow is that pGLO contains a gene for resistance to the antibiotic ampicillin, so the bacteria can survive in the ampicillin and Luria Broth (LB) provides nutriment for bacteria to grow. In order to prove that the pGLO plasmid can confer resistance to ampicillin, we compare –pGLO and LB/amp plate with +pGLO and LB/amp plate, and we can found the bacteria in the plate which has no pGLO plasmid cannot survive, and the bacteria in +pGLO plate could survive.
So pGLO plasmid has the gene for resistance to the antibiotic ampicillin (Timis oara, 2009). The existence of arabinose makes bacteria fluoresce under the UV light because it binds to the regulatory gene responsible for turning on the GFP biosynthesis pathway. Our results support past studies in that the bacteria cannot survive in the plate that contains –pGLO and ampicillin; and the reason is that the bacteria do not have the GFP gene in the pGLO plasmid that could resist to the antibiotic ampicillin.
However, there are some differences between our results and past studies in growth of bacteria in the plate without arabinose (Table 1). In my hypothesis, I said that the bacteria can only survive in the plate which has arabinose. Therefore, the result is that the bacteria can still grow in the plate which has +pGLO, LB and ampicillin and in the plate which has –pGLO and LB. AraC is an allosteric protein with two binding sites, the first binds arabinose causing a conformational change in the protein that allows the second site to recognize and bind to araI (Snyder and Champness, 1997).
That is to say, arabinose is just used to turn on the gene in the bacteria and the bacteria can still survive without arabinose. There are also some weaknesses in this experiment. First, there will be some miscellaneous bacteria in the air and some of them may pollute the bacteria in the plate. For example, if the miscellaneous bacteria pollute the bacteria that in the plate which contains +pGLO, LB, ampicillin and arabinose and bacterial cells of Escherichia coli (E. coli) died from these miscellaneous bacteria, we will get the wrong results and cannot explain the reason.
Second, it is difficult to control colony we obtained and get the same colony each time. For instance, if we pick up little colony at the bottom of one tube and pick up a lot of colony in the other tube, the results may lead to the wrong understanding. In conclusion, genetic transformations need a vector such as a phage or a plasmid, a receptor, such as bacterial cells and a genetic marker. Our experiment successfully showed that pGLO plasmids are really important and indispensable to transfer GFP gene and make the gene of the receptor expression completely. Literature cited Joshua Lederberg. 1994.
The Transformation of Genetics by DNA: An Anniversary Celebration of Avery, Macleod and Mccarty in Anecotal, Historical and Critical Commentaries on Genetics. The Rockfeller University, New York. Mosher Roy H. 2002. Using pGLO to Demonstrate the Effects of Catabolite Repression on Gene Expression in Escherichia coli. University of Illinois at Springfield. P 17 – 22. Timis oara, Romania. 2009. Entrapment of Fluorescent E. coli Cells in Alginate Gel. Lucrari stiintifice Zootehnie si Biotehnologii, vol. 42 (1). P 130-135 Weedman Donna. 2012. Attributes of Living Systems. 7th ed. Bluedoor,LLC. P 109 – 117