Week One

There are a vast number of bacteria species in the environment, the species in focus is Deinococcous.  The interest in Dienococcous has come about because of its desirable characteristics. The Dienococcous species are the most radiation-resistant micro-organisms that have been discovered and have genes that encode cell cleaning systems that eliminate organic and inorganic cell toxins (Gerber, 2015). This demonstrates it capacity to persist under multiple stressors and the efficiency to repair DNA damage. There is still much to be learned about the species of Deinococcous, thus this project will be testing Deinococcous Aquaticus’s susceptibility to the antibiotic tetracycline. 

Deinococcous Aquaticus is a gram-negative bacterium, so it appears pink under a microscope after a gram stain (BacDive, 2023). Gram-negative describes the structure of the cell wall. It appears pink because the peptidoglycan cannot retain the crystal violet, the first dye added, because it is under an outer membrane and lipoproteins. The outer membrane of the cell wall has protein pumps and lipopolysaccharides which may affect its susceptibility to the tetracycline. 

Tetracycline is a protein synthesis inhibitor. It works at the ribosomal level, inhibiting the 30S ribosomal subunit, preventing the aminoacyl-tRNA to the acceptor site on the mRNA ribosome (Shutter, 2023). The bacteria can no longer grow or replicate. There are two known mechanisms of tetracycline resistance, including ribosomal protection proteins and efflux pumps. This efflux pump has been found in Aquaticus. This bacterium has genes tetA and tetR, tetA codes for the efflux pump while tetR codes for the regulator of the pump. TetR binds to tetracycline allowing it to release the repression of tetA transcription. This system in Aquaticus will be explored this semester.

 

BacDive. (Updated 2023 Dec 14). Deinococcus Aquaticus PB314:Type strain: DSM 1876, KCTC 12552, NBRC 101311: Bacdrived:3882. https://backdive.dsmz.de/strain.3882#:~:text=text+Deinococcus%20aquatius%20PB314%20%7C%20Type%20strain,%2C%20NBRC%20101311%20%7%20BacDiveID%3A3882&text=Deinococcus%20aquaticus%20PB314%20is%20an,freshwater%20%with%20sediment%20of%20Gapcheon

Gerber, E., Bernard, R., Castang, S., etc. (2015, July) Deinococus as new chassis for Industrial Biotechnology: Biology, physiology and Tools. Journal of applied microbiology. https://www.ncbi.nlm.nih.gov/pmc/arcticles/PMC4682472/#ref-list-a.h.atitle

 Shutter MC, Akhondi H. Tetracycline. [Updated 2023 Jun 5]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2024 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK549905/#

Comments

Post a Comment

Popular posts from this blog

Week Eight

Image
  The next step to perform transformation is to collect the plasmid. E. coli containing pRAD1 was streaked on 50 ug/ml ampicillin LB plates and placed in the 37-degree Celsius incubator. After 24 hours in the incubator a gram stain should be performed to ensure the correct bacteria has grown; E coli, depicts gram negative rod s. A 20 ml 50 ug/ml ampicillin LB broth flask is inoculated with a colony from the plate and placed in the shaking incubator. The next day it is ready to be used for plasmid extraction. A procedure that breaks open the cell membrane, releasing genetic information , and discards the cell debris.   Materials:   -Microcentrifuge tubes   -Centrifuge   - pRAD1 E. coli culture   - Zymo research plasmid extraction kit     Protocol:   1. Add 600 ul of bacterial culture to a 1.5 ml microcentrifuge tube.   2. Add 100 ul of lysis bu ff er and mix by inverting the tube 4-6 times.   a. The next step must be c...
Read more

Week Seven

  This week marks the start of the transformation process of D einococcus caeni . Before we are able to perform the transformation procedures there are a number of things that must be prepared. Transformation occurs naturally in the environment under selective pressures that cannot accurately repli cated, thus the bacterial cells ability to be transformed must be included . We call these included cultures comp etent cells . Firstly, competent cells of   the bacteria in focus must be prepared. The comp etent cell are chemically prepared in the lab. Sodium chloride, or salt, is used to neutralize the negative charge of DNA and the phospholipid bilayer in the cell membrane, promoting the DNA to move closer to the cell. The cells are stored in the –80 degrees Celsius freezer to maintain the permeability of the cell membrane while the glycerol prevents the cells from freezing and lysing.     Materials:   -TGY broth   -Glycerol 60%   -DI wate...
Read more

Week Three

Image
Currently in the lab Deinococcus Canei is being sequenced. Though the genome has not yet been fully sequenced, it can be said that D. Canei is closely related to Deinococcus Aquaticus. With that, D. Canei susceptibility is tested this week to determine if it has an efflux pump, and if so, identify how useful it is against the antibiotic tetracycline. In the last test with D. Aquaticus the concentrations 0 ug/ml, 10 ug/ml, 100 ug/ml, and 1000 ug/ml. The only plate that had growth was the 0 ug/ml, the only plate that did not contain tetracycline. Since, these concentrations were bactericidal the concentrations used for this experiment were lowered to 0 ug/ml, 10 ug/ml, 50 ug/ml, and 100 ug/ml. Materials:  Liquid culture of D. Canei  Four flasks of 25 ml TGY with agar Tetracycline with stock concentrations of 2.5 mg/ml Hot plate Vortex Micropipette and corresponding tips Procedures: Inoculate a flask of 10 ml TGY with a colony from D. Canei plate Place in shaking incubator for 72...
Read more