Week 4 (9/26/22 - 9/30/22): Exploring New Kanamycin Concentrations/Plasmid Extractions

 Introduction: 

Kanamycin is a protein synthesis inhibitor that binds to the 30S subunit of the ribosome. It is an aminoglycoside that typically treats gram negative bacteria. Based on the Minimum Inhibitory Concentration (MIC) run from 9/14-9/16, kanamycin was chosen as the antibiotic of choice for D. aquaticus’ transformation. An additional MIC test was run from 9/21-9/23, with results showing that the concentration of Kanamycin is somewhere around 1.56 ug/ml. Because the results were not significant (mean absorbance was 0.27), additional MIC testing is required to find a more exact concentration of kanamycin to be used in the transformation of D. aquaticus. Plasmid extractions were done to help the BIO208 class. The pUC18 plasmid specifically is the plasmid that will be targeted during the extraction. This plasmid is specific to E. coli.

 

 

Methods: 

 

MINIMUM INHIBITORY CONCENTRATION

 

D. aquaticus has been inoculated and stored in a 100 ml flask in the 30^o C refrigerator. OD600 value for this D. aquaticus sample is 2.37.

 

250 ml of TGY media was prepared on 09/20. The TGY media was added to each of the 32 test tubes prior to starting the experiment. The distribution of test tubes are as follows: 10 test tubes in each run with a total of 3 runs, as well as 2 controls. The specific amounts of media added to each tube are shown in figure 1. The test tubes full of TGY media were autoclaved overnight on 9/26.

 

The 2 mg/ml stock solution of Kanamycin was prepared by Chad on 09/15 and stored in the 30^o C refrigerator. Kanamycin was made into a 1 mg/ml stock solution: 500 ul of the 2 mg/ml stock solution was added to 500 ul of PCR water. 

 

On 09/27, the autoclaved test tubes were lined up to prep for the MIC to be run. Kanamycin was retrieved from the refrigerator and was added to each of the test tubes using the P20 and fisher-brand micropipette. Amount of antibiotic added to each test tube is shown in figure 1. Each test tube was placed on the vortexer at level 6 for 10 seconds. After vortexing, 50 ul was removed from each test tube and deposited in a waste beaker. 50 ul of D. aquaticus was added to each test tube, and each test tube was vortexed again at level 6 for 10 seconds. 50 ul of D. aquaticus was also added into the positive control tube.  All 32 test tubes were then placed in the shaking incubator at 30^o C for 48 hours to ensure a proper growth curve. At the 20- and 46-hour mark, all the test tubes were removed from the incubator and read on the spectrophotometer at 600 nm to find the absorbance values. The spectrophotometer was blanked with the negative control, which only contains TGY media. Before reading the test tubes on the spectrophotometer, each tube was placed on the vortex mixer for 10 seconds at level 6.

 

Run	Volume of Antibiotic Added (ul)	Volume of Media Added (ml)	Concentration of Antibiotic (ug/ml)
1	15	4.985	3
2	13.75	4.986	2.75
3	12.5	4.988	2.5
4	11.25	4.989	2.25
5	10	4.99	2
6	8.75	4.991	1.75
7	7.5	4.993	1.5
8	6.25	4.994	1.25
9	5	4.995	1
10	3.75	4.996	0.75

Figure 1: Amount of antibiotic and media added into each tube to get the final antibiotic concentration. 

 

 

PLASMID EXTRACTION

 

Extractions performed on 9/27 (A, B, C, and C trial 2) were done using the Zyppy Plasmid Miniprep. Extractions performed on 9/28 (B trial 2 and C trial 3) were done using the GeneJET Plasmid Miniprep. The bacteria was added to the microcentrifuge tube, centrifuged for 30 seconds, and then the supernatant was discarded. This was repeated 3 times before adding in any solutions.

 

ZYPPY PLASMID MINIPREP                                                                                     

GENEJET PLASMID MINIPREP

Results:

 

MINIMUM INHIBITORY CONCENTRATION

Figure 2: Absorbance values of D. aquaticus after being treated with kanamycin. Absorbance was measured after 20 hours, 46 hours, and 66 hours.

 

	Contents	Absorbance After 20 Hours	Absorbance After 46 Hours	Absorbance After 66 Hours
Positive Control	5 ml TGY media	0.68	1.69	2.41
Negative Control	4.950 ml TGY media, 50 ul D. aquaticus	0.23	0.23	0.21

Figure 3: Absorbance of each of the controls used in the MIC experiment, along with the contents of each control.

 

Figure 4: Graph of the relationship between the concentration of kanamycin and the absorbance of D. aquaticus.

 

 

PLASMID EXTRACTION

 

Extractions were done initially with samples B and C. An extraction was done on A, as well as a repeat on sample B and C. Samples A and B both had high concentrations; sample A was 586.6, while the mean for sample B was 693.7. Sample C had a much lower concentration, with a mean of 51.7. 

 

Sample	Date	ng/ul	A260/A230	A260/A230
A	9/28	586.6	1.90	2.27
B	9/27	663.6	1.86	2.24
B trial 2	9/28	723.7	1.87	2.24
C	9/27	67.7	1.80	0.96
C trial 2	9/27	39.4	1.95	1.92
C trial 3	9/28	47.9	1.96	2.13

Figure 5: Concentrations of each pUC18 plasmid extracted from E. Coli, read on the NanoDrop on dsDNA setting.

 

 

Discussion: 

The MIC test shows a negative correlation between the concentration of kanamycin and the absorbance. Each time the test tubes were read on the spectrophotometer they were blanked with the negative control, so the spectrophotometer would be set to read ‘no bacterial growth’ as 0. Since values above 0 are considered to have bacterial cells in them, the results point to the MIC being around 1.75-1.5 ug/ml. These are the concentration values where we start to see some bacterial growth, with the mean absorbance for 1.75 ug/ml as 0.09 and the mean absorbance for 1.5 ug/ml as 0.25. For a more defined result, a MIC using a 96-well plate will be performed and absorbance will be determined using a microplate reader.

Performing the MIC with a 96-well plate will also give a more accurate absorbance value. Using a spectrophotometer risk the possibility of an inaccurate result because this machine is unable to tell the difference between dead of alive cells. This means that our expected concentrations of 1.5 ug/ml and 1.75 ug/ml could possibly only be giving us positive absorbance values because it contains dead D. aquaticus cells. The microplate reader will be able to determine the quality of the bacterial cells in the sample, resulting in a definitive MIC.

 

 

Plasmid extractions were performed using 2 different plasmid prep kits to see if there was any variability in the results. There was a difference in values, but overall both plasmid kits produced similar results. The 260/280 values from the Zyppy kit had a mean of 1.87, while the 260/280 values from the GenJET kit had a mean of 1.91. These are closed to the desired value of 1.8, however because they are slightly above this it means there is a chance of contamination by protein or a reagent. The 260/230 values from the Zyppy kit had a mean of 1.71, while the 260/230 values from the GenJET kit had a mean of 2.21. These results suggest that there is contamination in the Zyppy kit because most of the 260/230 values were below the desired value of 2. The GenJET kit produced results that were all above 2, supporting the idea that they are pure samples of the plasmid. The absorbance values for A and B show that there is significant growth in the samples, while the absorbance values for sample C are not high enough to indicate enough growth.