MACUB (2021) Conference

Abstract: Replication Protein A(RPA) is a single-stranded DNA (ssDNA) binding protein. This protein has many biological functions, specifically to repair DNA and activate cell checkpoint pathways. Thus, the inhibition of RPA prevents the division of cells. Given its ability to prevent DNA repair and the proliferation of cells, chemicals agents that inhibit RPA can potentially be a viable therapeutic against cancer. Chemical agents that can be potential inhibitors of RPA are currently being synthesized through organic synthesis. These chemical agents are to be characterized through nuclear magnetic resonance (NMR) and Mass spectroscopy analyses.

Abstract: Triple-negative breast cancer (TNBC) is an aggressive disease with limited treatment options. Single-walled carbon nanotubes (SWCNT) have unique properties including stability under various conditions and high surface area. Biomedical applications of SWCNT have the potential to expand cancer treatment options. Previous results from our lab have shown that TNBC cells have reduced rates of migration after treatment with dispersed SWCNTs. Our study will test the hypothesis that SWCNT treatment inhibits migration by altering expression of Matrix Metalloproteinases (MMPs), tissue inhibitors of Metalloproteinases (TIMPs) and PRPF4B. When PRPF4B, a pre-mRNA splicing factor kinase, is down regulated breast cancer cell migration is inhibited. MMPs and TIMPs have been reported as putative tumor markers and specific types have been implicated in breast cancer. All gene expression studies will be performed by real-time PCR. Anticipated data from our gene expression studies may help reveal that SWCNTs can be potential therapeutics for treating breast cancer.

Abstract:

Background: Protein vaccines are antigens. They are highly efficient in developing antibodies by the cells of recipients following intramuscular administration quickly. We have developed a novel, unique and virtual protocol for the production of RBD of the SARS-CoV-2 S-protein. The SARS-CoV-2 RBD-protein directly stimulates antibody production against SARS-CoV-2. Hence, it is a safer, fast acting, and effective vaccine against SARS-CoV-2 and also efficient for immune compromised individuals.

Methods: We will reconstruct a plasmid carrying RBD, FP and sfGFP cDNA in sequence in an orf, transform Escherichia coli, C2566H, carrying T7 RNA polymerase gene. The transformed cells will express RBD-FP-sfGFP fusion protein developing green fluorescent cfu. The RBD-FP-sfGFP fusion protein will be isolated from the transformed E. coli, cfu. The RBD-protein will be separated from the sfGFP by digesting the FP with an enterokinase specific for the FP. The RBD will be eluted using HIC. The eluent RBD will be confirmed by immunoreaction using BioVision Elisa kit and quantified using a spectrophotometer at UV280nm.

Results: The plasmid reconstruct will be selected by ampr cfu. In addition, the plasmid will also carry the T7 promoter controlling the expression of RBD-FP-sfGFP fusion protein. The transformed Escherichia coli will efficiently express the RBD-FP-sfGFP fusion protein. The highly efficient sfGFP fused with RBD-FP will turn the transformed cfu green. The RBD will get separated from the sfGFP by the FP specific enterokinase. Pure RBD protein will be produced by HIC and the eluate carrying the RBD proteins will test positive against SARS-CoV-2 antibodies present in the BioVision Elisa kit. The RBD content in the eluate will be quantified by the spectrophotometer at UV280nm.

Conclusion: A BioVision ELISA positive test detects presence of <10 pg RBD/ml of the sample. For the application of RBD protein as a vaccine, a larger sample can be produced following the same protocol, formulated following a standard procedure and safety protocols. The RBD-protein vaccine, once administered, will be recognized by the cells of the immune system of the recipient, and stimulate T and B lymphocytes to produce antibody against RBD and thus the SARS-CoV-2 virus. The RBD protein carry no potential to recombine with the genome of the recipient.

Abbreviations: ampr = ampicillin resistant, cfu = colony forming unit, HIC = hydrophobic interaction chromatography, ampicillin, FP = fusion peptide, orf = open reading frame, RBD = receptor binding protein, sfGFP= superfolder green fluorescent protein, S-protein = Spike protein, SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2.

