Week 12 (11/28/22 – 12/1/22): Could It Be? Almost!

Although this week resulted in some not-so-exciting news, we may have made some progression in our research. We ran another transformation on Deinococcus radiodurans, as per usual, and this time after allowing our plates to sit over the weekend in the incubator, our +Cm plates had actual growth on them, with colonies that were of the same shape and color as radiodurans should be when looking head-on at the plates. This alone was quite exciting because out of all the transformations we have run so far, this was the first time we saw this type of growth on our plates (at least growth that wasn't deep red and indicated Serratia!). 
It was quite exciting initially because it looked like we could have possibly actually done it this time! Alas, our gram stains unfortunately told us otherwise.

Now, what was interesting was that we had actually done a Gram stain after plating our competent cells to make sure that the cells were viable and that they were not contaminated, and they did seem just fine. However, when we finally did do the Gram stain on what we thought was our transformed cells, we saw none other than.... Contamination. Just let us back to redoing a Gram stain on our competent cells to make sure that there was nothing that we missed, and this time, although we did see growth of the proper bacteria, one of the slides from one of our competent cell plates did show contamination (if you look closely, you can see tiny purple dots alongside our purple tetrads (radiodurans) on plate/slide A2: 
Here are photos from the gram stains that we did on what we thought were our transformed cells: 

However, not all hope is lost! If you look at the Gram stain of slide B1, you can actually see tetrads! (Albeit maybe only about one or two of them). The little purple tetrad right in the middle of slide B1 is exactly what our transformed radiodurans should look like, and this is on a +Cm plate! So personally, I do believe that we got transformation but that whatever those other bacteria are overtook the growth of radiodurans, and although this contamination is not the best news, it does give a bit of hope after 4 months of work on this project!

We will be running one more transformation before the break ends, and for this one specifically, we are going asceptic and will be working under the fume hood to hopefully use the UV to help kill any bacteria on any item that we may have, and just prevent any other bacteria from getting in any of our material. 

As a final check, we went ahead and played it all for of our sets of competent D. rad cells from freeze back, because we have a theory that this is where our contamination could have been coming from this entire time! If this is the case, we'll have learned a solid lesson in asceptic techniques (and could have potentially saved ourselves some tears along the way :)) I guess we should see what happens when we do our gram stains next week!

Comments

Post a Comment

Popular posts from this blog

Week 11 (11/14/22 – 11/18/22): Inventory & Prep

Image
This week's focus was on preparing for getting what I can done within the next 4 weeks before it is time to wrap up the semester for the holiday break. One thing I did implement was creating a more streamlined process of keeping track of our inventory so that we can always know how much of what material (and of what bacteria) we have in stock at all times. Because of this, I created quite a bit of broth, both with and without agar, so we can have what we need for the next few weeks. The following was created:  - 200 mL of LB broth (will be used for any kind of inoculation of E. coli)  - 200 mL of TGY 3:3:1 broth (will be used for any kind of inoculation of Deinococcus radiodurans or Deinococcus aquatics)  - 200 mL of LB broth with agar (will have ampicillin added to it when creating plates and will be used to inoculate any of our E coli that have plasmids with the Ampicillin-resistant gene)  - 75 mL of LB broth with agar (will be used to inoculate regular E coli onto...
Read more

Week 3 (9/19/22 – 9/23/22): Back to the Drawing Board

Image
  This was the week where after running transformation and plating on Friday (a third attempt), we were hoping that we would have transformed Deinococcus radiodurans such that it would have shown antibiotic resistance via integration of the pRADZ3 gene into its own genome. However, this unfortunately didn’t happen, for reasons that we are unsure of (so far). When we checked our plates on Day #4, 5, there was still no growth, indicating no transformation. Because of this, it is time to go back to the drawing board and are looking over every piece of the transformation protocol, from inoculation of E. coli (with its necessary plasmid that will be extracted for transformation) and   inoculation of D. rad (which is the species of Deinococcus that will be used for transformation) to plating of the hopefully transformed bacteria.   Now, an interesting factor to consider while looking at steps to transformation [on paper] for a third time   is to consider a factor that ...
Read more

Week 13 (12/5/22 – 12/9/22): Preparation for One More Trial

Image
This week, since we only have one week left of school until the semester ends, we are in the wrap up phase to where we have discovered that we can do one more transformation run. This will be our last chance of the year to see if we can attempt transformation without contaminating any of our items, as this seems to me the most prominent issue that we have had throughout the semester. Now, what was interesting was that we decided to do gram stains of all of the bacteria that we used for a transformation that was immediately taken out of the shaking incubator on day two of transformation, right before we plated them. We actually thought that we would find contamination in at least one of these tubes, but alas, every single one of them was a perfect gramstain of just the bacteria we needed. Because of this, and because the gram stains of all of our competent cells from freeze back where sterile, we could only then conclude that contamination occurred on transformat...
Read more