Cell_Division_in_Bacteria

The overall purpose of this study was to determine and understand cell divisions in bacteria, specifically the septation process and the proteins involved. The researchers experimented to discover which proteins were involved in cell division and when. They focused on fts genes and their mutations to better understand the protein complexes involved in cell division of bacteria. Through confirmation of the localization of the proteins they support their idea that a large protein complex functions in septation. To support their hypothesis, they clone the fts gene and its mutations to view its localization activity The researchers want to understand what proteins were involved, and when they were involved, in cell division, they used PCR. PCR is a common technique in microbiology used to amplify a specific piece of DNA to make thousands to millions of copies of a particular DNA sequence. PCR is used to amplify ftsK fragments in this experiment, and therefore specific primers are necessary. They made specific primers FtsK1, FtsK3, FtsK4, and FtsK7; each primer keeps the same N-terminus intact, Ftsk1, and then made a different length, FtsK3 being the longest and FtsK7 being the shortest, in order to see what part of the FtsK protein targets . The N-terminus is the start of a protein. They created different lengths, having the same fusion junction with GFP, but varying on the 3’ end, in order to the amount of amino acids needed to target GFP to septum. The primer for FtsK1 included a Sac 1 site so that when inserted into the plasmid it would be inserted right in front of the promoter lacZ. The other three included an XbaI site at the desired end so that the GFP, also cut with the XbaI, would connect. Gfp is a gene that produces a protein, GFP, that when turned on will glow. Therefore, by tagging the FtsK, one can see what it is doing, and when it is doing it. In this case, GFP is turned on by the lac promoter. Once primers were created, in order to amplify the inserts, they used primer pairs in the reaction mixtures: FtsK1-FtsK4, FtsK1-Ftsk3, and FtsK1-Ftsk7; they were each paired with the FtsK-1 so that they would be placed in front of the promoter and would be able to glow. PCR mixtures also included template DNA, from JM105 diluted cells or BSP853, not mutated cells, Taq polymerase, a thermostable DNA polymerase, and Vent polymerase, a DNA polymerase with higher fidelity than Taq polymerase, so that continuous denaturation and replication can occur, leading to the amplification of the specific fragments. The DNA polymerase is used to read the template strand and synthesize another strand. The PCR process begins with the denaturation of the fragments and template DNA at 95 degrees C for 1 minute, followed by annealing at 50 degrees C for 2 minutes, and then polymerizing at 72 degrees C for one minute. This process was repeated 30 times to achieve the amplified fragments. The products were then purified and cut with SacI and XbaI, restriction enzymes, and put into the already cut pZG, which contains the GFP gene. Theproduct plasmids named pK3G, pK4G, and pK7G in relation to the different length primers used above. By using restriction enzymes, the plasmids are cut in specific places to create unique ends for that restriction enzyme. The GFP gene was attached to the C-terminus, opposite end of N terminus, of FtsK. The fragments and plasmids were ligased together. Moreover, fragments containing the ftsK44 mutation were also closed by using cells from TOE44, a mutated cell with dysfunctional FtsK, for their DNA template, and the primer pairs Ftsk1-FtsK4 and Ftsk1-FtsK3. These fragments, primers, and DNA template then participated in the same PCR process as the non mutated fragments; then named pK3(-)G and pK4(-)G, in relation to mutation and primers used. Expression of the GFP was also dependant on the lac promoter, but was regulated by the lacI^q gene. Once they had the plasmids, they expressed them in E. Coli cells. The researchers grew the cells in over various time intervals and temperatures, as well as in different environments. IPTG (isopropyl-beta-D-thiogalactopyranoside) is used as a mimic of allotatose which triggers the transcription of lac operon , which acts as a promoter for the FtsK-GFP, inducing expression. IPTG needed to detect expression. Live cells were then viewed using an fluorescence microscope to detect the Ftsk-GFP during cell division, could not be viewed with naked eye. As explained above, the researchers used a GFP-tagging technique that confirmed the proteins that were involved with septation. With this tagging strategy, they were able to visualize the workings of the different FtsK-GFP fusions. To test if functionality of fusions, they introduced them into the mutant strain TOE44. They saw complementation. Complementation occurs when new proteins added are able to recover the wild type, the non mutated form. When added the FtsK-GFP to the TOE44, which originally unable to replicate, was able to replicate because of complementation. However, a decrease in complementation was seen as size decreased: pK3G, the largest, complimented fully, pK4G partioally complimented, and pK7G, the smallest, did not compliment at all. These results show that at something essential for complementation lies between the base pairs of pK7G and pK4G. Furthermore, they examined the localization of the FtsK-GFP at the septum. They observed two populations, one with a dim, spread out fluoresce, and the other with a bright fluorescence concentrated at the center of the cell. This suggests that FtsK-GFP and FtsK travel to the septum later in cell division process. All three fusions localized, showing that although not all were able to compliment, they were all able to localize and target correctly. Since even the shortest fusion localized, it shows that just the first 15% of the protein are enough to target to septum, meaning that this segment’s job is to mid-cell targeting for FtsK. They also tested the mutant, FtsK44, and saw that is was defective in localizing to the septum. When placed in TOE44, they were unable to compliment. The mutant proteins also displayed uniform fluorescence, meaning that it cannot localize. This lack of localization could also be due to other factors, such as multiple mutations, poor PCR, poor expression, or degradation, but when further tested found that it was only a single base pair mutation, ruling out possibility to multiple mutations. The experiment also included the use of electrophoresis to make sure they obtained the correct size. Further tests, such as immunodectection, show that the lack of localization is due to the mutant’s inability to detect. To address issue of obligomerization, converts monomers to a finite degree of polymerization, the FtsK-GFP in TOE44 was observed under non-permissive conditions. They saw localization supporting the idea that the N-terminal domain is a true localizing domain. GFP is a useful tool in for this experiment. It allowed the researched to determine protein localization as well as which proteins are involved and if they localize to the septum, and further define the localization domains. Through this experiment, they built upon the evidence that key proteins are involved in the cell division of E. coli, and it is possible that it is localized to septum region. They concluded that FtsK-GFP is targeted late during septation and participated in actually constricting. The role of FtsK was also examined; one possibility is that is forms a seal at the last stage of septation, or that is seals the septum closed and helps keep DNA away from seal. The results also show a connection between proteins, for example FtsK can recognize FtsZ or another early player in division, and that FtsK depends on FtsA, as well as many other factors. In conclusion, this experiment furthered the findings on certain proteins involved in septation and the division of bacteria. As more proteins become know, the closer scientists get to a more concise understanding of the pathway of cell division apparatus. ^^should re write that. Idk somethings just funky. Haha mula agrees