Revised UV Exposure on Deinococcus xinjiangensis

 Introduction 

Previously,  D.xinjiangensis was grown in 1 TGY and 2 R2B broths and exposed to UV radiation in 10 intervals from 100,000-999,999μJ/cm² . Growth was shown in majority of energy levels for cells grown in TGY broth whereas no growth was shown for either of the cells grown in R2B. Some adjustments to find a threshold for the R2B cells include: narrowing the domain of energy in X100μJ/cm² ,  exposing in the same media , and shortening the duration of exposure. We're going to do a short experiment on 2 broths grown in R2B only to better understand a threshold for these cells.


Methods


To prepare, two R2B samples were grown overnight and normalized to an OD value of 1. Next 50μL were pipetted from these cells and exposed on parafilm to 50,000μJ/cm² for 2 minutes.  This step was repeated for the energy level of 100,000μJ/cm². Next, 20μL of irradiated cells were transferred into a tube containing 480μL of R2B , to make a total volume of 500μL. These 2 pairs were then inoculated two seperate R2A plates and incubated for 4 days at 30 degrees Celsius. Lastly, non-irradiated cells were also inoculated on R2A plates and followed the same incubation treatment. 


Results


Irradiated and non-irradiated cells post-incubation


Discussion

No growth of D.xinjiangensis was found post-UV exposure. However, contamination can be seen on the 50,000μJ/cm² R2B plate. Non-irradiated cells also showed growth, but contained a different dilution factor than the cells that were treated. In the future, it would be best to increase number of datapoints, and the domain of energy exposure should be 10 intervals between 100-50,000μJ/cm² . Additionally, all samples need to be diluted with the same dilution factor pre-inoculation. Lastly, the dilution factor needs to stay 1:10, so media needs to be decreased from 480μL to 180μL.







Comments

Post a Comment

Popular posts from this blog

Colony PCR Amplification of 7kb Plasmid from Deinococcus aquaticus and cmr from E. coli's pRAD1 Plasmid

Image
 Introduction  This semester, Leilani Boren, Micheal Dockham and I are transforming the 7kb plasmid from Deinococcus aquaticus with E.coli's chloramphenicol resistance gene via PCR amplification and Gibson assembly. Once assembled, the 7kb plasmid will be farmed and reintroduced into D. aquaticus. To do this, we must first prime D.aquaticus' 7kb plasmid as our vector and E. coli's chloramphenicol resistance gene as our antibiotic resistance gene via PCR.  DNA cloning works by inserting a target gene (cmr) into a plasmid (7kb) . Afterward, the plasmid is introduced into bacteria (E.coli) via transformation. Colonies will be screened via selective media (LB + chloramphenicol) and, once verified, will be used to farm recombinant plasmid DNA.  Typically, Plasmid DNA is manipulated via restriction enzymes which will cut at cloning sites to create an area for our fragment to be introduced and hybridized. However, we will be using the Gibson Assembly due to successes in combining
Read more

Restriction Enzyme on Deinococcus Aquaticus 7kb Fragment

Image
 Introduction Last week in lab, two largely concentrated samples of the 7kb fragment from Deinococcus Aquaticus was obtained. However, the gel showed this sample to be at 14kb. A theory for this is that two 7kb fragments are mineralized together. Therefore, we are using restriction enzyme to demineralize these fragments and have them appear at 7kb. If this isn't the case, we will use primers. Restriction enzymes are sequences that are recognized by restriction enzyme to cut plasmids. We cut at the restriction site xbaI (1188 base pairs) but this should show as 7kb on the gel.  Procedure A 1mL tube was filled with 68ul pcr water, 10ul cutsmart buffer, 2ul restriction enzyme , and 20ul of our plasmid isolation samples from the week prior (2mg). Once we have a 100ul total , we will incubate this tube on a heat block at 37 degrees celcius. Due to not knowing how long this enzyme will take to completely cut at all of the restriction sites, we will use 4 incubation time periods. These ti
Read more

Using New GelRed on Edited 7kb PCR Reactions and Extractions

Image
 Introduction  Recently, a heat gradient from 50 °C-66 °C   on the 7kb plasmid from D.aquaticus was ran and contained no visible banding. Due to an empty agarose gel, this may reveal that the template we're using ( boiled cells ) doesn't contain the 7kb plasmid. This would mean our primers aren't able to bind appropriately. Another possibility is that our annealing temperature. Therefore, the alternative bead-beating template will be used in a PCR reaction and run on the more sensitive GelRed agarose gel.  Additionally, a new lysing protocol involving proteinase k and lysozyme will be used to try to extract 7kb plasmid. A theory to test is that we've obtained 7kb plasmid and hadn't seen bands due to the previous dye needing >500ng of template DNA. This new GelRed should only need 5-50ng of DNA to bind visibly.  Methods To run LongAmp PCR products, PCR settings were set to the following: initial denaturing was set to 4 minutes at 94 °C, denaturing for 30 seconds a
Read more