S-STEM Scholar Aland Ali's Blog

 Introduction Deinococcus. sonorensis has recently been hypothesized to be transformable. This understudied species of Deinococcus was retrieved from the Sonoran desert and a nonarid soil from a Louisiana forest. It's unique in that it forms a plaque-like biofilm which makes the cells extremely difficult to lyse. A solution we've found to this is using R2A as a selective media which affects the morphology of this species. The changes after growth on R2A make the cells easier to work with. Due to being believed to have no plasmid, and no attempts at transformation, it may be an interesting objective to try and transform D. sonorensis. Our transformation will be using the cmr gene from pRAD1 found in E. coli.  Methods To begin, competent cells of D. sonorenis   were made with R2B. Afterwards, competent cells were thawed on ice and 100μL of cells were from each tube were transferred into three 1.5mL centrifuge tubes. Next, 9μL (~647ng) of cmr PCR product was introduced to th...

 Intro Previously, cells grown only in R2B were exposed to UV for 2 minutes at two varying energy levels. These energy levels were 50,000 and 100,000μJ/cm² . Dilution factors were not 1:10, and no growth was characterized on any irradiated cells post incubation. As a final attempt to see growth in R2A cells, we will shorten the duration of exposure to 1 minute, keep dilution factors 1:10, and have 8 intervals between the values of 200-25,000/cm² . These specific values will be 200, 400, 800, 1600, 3100, 6300,12500, and 25000μJ/cm² .  Additionally, previous urease tests yielded negative results which contradict the original paper in 2009 characterizing D. xinjiangensis. To confirm our findings , we will make more urease plates and reinoculate them.  Methods 3 broths of R2A were inoculated with D. xinjiangensis and grown overnight. Afterwards, they were normalized to an OD value of 1. 24 1.5μL centrifuge tubes were filled with 180μL of R2B. After sterilizati...

 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. Th...

 Introduction The Deinococcus genus is known for its robust ability to withstand radiation, extreme oxidation, and desiccation. D. radiodurans  is the prime example in this genus for this. Previously, we had done biochemical testing on D. xinjiangensis . Now, we are interested in seeing how UV resistant D. xinjiangensis is and if the media the bacteria is grown in may affect these attributes. We will be comparing culture grown in R2B and TGY broth before exposing them to varying degrees of UV radiation in the unit of X100μJ/cm²  using a UV-Crosslinker.  Methods To begin, 1 TGY and 2 R2B 20mL cultures of D.xinjiangenis were grown overnight and normalized to an A600 value of 1 in a 1.5mL centrifuge tube. To prepare for dilution, 20 1.5μL centrifuge tubes were filled with 450μL of R2B. 10 1.5μL tubes were filled with 450μl of TGY broth. Next, 50μL of the normalized samples were pipetted onto 3 separate parafilm squares labeled A1 , B1 and C1. Letters represent biologic...

Introduction Deinococcus xinjiangensis is a faintly pink, coccus shaped bacteria isolated from the Taklamakan desert located in Xinjiang, China. This species is understudied , with one paper based on the characterization of this species in 2009. This paper found that D, xinjiangensis was gram-positive, spherical, aerobic, non-motile, pink-pigmented and sensitive to desiccation and gamma radiation but highly UV resistant. We are interested in extending off of this paper by using more biochemical tests and UV resistance tests to further characterize D. xinjiangensis . We would like to do this because often, grand ideas are found in the most understudied of species. For instance, CRISPRS were randomly found in E.coli in 1987.  Biochemical tests may require making a special media that promotes a change in growth after inoculation. Others require a reagent that act as a visual cue to test for products of a metabolic reaction.  Some require both, but all test for biochemical react...

 Introduction Previously, transformation wasn't successful with Gibson assembly product. This led us to hypothesize that primers may have too much non-specific binding due to not having ideal annealing temperature and MgCl concentrations. To test this, we will run four PCR reactions. A pair will be ran at 59°C and at 61°C. 7kb template used previously will be ran with variable MgCl concentrations. This will confirm ideal MgCl concentrations as well as ideal temperature for annealing during PCR.  Methods Our first pair of Longamp PCR samples will contain 1M MgCl and 3M MgCl, Longamp Mastermix, and 2ul of template. Initial denaturing was set to 4 minutes at 94°C. Denaturing was set to 30 seconds at 94°C. Annealing temperature was set to 59°C for 45 seconds. Extension time is 6 minutes and 1 second at 65°C. Final extension was set to 10 minutes at 65°C with an infinite hold at 10°C.  Our second pair of Longamp PCR samples will contain 1M MgCl and 3M MgCl, Longamp Mastermix, ...

 Introduction  Previously, PCR amplification was run on 7kb plasmid extracted via the Zyppy Kit and cleaned up through the Monarch PCR & DNA Cleanup Kit (5μg). When viewed on a GelRED agarose gel, 3 bands were present, 2 were non-specific to the 7kb we wanted. However, we will continue to attempt Gibson assembly using 3 times as much vector to make up for 1/3 of our sample being the 7kb plasmid. Afterward, we will transform competent E.coli cells with the Gibson assembly product.  Methods  To attempt Gibson assembly, we began by diluting our 46ng/μl cleanup sample of cmr to a concentration of 11.5ng/μl. We diluted our sample to keep our vector and gene equimolar. Next, on ice , we added 9μl of our 7kb samples to 1 PCR tube each. 1μl of our diluted cmr sample was then added to both tubes, making a total volume of 10μl in each. Next, 10μl of Gibson assembly mastermix was added to each tube. Our positive control had 10μl of control reagent and 10μl of Gibson assembl...