Sp'23: Week 5 (2/13/23 - 2/17/23): Going for Transformation of Aquaticus

Before I came into lab this week, my lab mentor went ahead and took my two tubes of inoculated E. coli with pRad1 (tubes "2.10A" & "2.10B") in LB + Ampicillin [50 ug/mL] out of the incubator and placed them into the fridge, this way, when I came into lab on Wednesday, I could go ahead and check the growth of my E. coli. After vortexing each tube and doing a nanodrop (OD600) reading, the following results were obtained: 

(Figure A) The above shows the two tubes ("2.10A" & "2.10B") that I did OD600 readings on, each of which showed strong growth of 2.28 & 2.20 for Tubes 2.10A & 2.10B respectively (ideally numbers of at least 1 are preferred). 

After this, I ran a plasmid extraction on each of the two tubes using the Zippy kit (Box B) ("ZymoPURE Plasmid Miniprep Kit"). This was the same type of kit but a different box used (last week I used Box A). The results are shown below: 

(Figure B) These are the results obtained from the dsDNA nanodrop readings from Tube 2.10A (first row, concentration of 80.0 ng/uL) and Tube 2.10B (second row, concentration of 75.8 ng/uL). 

(Figure C) This shows the Zippy (Box B) extraction kit / setup that I used for running extraction of [ideally] pRad1 with E. coli from Tubes 2.10A & 2.10B. 

The dsDNA numbers obtained were not ideal, as we are hoping to work with pRAd1 concentrations of at least 200 ng/uL for transformation of Deinococcus Aquaticus. One theory I have as to why our numbers were so low is that the E. coli used to extract pRad1 was obtained from plate "2.1_" which did not have the added Ampicillin stressor (with a concentration of 50 ug/mL). Now, because these E. coli from the non-Ampicillin plate were inoculated into Tubes 2.10A & 2.10B, each of which held the mic level of 50 ug/mL of Ampicillin, and even though both tubes had strong growth of E. coli, it's possible that them coming from a non-Ampicillin plate affected the plasmid or the E. coli, or that they were affected during the extraction process more so because they hadn't originally been inoculated from an Ampicillin-positive plate. 

(Figure C) These two 2mL microcentrifuge tubes ("2.15A*" and "2.15B") hold the extracted plasmid. Tube "2.15A*" showed a concentration of 80 ng/uL with the dsDNA nanodrop reading, while "Tube 2.15B" showed a concentration of 75.8 ng/uL). On the sides of the microcentrifuge tubes are written the original amounts obtained (each extraction resulted in 25uL of plasmid), along with amounts of plasmid used. Example: Tube "2.15A*" shows 2uL of plasmid used for the nanodrop reading, leaving 25uL of plasmid (this will be the tube that will be used for transformation), while Tube "2.15B" shows that 2uL were used for a nanodrop reading, with the final 25uL used for the XbaI digest and gel run. 

Either way, I proceeded forward and ran an XbaI plasmid digest on Tube "2.15B," and because of the original concentration obtained (75.8 ng/uL), ended up needing to use the entire tube for the digest. This is due to the fact that the XbaI digest calls for a 50uL solution that is made of the following: 

• 1 uL restriction enzyme (XbaI) 
• 5 uL of the "10x NE Buffer" 
• 1 ug of plasmid (which, with a concentration of 75.8 ng/uL, results in 13.2 uL needed for the 50uL solution) 
• PCR water (enough to total 50 uL of solution, which, in this case, would be 30.8 uL of PCR water). 

However, the smallest dose I can obtain with the micropipettes available in the lab is 2 uL (using the red 2-20 uL micropipette). Because of this, I needed to double my concentration of the above ingredients, creating a 100uL solution (this way, I can draw 2 uL of the XbaI restriction enzyme for the enzyme digest). This required 26.4 uL of plasmid needed from Tube "2.15B," of which I only had 23 uL left. So I used what I could (which was the rest of Tube "2.15B") and created a 100 uL solution for the XbaI digest using the following ingredients and dosages: 

• 2 uL restriction enzyme (XbaI) 
• 10 uL of the "10x NE Buffer" 
• 23 uL of plasmid 
• PCR water (enough to total 100 uL of solution which, in this case, was 65 uL of PCR water). 

(Figure D) This is the XbaI digest kit used that contains the XbaI restriction enzyme and the "10X NE Buffer," both of which are sensitive to temperature and have to be kept on ice when running the XbaI plasmid digest protocol (as they are otherwise kept in the -20 degree Celsius freezer). 

