Week 7 (10/17/22 — 10/21/22) Constructing a New Plan

Introduction:

This week involved evaluating MIC results from the previous week, as well as preparation/planning for a more definitive result next week. Because the results from our MIC were finalized on a day we do not come in to the lab, an additional test needs to be performed to insure we are working with D. aquaticus at the time that the growth curve should be sufficient (at 48 hours).

Methods:

TGY (3:3:1 ratio) was made on 10/18 and autoclaved overnight. 

D. aquaticus sample from 09/06/22 was inoculated onto a plate, creating 2 plates with a normal streaking pattern, and 2 plates done as a 4 way streaking pattern. Plates were stored in incubator. 

The 96-well plate was removed from the microplate reader on 10/18, and the results were analyzed.

Figure 1: Concentrations of kanamycin in each well, in ug/ml. Note that 96-well plate is upside down. Rows F-H have the same concentrations. Row C contains the negative controls (containing 250 ul TGY),  and row C contains the positive controls (containing 2.5 ul of D. aquaticus and 247.5 ul of TGY). Note that plate was inoculated upside down due to personal error. Results are still accurate.

Results: 

Inoculated plates were collected on 10/20, and because the incubator temperature was lowered after plates were added, there was no growth on any of the 4 plates. Additional plating was done on D. aquaticus on 10/21 by Kaylee, Jayce, Melanie, and Anh.

Figure 2: Minimum Inhibitory Concentration results of kanamycin with D. aquaticus after 48 hours, resulted on 10/15. Row H has antibiotic concentrations increasing from 0.75 ug/ml to 1.75 ug/ml. Rows F and G are duplicates of row H, making there 3 runs total. All samples in row C are positive controls, containing TGY and D. aquaticus. All Samples in row A are negative controls, containing only TGY.

Discussion: 

Results point to the MIC being at 1.50 ug/ml, but this is not fully supported. An additional MIC test will be run next week, with kanamycin concentrations varying between 2 ug/ml to 0.75 ug/ml. 2 ug/ml is added because the result in F12 shows a growth curve at 1.75 ug/ml. Adding 2 ug/ml concentration back to the MIC will provide a result that shows no growth. This will allow for better analysis of the MIC, because these recurrent results could point to the MIC being at 1.75 ug/ml.

MIC testing will be performed throughout the next week (10/24-10/28). 10/24 will be the day to perform the MIC test by adding the TGY, kanamycin, and D. aquaticus to the 96-well plate. 48 hours later on 10/26, the plane will be removed from the microplate reader, and 25 ul will be taken out of each tube and added into a test tube that already has 225 ul of TGY, making a 1/10 dilution. This will be done for all 24 wells that were occupied, and it will incubate in the shaking incubator at 30o C for 48 hours. On 10/28 the test tubes will be analyzed using the nano-drop to evaluate the OD600 values. If performed correctly, this will give us the correct MIC for D. aquaticus. 

Further MIC testing may be done in attempts to analyze the effects of other antibiotics on D. aquaticus. All antibiotics in the lab were collected and documented with the intent of performing additional MIC testing. Transformation of D. radiodurans must be completed before we move on with our transformation of D. aquaticus, so experimenting more with different antibiotics will help us determine the best choice of antibiotic for our bacterium. There are 13 additional antibiotics we can perform additional MIC testing on. This could be of benefit because there is no known kanamycin resistance gene on pRAD1, the plasmid we are going to be working with for our transformation.