Inoculation of Deinococcus Radiodurans for Future 7kb Plasmid Research

Introduction


Last semester in lab, a plasmid isolation was done on Deinococcus Aquaticus. when this plasmid was run on a gel, a 7kb strand was visible.  No information was known about this mystery plasmid. Since then, our sample has denatured or was discarded accordingly. This semester, we (Aland Ali, Anh Nguyen, Rhiannon Giesegh, Mike Agneskiy) plan on farming and growing this plasmid to discover more of its characteristics. However, this week mainly consisted of a “bootcamp” of sorts that provided a refresher course of lab protocols, safety, storage, hygiene, and overall etiquette. Due to this, not much time was spent on beginning our projects until the end of the week. This time was spent creating TGY Agar plates and inoculating Deinococcus Aquaticus from freeze bag to farm our future plasmid. 


Procedure


To begin, 8 TGY Agar plates were created, labeled, and left overnight to ensure proper gel-like consistency. Next, Deinococcus Aquaticus from a freeze bag was acquired and set in an ice bucket to maintain more ideal temperature. This ice bucket, along with sterilized disposable inoculating loops were brought into a sterile environment containing a biosafety cabinet.  Surfaces of tables, plates and materials were sprayed with ethanol and wiped with standard napkins. The blower of the biosafety cabinet was turned on, the covering was lifted, and items were brought into the cabinet. It is imperative that once hands are in the cabinet, they do not come out (as it increases risk for contamination), Therefore, a second team member with sterile gloves assisted in the transfer of materials to the biosafety cabinet. Once all items were in the cabinet, inoculation of Deinococcus Aquaticus was continued as normal using a variation of spreads onto the 4-5 labeled TGY agar plates. This consisted of opening a disposable loop package from opposite end of loop, obtaining some of the Deinococcus Aquaticus sample once warm enough, and swiftly spreading the sample onto our TGY agar plates. New loops were used for each plate. Once complete, surfaces were cleaned, materials were discarded, and the UV light for the cabinet was turned on for >15 minutes to ensure sterilization and plates were stored in an incubator.  


Results 

Results of growth of Deinococcus Aquaticus are pending but should come by next week. Unused agar plates were placed in a refrigerator.





Discussion

As we wait for our samples to incubate, LB media was made for future inoculation of these potential colonies. Colonies are expected to be orange in appearance if all goes well. These colonies will be transferred aseptically to tubes of LB media and grown accordingly. The biosafety cabinet will not be used for these procedures as they are not our primary source of Deinococcus Aquaticus. 


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