Abstract: In biotechnology and molecular biology, different model organisms are used for experimental purposes. The most common of these model organisms is Escherichia coli (E.coli), as it is non-pathogenic, has a short generation time, its genome is well understood, and can easily be manipulated for cloning and transformation purposes. While working with E.coli or other microorganisms, contamination may become a big problem and can ruin long hours of work. Thus, it is extremely important to master sterile techniques and learn correct procedures that will aid in designing further experiments with bacteria. In this presentation, we aim to review the details of sterile techniques commonly used in a biotechnology laboratory setup. We also aim to discuss procedures and specific examples, about qualitative (streaking) and quantitative (serial dilution) methods for obtaining single bacterial colonies. For our presentation, we will focus on two bacteria- E.coli and Serratia marcescens (S.marcescens) and present distinct illustrations about the quadrant method of streaking, common problems that first-time college students make during such a laboratory procedure. Furthermore, we will present specific quantitation examples to demonstrate serial dilutions using stock cultures of E.coli and S. marcescens grown overnight. Overall, the presentation will revisit important concepts and techniques about handling microorganisms commonly used in biotechnology.

Abstract: DNA fingerprinting is a technique that can examine variations and unique patterns in DNA. The technique involves identifying similar variable number of tandem repeats (VNTR) between different DNA samples. VNTR are sequences of DNA that are repeated multiple times in our genome at particular loci. Forensic analysis of DNA fingerprints checks different loci on the human genome to find variations or similarities in the VNTR patterns and then help link DNA found at a crime scene to a possible suspect. VNTR are polymorphic meaning they have many forms and is therefore difficult to identify similar VNTR patterns between DNA samples in a classroom setting. In our experiment, we determine the presence or absence of the dimorphic alu element on chromosome #16 PV92 locus amongst 15 students. Alu elements are similar to VNTR and are considered “jumping genes” which are repetitive DNA elements that can inserted hundreds of times in the genome. The dimorphic alu element on chromosome #16 PV92 locus is 300 bp and can only be present or absent on this chromosome. To identify its presence/absence, we designed forward and reverse primers that would flank the alu element 400 bp away. In our experimental method, we the students extracted genomic DNA from their cheek cells, added the primer mix and then amplified the DNA by PCR. The PCR samples were then run on a gel by electrophoresis. 4 students were homozygous +/+ and had the alu element on both chromosomes. 9 students were homozygous -/- and did not have the alu element on the chromosomes. 1 student was heterozygous +/- and had the alu element on one chromosome but not both. These results can highlight whether particular students who possess or lack the alu element are related to each other. The different ethnic backgrounds of these students can also provide evidence that the alu element appears in different geographic locations around the world and in different populations. It can also represent evolutionary characteristics of alu elements.

Abstract: Enterococcus spp. are gram-positive bacteria that colonize human and animal intestines. They can survive in aerobic or anaerobic environments as well as in extreme conditions, making them ideal microorganisms for testing of fecal contaminated environmental water. Rapid detection of Enterococcus spp. is needed to reduce the risk of exposure to contaminated water. There are several ways to test for the presence of Enterococcus spp. such as the Enterolert enzymatic assay or microbial DNA amplification by Polymerase Chain Reaction (PCR). Most tests are expensive or require special instrumentation like a thermocycler. We sought to explore the usage of an alternative method to PCR called Loop-mediated isothermal amplification (LAMP). Using LAMP, we analyzed for the presence of Enterococcus spp. in the water around New York City. The advantage of LAMP reaction is that it is inexpensive, requiring only a water bath between 60-68o C, 4-6 specialized primers and a processive Bst DNA polymerase. To make the assay rapid, we use DNA prepared by the boiling method instead of traditional chromosomal extraction and employ a colorimetric assay to detect positive LAMP reaction. Our preliminary data show that water sample contaminated with Enterococcus spp. can be detected using common household items and our own do-it-yourself detection apparatus

