On Monday in molecular biology, Dr. Peng discussed translation in bacteria and eukaryotes. The third test in that class was on Wednesday and covered site-specific recombination and translocation of DNA, transcription mechanisms, RNA splicing, and translation.
In biochemistry on Tuesday and Thursday, we learned about lipids’ roles in membranes, including the phospholipid bilayer of biological membranes and the fluid mosaic model. In bacterial physiology, we began with the structure of microbial communities, particularly biofilm formation. We continued with cell-to-cell communication systems, such as quorum sensing, which allows bacteria to sense how many of their own kind are in the population. Quorum sensing is used to coordinate varied activities such as competence, virulence, biofilm formation, sporulation, and bacteriocin production. We also discussed symbiosis such as mutualism, cooperation, commensalism, predation, and parasitism.
On Friday, the bacterial genetics lab met in the computer lab to analyze the Sanger sequences of the RFLP assay. The data did not work out as well as expected, probably because the PCR product was not incubated long enough with the restriction enzyme and the enzyme did not cut all the sequences. We have the final test for the lab next week, and then the class will be completed.
In molecular biology on Monday and Wednesday, Dr. Peng lectured about translation in prokaryotes and eukaryotes. He discussed the structure of tRNAs as well as tRNA charging by class I and class II aminoacyl-tRNA synthetases. We also learned about ribosome structure and translation initiation, elongation, and termination.
In biochemistry on Tuesday and Thursday, Dr. Popescu discussed lipids, including sections on the biological roles of lipids and structure and properties of storage, membrane, and signaling lipids. We learned about all 6 classes of lipids – fatty acids, triacylglycerols, wax esters, glycerophospholipids, sphingolipids, and isoprenoids. On Tuesday in bacterial physiology, we learned about bacterial two-component signaling systems such as the Arc, Nar, Che, Pho, Bvg, Agr, Spo, PhoQ/PhoP, and EnvZ/OmpF systems. We also began to discuss microbial communities and environmental stresses such as heat shock response, SOS response, and oxidative stress responses.
Bacterial genetics lab was cancelled on Friday because the Sanger sequencing results weren’t ready. We had planned to do computer analysis of the results.
On Monday and Wednesday in molecular biology, Dr. Peng lectured on RNA splicing mechanisms and RNA editing. He reviewed important proteins and nucleic acids in the spliceosome such as snRNPs (small nuclear RNA paired with protein). He also went over the three ways of splicing an RNA – nuclear pre-mRNA, group I introns, and group II introns. We then moved on to alternative splicing and RNA editing such as site-specific deamination and RNA-directed uridine insertion/deletion.
On Tuesday and Thursday in biochemistry, Dr. Popescu began the chapter on carbohydrates and glycobiology. We started with basic structures of monosaccharides including open chain and ring forms and moved to disaccharides formed via glycosidic bonds, before looking at structures and metabolism of common polysaccharides including glycogen, starch, cellulose, and chitin. We concluded the chapter discussing the biological function of glycoconjugates – glycolipids, glycoproteins, and proteoglycans. In bacterial physiology, we concluded the section on metabolism by learning about the tricarboxylic acid cycle (TCA cycle), the electron transport chain, oxidative phosphorylation, and the 5 types of fermentation in bacteria (lactic acid, ethanol, butyric acid, mixed acid, and propionic acid fermentation).
On Friday in bacterial genetics lab, we performed plasmid preparation to separate the plasmids from the E. coli and chromosomal DNA and proteins. Dr. Brown also lectured on Sanger sequencing in preparation for analysis of our sequences.
The Monday before fall break, I had a test in molecular biology on DNA mutability and repair, homologous recombination, site-specific recombination, and transposition of DNA. The Monday and Wednesday after fall break, Dr. Peng reviewed transcription mechanisms in bacteria and eukaryotes.
In biochemistry on Tuesday, we had a review for the test on Thursday. The test covered protein metabolism, enzymes, nucleotides and nucleic acids, regulation of gene expression, and DNA-based technologies. In bacterial physiology, Dr. Roberts reviewed bacterial metabolism, including the Embden–Meyerhof–Parnas (EMP) pathway, the Entner–Doudoroff (ED) pathway, and the pentose phosphate pathway (PPP), focusing on key intermediates, enzymes, and regulators.
