To grow the bacterial culture, use 10 ml of liquid LB growth media for incubation. 500 ml of the bacterial culture is allowed to grow overnight at 37°C. It is later shaken vigorously to increase the OD600 to 0.5, which means that time equals zero. At time zero, 1 mL of the culture is transferred into a 1.5 mL centrifuge tube and centrifuged for 5-10 minutes to obtain a pellet. The supernatant should be discarded. The centrifuge with the bacterial pellet is labeled “G0” and stored at -20°C. The culture is induced with 1 Mm of IPTG and allowed to keep growing. After 3 hours past induction, 1 mL of the culture is pelleted into a different 1.5 mL centrifuge tube, and the bacterial pellet is labeled “G3.” The centrifuge with G0 bacterial strain needs 15 mL of the strain to be collected and to be pelleted into the centrifuge tube. The last pellet is labeled “G3-15 mL.” Finally, both the G3 and G3-15 mL are to be stored at -20°C.…
The results show that tea tree and eucalyptus have about the same antibacterial activity as the positive control (ampicillin) used in this experiment. Oregano and colloidal silver’s zone of inhibition were closer to h2O, which concludes that at the amount used for both there is no antibacterial…
theory that the buds would float towards the dull chamber gives to not be right, and we have…
First, the unknown sample from experiment 2 was boiled with just enough water dissolve it completely. After all of…
Me and my lab partner, obtained a mixture of a un known proportion from the instructor and then flow the guide line in our lab manual to separate the mixture by applying the separation method motioned in our lab manual pages 33-40 . In this experiment, the separation methods were decantation, filtration and evaporation and extraction and each is explained below:…
Actions Taken: I labeled six test tubes in accordance with their respective Sample number and placed them on the test tube rack. I then placed a pea-sized amount of each sample in their corresponding test tube. Following that, I utilized the graduated cylinder to measure and transfer 5 mL of distilled water into each of the six test tubes. I then placed a rubber stopper on Sample #1, and shook vigorously to determine its solubility. I repeated this with each sample, and then recorded my results in Data Table 1.…
The purpose of this experiment was to show the genetic transformation of E. coli bacteria with a plasmid that codes for Green Fluorescent Protein (GFP) and contains a gene regulatory system that confers ampicillin resistance. A plasmid is a genetic structure in a cell that can replicate independently of chromosomes. In this lab, the Green Fluorescent Protein, which is typically found in the bioluminescent jellyfish Aequorea Victoria, was cloned, purified, and moved from one organism to another with the use of pGlo plasmids. It was hypothesized that if bacteria that were transformed with +pGlo plasmids are given the gene for GFP, then transformed cell colonies will be located on the LB/amp/ara and LB/amp agar plates. Cells that have been transformed with +pGlo plasmids have the ability to grow in ampicillin plates, and the arabinose sugar allows the colonies to be visibly fluorescent under ultraviolet light. The GFP is able to resist ampicillin because of the Beta- Lactamase protein that is produced and secreted by the bacteria that have been transformed to include it in their plasmids. Arabinose is a carbohydrate, normally used as a source of food by bacteria. Bacterial colonies are not able to grow on –pGlo plates because they are sensitive to ampicillin. In this lab, I will move the GFP gene from one organism to another with the use of pGlo plasmids.…
In the experiment involving varying pH levels, E. faecalis, S. epidermidis, L. lactis, and L. casei were subjected to different pH levels and then were allowed to incubate in order to determine the minimum, maximum, and optimum pH levels for growth of specific bacterial species. It was found that lower pH levels between 2 and 4 inhibited or promoted little to no growth for E. faecalis, S. epidermidis, and L. casei. It is evident that these bacteria were resistant to alkaline environments, however, it should be noted that certain mechanisms must have been acquired or involved in maintaining equilibrium within the bacterial cell. High alkalinity generally leads to physiological problems within microbes including the disruption of plasma membrane…
The purpose of this lab is to successfully infiltrate E. coli bacterial cells with a pARA-R plasmid that is antibiotic resistant and has the rfp gene, or red fluorescent protein. This can be verified if the E. coli obtains the characteristics of the plasmid when it enters. To start, three Petri plates containing agar are needed. On each plate there is a control group and a treatment group; the treatment group being the one with the plasmid. Before the plasmid is put with the E. coli, first the bacteria are “stressed out” by warming them up in a hot water bath and cooling them down very rapidly in ice. The first plate consists of Luria Broth (LB), the second plate consists of LB and the antibiotic ampicillin (amp), and the last one contains LB, amp, and the sugar arabinose (ara). The bacterial cells are subjected to a heat shock and then are placed onto the three plates. The plasmid is spread on to only half of the first two plates, on the sides of the treatment group. Half of the E. coli get the plasmids and the other half do not (that side being the control group). On the third plate the plasmids are spread on the whole plate. The bacteria are left in an…
The laboratory exam will be given on Tuesday December 4th (sections 0375 and 3252) or…
3. Took sample from pump and examined it under a microscope to only to discover contaminants.…
4 a. What test will you perform to determinate when you have added sufficient NaOH solution in Part II?…
Carson, V. (2013). Microbiology Lab (1st ed.). Department of Cell Biology, Microbiology & Molecular Biology. University of South Florida.…
b. To become familiar with the concept of using green fluorescent protein (GFP) as a molecular tag for studying gene expression in bacteria and other organisms.…
Abstract: Application of fluorescent fusion proteins to the field of expression and interaction proteomics as a means of dynamic imaging proteins in vivo has allowed for rapid advancements in biotechnology research. Production of such proteins first involves the insertion of a given protein-coding gene transcriptionally in-frame with a fluorophore sequence under the control of a single promoter and terminator. With reference to this experiment, successful BamHI and PstI digestion of both pHSG-WZB donor (coding for wzb tyrosine phosphatase) and pT5(6H)CFP recipient vectors allowed for ligation formation of pT5WZB(6H)CFP (coding for desired fluorescent fusion protein). Transformation into competent E.coli and plating against TE buffer and pHSG-WZB controls experimentally disputed theoretical expectations as ampicillin resistance (50725 cfu/mL) and fluorescence (93%) along with kanamycin resistance (66800 cfu/mL) and non-fluorescence were displayed, suggesting ineffective fragment ligation. Using effectively ligated transformants, screening for positive clones via plasmid extraction and PstI and XhoI digestion displayed two expected fragments; pT5 (experimental: 2339 bp) and cfp-wzb (experimental: 1198 bp). SDS-PAGE analysis of auto-induced positive clone cell lysate revealed the expression of the cfp-wzb fusion protein (experimental: 44.3 kDa). Using a hexahistidine tag, successful fusion protein purification was achieved via nickel affinity chromatography and confirmed by SDS-PAGE. Finally, para-nitrophenyl phosphate assay of tyrosine phosphatase activity allowed for determination of wzb kinetic properties such as Km (6.34 mM) and Vmax (0.0644 µmol/min/mg). Gaining an experimental competency in formation, detection and use of specific fusion proteins will ultimately allow for the…