Week Six

There are over 100 species in the Deinococcus genus that have been identified. Interest in this genus has grown as they are polyextremophiles, including the phenotype of radio resistance, and have potential bioremediation genes. These genes are capable of the degradation of complex waste mixtures, organic solvents, heavy metals and radioisotopes. For example, the Deinococcus radiodurans is the most characterized and has attracted interest for degrading radioisotope. Deinococcous geothermalis is another species that has been under the scope for removal of radioactive waste in higher temperatures. However, there is a limited amount of research that has been conducted on other species. As of now only a hand full of species have been successfully transformed, a standard laboratory technique used to integrate exogenous DNA.  Deinococcus radiodurans was the first to be transformed. However, the transformation protocol requires a microgram quantity of the specific plasmid.  

Escherichia coli is a well-researched bacterium and has become the basis for many standard laboratory procedures. For example, the pRAD1 plasmid, contained by E. coli, is most often used to determine transformability of other bacteria species. A standard protocol has been created for the transformation of D. radiodurans with one microgram of the genetic material. This amount of DNA can be difficult to collect, thus it would be beneficial to design a protocol that produces successful transformation with less genetic material.  

There is relatively little information on D. caeni. It was isolated in active sludge at a water treatment plant. Active sludge is a process in water treatment plants which utilize specific bacteria to degrade pollutants in the water. Thus, D. caeni is another species in the genus that has bioremediation potential. The capacity for transformation in D. caeni is unknown at this point in time. To determine its capacity D. caeni will be transformed using pRAD1.  

 

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