In bacterial genetics lab on Friday, we prepared a restriction fragment length polymorphism (RFLP) assay on our PCR products.
In molecular biology on Monday and Wednesday, Dr. Peng discussed DNA damage and repair mechanisms including nucleotide excision repair, non-homologous end joining, and translesion synthesis. For the honors paper in this class, I decided to write about the molecular biology of preeclampsia, as I have previously researched the pathophysiology of the disease. In biochemistry, Dr. Popescu reviewed DNA technologies beginning with the mechanisms of cloning. On Thursday, she continued with reviews of polymerase chain reaction amplification of DNA, electrophoresis separation of DNA, and DNA genotyping. The second part of the lecture was about eukaryotic gene expression in bacteria.
In bacterial physiology on Tuesday, Dr. Roberts handed back quizzes and midterm grades, answered any questions about any lecture material, and listed important topics for the test on Tuesday. On Thursday, four groups presented papers titled Antimicrobial effects of silver nanoparticles, Capsule switching and antimicrobial resistance acquired during repeated Streptococcus pneumoniae pneumonia episodes, Effects of fermented sumach on the formation of slime layer of Staphylococcus aureus, and Membrane lipoteichoic acid of Streptococcus pyogenes and its stabilized L-form and the effect of two antibiotics upon its cellular content. These papers in addition to all the others presented in class will be on the test on Tuesday, so I have read through the papers again during my studying for the test.
In bacterial genetics lab on Friday, we selected colonies that had the recombinant plasmid. The microbial DNA, if taken into the plasmid, would have disrupted the gene for X-gal utilization. When transformed into E. coli that were plated on an agar with X-gal, the E. coli with non-recombinant plasmids would grow into blue colonies, since they could utilize the X-gal. The E. coli with recombinant plasmids, since they could not utilize X-gal, grew into white colonies. My lab partner and I had fewer colonies than the rest of the class, so we selected all our colonies, while the rest selected white colonies.
In molecular biology on Monday and Wednesday, Dr. Peng discussed DNA replication. We reviewed the full process of replication as well as how it is regulated. In bacterial physiology on Tuesday and Thursday, we covered membrane bioenergetics, starting with a section on transport mechanisms (diffusion, solute transport, and group translocations). A section on protein transport followed, which covered the Tat and Sec secretion systems in E. coli. The final sections were bacterial respiration, proton potential, and the electron transport chain.
Biochemistry class on Tuesday was an introduction to enzymes. One thing that I did not remember well was the catalytic mechanisms of enzymes, which include acid-base, covalent, metal ion, and electrostatic catalysis mechanisms. Another topic I have previously struggled with is enzyme kinetics, so I will make sure to study that in detail before the next test. On Thursday Dr. Popescu discussed nucleotides and nucleic acids. None of the information in that lecture was particularly new or challenging since we have already discussed nucleic acids in molecular biology and bacterial physiology.
On Friday I was traveling to a wedding in Colorado, so I was unable to go to bacterial genetics lab or communications. My lab partner conducted PCR product cloning on two of our PCR products from last week (the two that showed strongest bands on the gel electrophoresis). She started with ligation of the PCR product, then transformed the plasmid into E. coli.
This week began with a review in molecular biology in preparation for the first test on Wednesday. The test included DNA and RNA structure, the genome structure and organization, and techniques used in molecular biology labs. On Tuesday morning, I had another review in biochemistry I for the first test on Thursday. Needless to say, I spent a lot of this week studying for both of these tests.
On Tuesday afternoon, two of my bacterial physiology classmates and I presented a PowerPoint summarizing a research paper. We were the first group to present, and I am relieved that I don’t have to worry about doing that later in the semester when there is more going on in other classes. Our paper was titled “Inhibition of Bacterial Cell Division Protein FtsZ by Cinnamaldehyde.” On Thursday, Dr. Roberts started a lecture on membrane bioenergetics, though we only got part way through the lecture on transport through bacterial cell membranes.
I took the two tests on Wednesday and Thursday, and finished up the week in bacterial genetics lab and communications on Friday. In the lab, we ran a gel electrophoresis to visualize the DNA we amplified last week. We also prepared our samples to run in a nested PCR, which will amplify only parts of the genes that were amplified last week. This is sometimes necessary because, while the 18S RNA genes are highly conserved between species, there can still be some differences that interfere with amplification of the gene.