(Figure E) Here are the settings used on the PCR Thermocycler that have been used each time (standard XbaI digest protocol) to finish running the XbaI digest. This time, I used the Thermo Cycler machine named "Franklin." 

After finishing the process of running an XbaI plasmid digest on our extracted plasmid from microcentrifuge Tube 2.15B, gel electrophoresis was run using a 1% gel (made by creating a solution of 300 mg "agarose" + 30 mL "1X TAE buffer"). Typically, this type of gel run takes approximately 35 minutes to finish, but after 45 minutes, the gel run wasn't even halfway through. This could have been the result of perhaps expired buffer, or even incorrect measurements of ingredients used to make the gel. Either way, due to time constraints, the gel needed to be stopped after approximately 45 minutes. But, lo and behold, after verifying with my lab mentor, the gel showed that we more than likely did have pRad1 plasmid to work with: 

(Figure F) The gel run only used two columns: Column 1 (left-most column) shows the 1kB ladder with which to compare the electrophoresis gel run of the [ideally] extracted pRad1 plasmid. Column 3 shows the digested plasmid, which, even though there were 2 lines (potentially three), shows that I likely had extracted pRad 1, which is 6,809 base pairs long, and is seen in the proper location according to the ladder. 

The results of the gel run (seen in Figure F above), gave my lab mentor and I the green light to proceed with the lab's first ever attempt of the transformation of Deinococcus Aquaticus. 

Now, my lab mentor did run Day 1 of Transformation the next day, which includes the combination of the pRad1 plasmid (from "Tube 2.10A") with 100 uL of competent Deinococcus Aquaticus, along with the heat shock, followed by incubation. His part resulted in having three separate Erlenmeyer Flasks, each of which held 10mL of [ideally transformed] Deinococcus Aquaticus using the pRad1 plasmid. Each of these flasks specifically contained the following: 

• "[2.17(A)] 2-14-23 K.A. TGY Broth 10mL / J. Hill NC DAQ" = 10mL of the negative control (D. Aquaticus with no plasmid added) 
• "[2.17(B)] 2-14-23 K.A. TGY Broth 10mL / J. Hill DAR2 (10)" = 10mL of the experimental control containing D. Aquaticus [ideally transformed] with 10 uL of the pRad1 plasmid added from Tube "2.15A" (concentration of 80 ng/uL) 
• "[2.17(C)] DB 2/14 TGY 10mL / J. Hill DAR1 (12.5") = 10mL of the experimental control containing D. Aquaticus [ideally transformed] with 12.5 uL of the pRad1 plasmid added from Tube "2.15A" (concentration of 80 ng/uL) 

(Figure G) Above are each of the 3 Erlenmeyer flasks containing 10mL of D. Aquaticus that hopefully been transformed. They include Flask "2.17(A)," the negative control, Flask "2.17(B)," one of the two experimental controls, and Flask "2.17(C)," the other experimental control. 

The following day, after an incubation period of approximately 17 hours, I ran Day 2 of Transformation, which included inducing the environmental Chloramphenicol stressor by adding 100 uL of [ideally transformed] D. Aquaticus to different plates. I added 100 uL from different flasks above to different TGY plates: 

• Plate "(A) 2.17~" (with 3 ug/mL of Chloramphenicol) had 100uL added to it from E. flask "2.17(A)" 
• Plate "(B)[1] 2.17~" (with no Chloramphenicol) had 100uL added to it from E. flask "2.17(B)" 
• Plate "(B)[2] 2.17~" (with 3 ug/mL of Chloramphenicol) had 100uL added to it from E. flask "2.17(B)" 
• Plate "(C)[1] 2.17~" (with 3 ug/mL of Chloramphenicol) had 100uL added to it from E. flask "2.17(C)" 
• Plate "(C)[2] 2.17~" (with no Chloramphenicol) had 100uL added to it from E. flask "2.17(C)" 
• Plate "(C)[3] 2.17~" (with 3 ug/mL of Chloramphenicol) had 100uL added to it from E. flask "2.17(C)" 

  

(Figure H) These are the plates created from Day 1 of Transformation. Each of these were inoculated under the hood to maintain aseptic techniques.. 

After doing this, all 6 plates were placed into the 30 degree Celsius incubator for growth over the weekend. They will be checked four days later, on Tuesday 2/28, for growth and hopeful transformation!