S-STEM Scholar Elijah Diaz Blog

This week, we checked the growth of transformation plates from the prior week, but no growth was observed. The control plates showed only pigmented growth, and the plates exposed to UV radiation on normal TGY also did not show any growth. Additionally, rehydrated normal D. rad. cells for the NASA project failed to grow. To address these issues, we planned and researched a new transformation protocol and successfully identified one specifically designed for D. rad. The protocol is as follows: 1. Take 5 mL of fresh D. rad. growth (broth) and transfer it into a 15 mL tube. 2. Add 1 mL of CaCl_2 to achieve a final concentration of 30 mM. 3. Incubate for 80 minutes at 30°C, then transfer 1 mL to a 1.5 mL tube. 4. After incubation, add 10 µL of plasmid and hold on ice for 30 minutes. 5. Dilute each tube with an additional 9 mL of TGY broth and incubate for 18 hours at 30°C. Using this new protocol, we performed three transformations. We also prepared one plate of normal TGY with two sampl...

This week, we rehydrated D. rad. growth for the NASA project and plated it onto respective plates for desiccation. A Gram stain of 3-way D. rad. from a plate confirmed it was free of contamination, and this culture was inoculated into a new TGY Kanamycin broth. We also passaged 4-way E. coli into fresh broth and performed three plasmid extractions from the culture. Successful growth of 3-way D. rad. in broth was verified by Gram stain, and a Gram stain of 4-way E. coli confirmed its growth in broth was also free of contamination. On 11/12, we conducted a gel on the plasmid extractions, which showed strong bands around 10,000 base pairs, as expected. Three transformations were performed, though contamination was found on one of the transformation plates. To address this, we re-inoculated the transformations and exposed them to 200 microjoules of UV for five minutes. Additionally, we conducted DNA extraction on 3-way D. rad. four times, with three of the trials yielding very favorable re...

This week, we inoculated 3-way D. rad. from a plate into TGY broth with 8 µg/mL Kanamycin but continued to encounter difficulty with its growth in the broth. We passaged 4-way E. coli into a new LB ampicillin broth, but a Gram stain revealed signs of contamination. To address this, we inoculated new LB ampicillin broth with 4-way E. coli from a freezeback and prepared 250 mL of LB Agar. A Gram stain of the new 4-way E. coli growth confirmed it was free of contamination. For the NASA project, desiccated D. rad. was unable to grow, so we inoculated normal D. rad. into TGY broth and R2B for desiccation. A Gram stain of this D. rad. confirmed it was also free of contamination.

This week, we inoculated 4-way E. coli into LB broth with 50 µg/mL ampicillin and performed a Gram stain on the growth, confirming it was free of contamination. The growth was then passaged into another LB broth with 50 µg/mL ampicillin for further experimentation. We also redid the NASA project run by inoculating D. rad. onto R2B and TGY broths. Additionally, we performed four plasmid extractions, but the nanodrop readings from these extractions were not favorable. Due to the less-than-ideal nanodrop values, these plasmid extractions were ultimately not used.

This week, transformations performed on 10/16 and plated on 10/17 were not successful. In preparation for a new run of transformations, we made two 250 mL batches of TGY Agar. Using these, we poured 10 TGY Chloramphenicol plates with a concentration of 3 µg/mL and 10 TGY Kanamycin plates with a concentration of 8 µg/mL. Additionally, we ran a gel on three plasmid extractions, but the results did not look favorable.

This week, we conducted two Gram stains on potential 4-way D. rad. growth observed on a Chloramphenicol plate and passaged this growth onto a new Chloramphenicol plate. We prepared 250 mL of TGY Agar and poured 4 TGY Chloramphenicol plates with a concentration of 3 µg/mL and 5 regular TGY Agar plates. A Gram stain was performed on the new Chloramphenicol plate with potential 4-way D. rad. growth, and the growth was passaged again onto another new Chloramphenicol plate. However, a subsequent Gram stain on this most recent passage revealed that the transformation was unsuccessful and contaminated. We planned to perform new transformations and are considering exposing plates to UV light immediately before incubation to improve results.