Abstract: Semiconductors are available in hardware, for example, PDAs, tablets, PCs, and watches, and so forth Furthermore, most semiconductor gadgets use silicon as semiconductors and metal electrodes. These gadgets show extraordinary execution however need lightweight, adaptability, and straightforwardness, which are vital variables for cutting-edge hardware. Natural semiconductors, then again, show this load of properties and are considered appropriate materials for adaptable and transparent electronic devices. Organic semiconductors, on the other hand, exhibit all these properties and considered suitable materials for flexible and transparent electronic devices. Pristine C60 and its derivatives have been used for solar cells, superconductors, ferromagnets, photoelectric switches, magnetic tunnel transistors, field-effect transistors (FETs). As a high-performance organic semiconductor, C60 exhibited one of the highest electron mobilities among n- channel materials for FETs. C60 single-crystals have been widely grown from vapor or solution C60 has been known as an outstanding electronic material, composed of 60 fully conjugated p-electrons within each molecule. Compared to vapor growth, solution methods provide a platform to easily control the crystal growth of C60. For example, by simply changing the concentration of the solvents, the shapes of the crystals were varied from irregular, needles, and clumped . The crystal shapes were shown to be associated with the structure and concentration of the solvents used. We, therefore, focused more on a solution growth method and controlled the growth parameters in terms of crystal alignment, shape, and dimensions to accommodate obtaining single-crystals of C60. This research comprises of l growing C60 crystals using silicon a substrate. Single-crystals of C60 were grown using the vapor technique and hot plate technique was used to transfer a small layer of silicon wafer onto large silicon wafer (300um). Using both an electron microscope and optical microscope, C60 crystals were observed for length, size, and density. Measured data shows that our best crystals had length of 10um. Obtained results show that we successfully grew crystals in different conditions, which give promise to using C60 as an organic semiconductor. We describe many processing methods such as Bis(trimethylsilyl)amine (HMDS) , Physical Vapor Deposition System (PVD) and Hotplate to grow different sizes arrays of aligned C60 single crystals. Our aligned C60 single-crystal needles and ribbons show(10um). By performing various experiments under different conditions to see what would work the best, we were able to successfully grow crystals of different sizes, different density with different directions. This experiments involved the use of hotplate and our results show that 500C has more closely aligned crystals than any other temperature and at 30 degrees, the result was less crystals . Using the HMDS method, 30 degrees was not very favorable because there were residue left but at 50 0C, there is a significant change as the crystals did not leave as much residue and are more aligned and lastly, using the (PVD)Physical Vapor Deposition System might not necessarily be a great solution to growing bigger crystals because as the temperature got higher, crystals were evaporating. We hope in the future to use this data to better get perfectly aligned C60 Crystals.

Abstract: The nuclear exosome is an indispensable quality control mechanism that can oftentimes co-transcriptionally regulate the stability of nascent RNAs and have a significant impact on cellular processes like splicing, export, and recombination. The helicase MTR4 is a well-known component of this complex and functions to make RNAs amenable to degradation by the exosome. Given that the exosome itself has many functions in the cell and the RNAs (pre-mRNA and non-coding) it regulates the levels of both have a significant impact on cellular programs like differentiation, it is imperative to have an efficient system to stably introduce knockdown of key exosome subunits like MTR4. This paper reports the successful construction of a lentiviral plasmid containing tetracycline inducible anti-MTR4 shRNA, its delivery into HEK-293T cells, as well as the robust knockdown of MTR4 and accumulation of target eRNA following induction. Moreover, the findings in this paper demonstrate that under the regime of MTR4 knockdown, eRNAs accumulate around actively translating ribosomes to a greater extent. Ultimately, the generation of this stable MTR4 knockdown cell line will allow us to circumvent many of the inconveniences associated with traditional transient siRNA transfection, and make future endeavors trying to decode the precise mechanism in which non-coding RNA impacts differentiation phenotypes at a post-transcriptional level more manageable.