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

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 a plate) 

- 75  mL of TGY 3:3:1 broth with agar (will have chloramphenicol added to it when creating plates and will be used to inoculated our D. rad that has been transformed with the pRad1 plasmid and which has the chloramphenicol-resistant gene) 

- Two more sets of 75 mL of TGY 3:3:1 broth with agar (will be used to inoculate regular Deinococcus radiodurans and regular Deinococcus aquaticus) 

Out of this, later on in the week we were able to create 7 different plates (labeled A-G) onto which we will be inoculating our various bacteria next week (we even had enough broth to create a 4 extra sets of plates). Here are the plates made along with what specific bacteria will be inoculated on them: 

A) LB plate + Ampicillin [ug/mL] (will be used to inoculate E coli with the pRad1 plasmid) 

B) LB plate + Ampicillin [ug/mL] (will be used to inoculate E coli with the pRadZ1 plasmid) 

C) LB plate + Ampicillin [ug/mL] (will be used to inoculate E coli with the pRadZ3 plasmid) 

D) A regular LB plate (without any antibiotics) (will be used to inoculate regular E coli) 

E) TGY 3:3:1 plate + Chloramphenicol [ug/mL] (will be used to inoculate D. rad that has already been transformed previously [by our lab mentor Jonathan] with the pRad1 plasmid) 

F) A regular TGY 3:3:1 plate (without any antibiotics) (will be used to inoculate regular D. radiodurans) 

G) A regular TGY 3:3:1 plate (without any antibiotics) (will be used to inoculate regular D. aquaticus) 

We also had enough to create 1 extra [regular] LB plate and 3 extra [regular] TGY 3:3:1 plates. 

On another note, in terms of our  transformation of Deinococcus radiodurans with the pRad1 plasmid (which is 6,809 base pairs long), we did run another transformation this week and are hoping to see results next week. However, one disappointing thing was that when we ran day one of transformation, which includes combining the plasmid with the competent d-rad cells along with allowing the solutions to sit nice followed by inoculation with agitation every 15 minutes, our growth the next day showed negative numbers (which isn't the best) via the OD600 reading (and blanking with TGY) on our nanodrop, although upon visual inspection, our lab mentor notice that we might have some potential growth. 

If there were no growth whatsoever, the numbers should have actually been zero rather than negative numbers, which to me means one of two things: 

1) There was, in fact, no growth, and the cause of the negative numbers could be that the TGY that the D. rad was inoculated in was almost 2 months old (and we blanked with TGY set aside in an eppendorf tube for the purpose of being used as a blank for the nanodrop -- but we're not quite sure how old that TGY was or who made it -- which could account for the difference in readings) 

Or... 

2) We did have some type of growth, but the difference between the TGY that we used to inoculate and the TGY set aside for blanking the nano drop caused the negative readings. If this was the case, and since the OD600 setting is based off of density, the TGY used to blank could have had a higher density due to a difference in ratio of ingredients used to create it (tryptone, glucose, and yeast), or it's possible (though unlikely due to visual inspection and aseptic keeping of it) that the TGY was contaminated.

All in all, we still ran the full process of transformation and plated on Friday morning. The hope is to see growth by Monday or Tuesday of next week. If we don't see any growth, we are running another transformation either way next week on Tuesday (with part 2, plating, to happen on Wednesday morning, and with the hopes of seeing growth the following week, which would indicate transformation).