S-STEM Scholar Micheal Dockham's Blog

 Began performing "carnage test" on aliquoted D. ficus culutres stored in the -80C Trest was to find optimal settings with the machine, based on our theory that the more "carnage" the higher the likelihood of transformation success.      40ul from -80C were thawed and placed in the cuvette for electroporation.          Settings were:               3000V               25uF              Infinite resistance, which uses then natural resistance of the cells versus a dialed in resistance.               Cells were then diluted and plated, placed in the 37C for 24 hours and observed the next day               No "carnage" was observed and plate had full growth. Later in week performed experiment again on 2 samples with different settings, to observe any ...

 Semester begins.  Week was mostly spent making media and prepping for upcoming projects. Monday 8.12 Made media for the week     200ml TGYB     250ml TGYA Tuesday 8.13     Inoculated D. Ficus plates from freeze back Wednesday 8.14     OD values were taken, however values were too low and culture was left to grow.     Started prepping for Glycerol Washing. Friday 8.16     Performed glycerol wash on roughly 400ml of  D.ficus culture for future electroporation experiments     Created 40ul aliquots as well as many 100ul+ aliquots as well  

 Tuesday 5.28 Prepped TGY broth for cultures (5x 20ml) Prepped TGY agar for plates (300ml) Discussed experiment and transformation with Dr. Tuohy Experimental Plates, (Predicted Result) TGY, (+) TGY +cmr, (+) Control Plates TGY, (+) TGY +cmr, (-) Wednesday 5.29 Poured TGY plates - 6 Poured tgy +cmr 3ug/mL - 2 Performed Transformation protocol - pRAD1 one uptake into D. ficus

  The flexibility of UV-inducible mutation in Deinococcus ficus as evidenced by the existence of the imuB–dnaE2 gene cassette and generation of superior feather degrading bacteria Introduction This era of the genome has brought in an unprecedented opportunity to engineer microorganisms for a multitude of diverse biotechnological applications. To accomplish this, the work and research requires rigorous scientific approaches rooted in methodical experimental design and execution.  This paper explores the SOS pathway of TLS polymerase, mutagenesis and the generation of superior keratin breakdown pathways. Where polymerase I-III can stall at UV damage such as thymine dimmers in DNA strands, polymerase IV/V or TLS, Translesion polymerase, is able to move along and repair the damage, however by doing this it also induces mutation, a method of survival to continue the replication cycle of DNA. The team then continues to explore these genes by performing knockouts to deduce that the i...

 5.7.24          Spent majority of the lab prepping and running gel to confirm the presence of pRAD1 plasmid in plasmid extraction performed the week before hand. Electrophoresis Gel - 30ml @1% with new dye     2ul - Plasmid     8uL - PCR H20     2uL - Dye     6uL - 1Kb Ladder Gel ran @ ~70mA for just under an hour.  This left us with an unusual band amount of 4. Where typically we see 3 bands, a supercoiled plasmid, linear plasmid and circular plasmid, here we see four. Went home to research and worked with team to and decided to check plasmid extraction kit for contamination.  It appears I have a multimer, and have been suggested to linearize my plasmid via restriction site and then run a gel again. Will approach team with this idea next week.  5.8  Plasmid extraction kit contamination results.  No contamination found in reagents. 

 This week has spent planning and prepping for the transformation of D. Ficus.  4.29 TGY media for plates was too concentrated agar wise, remade to 500ml from 250ml and re-autoclaved. 4.30 Poured 14 TGY plates for future inoculation.  2 TGY plates were inoculated w. D. Ficus cells were from February, may not grow. Need to observe.  Made 5x flask of 50ml of LB broth for future E. Coli inoculations.  .1ml/flask of amp(25mg/ml) 5.1 Inoculated 2x 20ml TGY flask with D. Ficus after positive gram stain results Inoculated 2x 50ml LB flask with E.Coli, added 100ul of 25mg/ml amp. Discussed further plans of plasmid extraction and component cell protocols and material request. Planned for transformation next week. 5.2     Gram stained all cultures, both E. coli and D. Ficus All good results Took OD values of chosen cultures.  E. Coli 2 OD 600 = 2.10 D. Ficus 2 OD 600 = 2.93 Held Biotech Club Meeting Prepped for Plasmid Extractions and Component Cell Protoco...

 This week has been spent in preperation for the ANAS conference that is taken place this weekend.  Weve spent the last week or so finishing up experiments, writing abstracts and drafting our poster that will be printed and displayed at the event.  I have mostly contributed to the formatting and designing of said poster, while the others have contributed greatly to the actual experiment. This has been great prep for the next and future conferences, as I will be presenting my own research and will have to go through this process again.