Our hypothesis was that the transformed solution with no plasmid DNA and inclining would produce no bacteria colonies, as it wouldn’t be able to row without the gene for inclining resistance. Also, the transformed solution with just LB and inclining would produce bacteria colonies but the transformed solution with LB/inclining/Rabbinate would produce glowing bacteria colonies (as Rabbinate allows the GAP gene to be expressed, but in both cases bacteria colonies would be present because of the gene of resistance to the antibiotic, inclining).
We essentially made the required transformed solutions–and the controls–swiped them on the agar plate, and then observed to see whether or not bacteria colonies grew and whether or not they glowed. Our data fully purported our hypothesis. We can thus conclude that bacteria can take in foreign DNA through the process of transformation and that this foreign DNA can fundamentally change the bacteria (ex: making it glow). Future research can involve inserting other pieces of DNA into bacteria from different organisms, making the bacteria take on various phenotypes characteristics.
Background Information: Genetic transformation is one of the most important processes in biotechnology. Essentially, genetic transformation involves the process where a cell (in this lab, a bacterial cell) takes up foreign DNA from its surroundings and incorporates it onto its own DNA. This gene transfer is accomplished with the aid of a plasmid, a naturally-occurring small piece of circular DNA in bacteria. Bacteria can efficiently adapt to new environments by transferring plasmids with beneficial genes aiding survival to other bacterial cells. In biotechnology laboratories, plasmids are transformed.
Essentially, restriction enzymes are used to make cuts in the plasmid?the ‘vector’–and in the DNA sequence containing the gene to be transferred to the bacteria. Then, this gene to be transferred is forged into the plasmid (in this lab we used previously add plasmids called pogo, containing a bluestocking gene and a gene for inclining resistance). Plasmids are then ‘forced’ into bacterial cells using a process called “heat shock,” where small pores in the bacteria open up. In the end–in order to find out which bacteria received the new gene–the transformed solution can be incubated on agar gel.
The transformed bacteria will exhibit certain unique characteristics (in our case actually growing, or glowing if Rabbinate was present). Materials and Methods: For this experiment, the following materials were used: two micro test tubes, trailer transfer pipettes, 500 Pl of Transformation Solution (Cacao), an ice bath, a foam rack for the tubes to keep them afloat in the ice-bath, non-pathogenic E. Coli bacteria, sterile loops, pogo DNA solution, four specially-prepared agar plates, LB-broth, and a water bath set at 42 degrees Celsius.
First we labeled one micro test tube ‘+DNA’ and the other ‘-DNA’ Then we transferred 250 Pl of Transformation Solution (Cacao)–using a sterile pipette- into each micro test tube, and placed the tubes in a foam rack, which floated on an icy water bath. Next, we used two sterile loops to transfer E. Coli. Bacteria from the starter plate to each of the tubes (‘+DON and ‘-DNA’ tubes). Now that we had added the required bacteria, we proceeded in our experiment by adding the pogo DNA solution–using a new sterile loop–to the micro test tube labeled ‘+DNA. The next part of the experiment involved the heat shock process, where plasmids were fused into bacteria. We incubated both micro test tubes on ice for 10 minutes. Then, we transferred both of them into the water bath set at 42 co for exactly 50 seconds, making sure to place them back on ice after the 50 seconds were ‘up. After letting the tubes incubate on ice for 2 minutes, we used a sterile pipette to add 250 Pl of LB broth to the ‘*DNA’ tube. We repeated this process with the ‘-DNA’ tube. We let these tubes sit for ten minutes at room temperature.
Finally, each transformation solution was prepared. Using new sterile loops for each plate, we spread the suspensions evenly around the surface of the agar of each of their four respective plates (plates were labeled +DNA LB/amp ; +DNA LB/amp/era ; -DNA LB/amp ; -DNA LB for what they contained or didn’t contain [Rabbinate, inclining, LB]). Lastly, we put our plates in an incubator so bacteria loud grow, and observed them the next day under UP light. Results: The nature of this research involved fusing pogo plasmids containing the GAP glowing gene and a gene for inclining resistance into E.
Coli bacteria, only some of which would take in this plasmid. The solutions having transformed bacteria DNA from the plasmid (+DNA) and the solutions where its bacteria did not receive plasmid DNA (-DNA) were spread on agar plates; the plates were then observed in the hopes of drawing important conclusions about gene transfer and transformation in bacteria. In the agar plate with LB/Amp, where the solution tit plasmid DNA combined into bacteria was spread, transformed white bacteria colonies were present, but no glowing under UP light was observed.
In the agar plate with LB/Amp/Era, where the solution with plasmid DNA combined into bacteria was spread, transformed white bacteria colonies were present that glowed bright green under UP light. In the agar plate with LB/Amp and -DNA, no bacteria were present. In the agar plate with just LB and -DNA, a bacteria lawn was present (bacteria were everywhere, but they did not glow). Discussion: Our experiment was designed to observe the genetic transformation of non- tightening bacteria (E. Coli) via pogo plasmids fusing into the bacteria.
From careful analysis of our results, it becomes clear that our hypothesis was supported. Originally, we hypothesized that the only bacteria that would glow would be the bacteria that had received the plasmid DNA–that had been transformed–but that had also been cultivated in Rabbinate. Also, we predicted the solutions with just the bacteria–not the plasmid DNA in addition to the bacteria–would NOT grow if inclining was present in the growth medium, but would grow if inclining was absent from the growth medium (agar). Our results confirmed our hypothesis.
Clearly, when analyzing our experiment, our hypothesis was supported by our results because it makes sense scientifically. First of all, any bacteria growing on the ‘+DNA’ plates must have been transformed, since the plates have inclining, an antibiotic. In being transformed, bacteria received the gene for inclining resistance from the pogo plasmid. Thus, the only ones that grew were the ones that had the inclining-resistance-gene (which they received from the plasmids). Although all transformed bacteria will grow, only the bacteria present n rabbinate will glow under UP light.
This Rabbinate is a sugar which turns on the KEEP glowing gene. Furthermore, even bacteria which had not received the plasmid genes (‘-DNA’), and thus could not glow, obviously would grow on a medium with LB, favoring the growth of bacteria. However, if inclining is present, non-transformed bacteria will not grow, as they would not have received the inclining-resistance-gene from the pogo plasmids. From this lab, we can conclude that bacteria–through the process of transformation–are able to take up foreign DNA (such as in the form of a pogo lassie) and express it in phenotypes characteristics (such as glowing).