This week, we poured 10 TGY plates with 8 µg/mL Kanamycin and inoculated 3-way D. rad. into 25 mL of TGY with 8 µg/mL Kanamycin broth. We performed three transformations using previous plasmid extractions, but unfortunately, the transformations did not succeed. To check for contamination or other issues, we Gram-stained 3-way D. rad. from older broth stored in the fridge. Additionally, we rehydrated desiccated D. rad. cells using a rehydration buffer. We also passaged 4-way E. coli into fresh broth and inoculated more normal D. rad. in preparation for the NASA project. Finally, we performed three additional plasmid extractions on 4-way E. coli. The nanodrop values for these extractions are included via image.

This week, we inoculated 3-way D. rad. into TGY with 8 µg/mL Kanamycin broth on Monday but encountered issues with its growth in the broth. DNA sequencing efforts did not yield favorable results, as the equipment used was previously used too many times. In preparation, we made various media. Additionally, we inoculated normal D. rad. into TGY broth and R2B in preparation for the NASA project. To ensure no contamination, we performed Gram staining on both cultures. Both of which looked free of contamination.

This week, we performed three transformations: one using plasmid extraction A2, one with B2, and a control transformation with tube C2. We poured 20 plates in total, including 5 with 3 µg/mL Chloramphenicol TGY, 5 with 8 µg/mL Kanamycin TGY, and 10 with normal TGY. We passaged 3-way D. rad. into fresh TGY broth and performed DNA sequencing on the 4-way E. coli plasmid extraction, with results expected the following Monday. Additionally, we planned to carry out genomic DNA extraction on 3-way D. rad. in preparation for future experiments.

This week, we poured 10 R2A plates and noted that the transformations performed on 9-12 and inoculated on 9-13 were unsuccessful. We tested Kanamycin’s efficiency on normal DRAD while also inoculating the most recent competent cells onto TGY with Kanamycin. To prepare for upcoming experiments, we planned additional plasmid extractions and made more media, including R2B and other types. We attempted to plate the previous transformation onto a TGY, Chloramphenicol, and Kanamycin plate and passaged 4-way E. coli into fresh LB ampicillin broth.

Having seen no success with our failed transformations, our group decided to begin considering a second project to work on simultaneously with our mutagenesis project. This project's goal is to test the limits of D. radiodurans' ability to repair its own DNA after extreme desiccation and radiation exposure. In addition to project planning, we inoculated the 3-way culture onto 8 µg/mL Kanamycin TGY broth and prepared 200 mL of TGY agar. We inoculated competent cells and the 3-way culture onto fresh plates and verified nanodrop readings for both. Additionally, we prepared new competent cells and performed three transformations using the most recent plasmid extractions.

The beginning of our week, Tuesday, saw us running a gel on our plasmid extractions from the prior week. The gel showed our plasmid to be the correct size, around 9000bp. The samples looked slightly dirty however, but still far better than the prior ones. We then conducted three plasmid extractions using our plasmids. These transformations, unfortunately, were again unsuccessful. In addition to being unsuccessful, our plated transformation plates showed contamination. We again gram stained our competent cells to esnsure this was not the source of contamination. These gram stains again showed no contamination. We were able to get successful growth of our 3-way D. rad by first inoculating from freezebac onto normal TGY plates free of antibiotic, then passaging that growth onto our typical 8µg/ml kanamycin plates. From the antibiotic plates, we then were able to get successful growth in antibiotic broth. The end of the week saw our group making new media for preparation of the following ...

Having ran linear PCR on our primers the prior week, we began the week with running a gel on the PCR products in addition to our most recently made plasmid. Our primers looked sub-optimal on our gel, but this was most likely due to an error in determining the annealing temperature for our reverse as opposed to bad primers. The plasmid however, looked quite bad. Despite it's nanodrop values being optimal, the sample on the gel looked quite dirty. Despite this, three transformations were conducted. These transformations, as expected, were unsuccessful. They appeared to show contamination. This raised concerns about our D. rad competent cells being possibly contaminated. We performed gram stains on these cells and they did not show any signs of contamination. This week we also observed our 3-way unable to grow onto 8µg/ml kanamycin plates from freezebac. We attempted to again inoculate onto antibiotic plates, having to wait until the following week to see results. After our plasmid sh...

Being the first week back of the semester, the beginning of this week consisted of primarily prep work. On the first day back, our group took inventory of our supply of media in order to determnine what media needed to be made. We gram stained our 3-way D. rad as well as our 4-way E. coli freezebacs to ensure we were working free of contamination. We then inoculated both bacteria samples onto fresh media. Due to struggles with transforming our D. rad, we planned and ran linear PCR to confirm our primers. In addition to this, compotent cells of D. rad were created for future transformations.