FtsZ protein determines the point of cell division in bacteria by polymerizing into fibers that form the Z-ring, or the site of cell division. Cinnamaldehyde has previously been shown to have bactericidal effects while being non-toxic to humans. The authors studied the effect of cinnamaldehyde on FtsZ protein binding. To show that cinnamaldehyde inhibited the GTPase activity of FtsZ, a light-scattering assay and GTP hydrolysis assay were used. Electron microscopy of FtsZ showed that cinnamaldehyde inhibited the formation of protofibrils, which inhibited the formation of Z rings, and resulted in longer cells, as was shown in E. coli cell. Additionally, the binding of cinnamaldehyde to FtsZ was shown through favorable enzyme kinetics, epitope mapping of cinnamaldehyde, and in silico molecular modeling of binding.
As explained in the paper, compounds targeting FtsZ have the potential to be very useful as antibiotic agents with a new mode of action. Cinnamaldehyde can also inhibit methicillin-resistant Staphylococcus aureus (MRSA) and other antibiotic resistant bacteria, so it could be used as a last resort against infections. Therefore, this research is very relevant to today’s scientific research. The experimental approach is also sound, in my opinion, because they use multiple assays/approaches for measuring each aspect of the FtsZ/cinnamaldehyde interaction the paper was focused on.
Future research in this area should include compounds that are stable in air, since cinnamaldehyde can be oxidized to cinnamic acid, which does not have the same bactericidal properties; this would be prohibitive to developing cinnamaldehyde as a useful antibacterial agent. However, since the authors of this study explored the binding site structure of cinnamaldehyde, similar drugs could be designed that would bind to FtsZ to block polymerization. Cinnamaldehyde could be a good starting point for future research.
Domadia, Prerna, et al. “Inhibition of Bacterial Cell Division Protein FtsZ by Cinnamaldehyde.” Biochemical Pharmacology 74. (2007): 831-340. ScienceDirect. Web. 16 Sept. 2016.
This week I continued working in the Department of Plant and Soil Sciences in Dr. Paul Tseng’s lab. I am currently performing plate assays on annual bluegrass (Poa annua) seedlings to determine the effects of different rates of herbicide (foramsulfuron). I am using four different biotypes of annual bluegrass collected from golf courses in Alabama and Mississippi. On Monday, I sterilized seeds of each of the four biotypes and transferred them to a plate with Murashige and Skoog medium to germinate. Next Wednesday, I will be transferring the seedlings to new plates of MS media before innoculating with herbicide.
On Monday and Wednesday afternoon I continued learning about genome structure in molecular biology. On Tuesday and Thursday, we finished the information for test 1 in biochemistry, which covered the function of globular proteins such as myoglobin, hemoglobin, antibodies, and the mechanism of muscles. Later in bacterial physiology, we reviewed questions that were commonly missed on the test last Thursday, and had a quiz this Thursday.
In bacterial genetics lab on Friday morning, the instructor lectured on primer design in detail for our test next week. We then prepared a polymerase chain reaction mix with various primers and our two DNA samples isolated from the amoebas.
This week began with discussion of the genome in molecular biology. Dr. Peng lectured first on the structure of the genome which involves DNA wrapped around histone proteins to form chromatin. We then learned how the chromatin is regulated to allow access to the DNA that is to be transcribed into RNA.
In bacterial physiology, we finished the lectures on microbial growth and reproduction, focusing on ways to measure growth, the physiology of growth, cell division, and growth kinetics. One of the things I found most interesting in this lecture was the Min system, which works with other regulatory systems to regulate the location of cell division. Min proteins oscillate between the cell poles, blocking the access of FtsZ, a protein which forms a ring at the division site. Since the Min proteins are focused on the cell poles, FtsZ is free to start the septum at the middle of the cell, resulting in two similarly sized daughter cells. The first test in this class was on Thursday, so I spent the first part of the week preparing for it.
On Friday, I went to my bacterial genetics lab, where we began with a brief discussion of PCR primer design before extracting DNA from unknown amoebas. Friday afternoon consisted of scientific communications followed by an extra band rehearsal for the football game on Saturday.