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Week 4/22

Introduction This week, the results for the UV test done on D. xinjiangensis were observed and a plasmid extraction was performed on D. sonorensis. Procedure The plasmid extraction was performed as follows:  -Add 600 µl of bacterial culture grown in LB medium to a 1.5 ml microcentrifuge tube  -Centrifuge the tube for 30 seconds at maximum speed and discard the supernatant -Add 600 μl of TE or water to the bacterial cell pellet and completely resuspend it -Add 100 µl of 7X Lysis Buffer to the tube and mix by inverting 4-6 times and proceed to the next step within two minutes -Add 350 µl of cold Neutralization Buffer to the sample and mix thoroughly until the sample turns yellow -Centrifuge the tube for 2-4 minutes at 11,000 – 16,000 x g -Transfer the supernatant (~900 µl) to the Zymo-Spin IIN column without disturbing the cell debris pellet -Place the column in a Collection Tube and centrifuge for 15 seconds -Discard the flow-through and place the column back into the collectio...
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Week of 2/21

Introduction This week in lab, we performed a gram stain on bacteria D.Ficus. Being that it was my first week in the lab, I needed to learn proper labeling and basic techniques used in this lab. The purpose of gram staining is to verify the inoculated colonies have no contaminants. The gram stain allows for differentiation between gram-positive and gram-negative cells. The first two dyes used in a gram stain, violet and iodine, form the CV-I, or crystal violet complex. The crystal violet complex cannot be easily rinsed from gram-positive bacteria because the thick peptidoglycan wall traps it. Gram-negative bacteria, however, have a thin layer of peptidoglycan in their cell wall, which cannot hold the dye. The final dye, safranin, is simply meant to stain the gram-negative cells so they will still show up under the microscope despite having been rinsed of the CV-I complex. Since D.Ficus is a gram-positive bacteria, the expected result is that all of the cells on the slide will be violet...
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Week of 4/29

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  Introduction This week was spent on researching and beginning the design for my own project, to be started in the upcoming fall semester. I have been asked to turn my focus towards CRISPR. In recent experiments, D. xinjiangensis failed to withstand ultraviolent radiation at a concentration of  30000µJ/cm 2  . My idea is to utilize CRISPR to edit the genes of D.xinjiangensis to have a more efficient DNA repair mechanism. Body CRISPR DNA Repair Notes - homologous recombination: nucleotide sequences are exchanged between two identical DNA molecules (this is a DNA method) - mutagenesis: process by which an organism's DNA changes, resulting in a mutation -DDR- DNA damage response - PprI-DdrO: a. DdrO represses transcription of DDR genes before stresses occur b. PprI is activated after sensing of DNA damage signals c. PprI cleaves DdrO through an unclear mechanism d. DDR genes unregulated until damage is repared - Proteins involved in Deinoccoccus DNA damage response: PprI,Pp...
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