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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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Week of 2/26

 Introduction This week, we performed a successful inoculation of D. aquaticus from plate to broth. The broth was used later in the week for DNA extraction using the Zyppy Plasmid Miniprep Kit. Procedure The following steps were followed to inoculate D. aquaticus from plate to broth: - A flask of appropriate size should already have media in it and be labeled with initials, date, bacteria name, and type of media. The flask should have been autoclaved and made ready for inoculation. - Gather a bunsen burner, an inoculation loop, and a plate. - Heat the inoculation loop over the bunsen burner flame to sterilize the loop, and let it cool for about 20 seconds. - Open the flask and have the lid in between your fingers underneath the loop. - Swipe the lip of the flask over the bunsen burner flame to sterilize. - Open the plate just enough to collect one colony with the inoculation loop. Close the plate. - Place the loop with the colony into the flask and sweep it around to spread the bac...
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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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