Week Two

Deinococcus Aquaticus suseptability to tetracycline was tested by streaking agar plates coated with a soft agar containing increasing levels of tetracycline; 0 ug/ml, 10 ug/ml, 100 ug/ml, and 1000 ug/ml.

As states previously, Deinococcus Aquaticus has an efflux pump embedded in its cell membrane. This pump works with the two genes tetA and tetR. The first gene tetA codes from the pump itself, while the second tetR regulates the function of the pump. With this structure in D. Aquaticus’s membrane suggests that it may have a resistance to the antibiotic tetracycline. Tetracycline is a protein that synthesis inhibitor, preventing the aminoacyl-tRNA from bringing to the active site of mRNA. This stops the bacteria from growing and replicating. 

 Materials: 

·      Solid Agar Plates

·      Soft agar

·      Liquid Culture of D. Aquaticus

·      Hot plate

·      Bunsen Burner and striker

·      Inoculating loop

·      Vortex

·      Micropipette and corresponding tips

Procedures:

1.     Inoculate a flask of 20 ml TGY media with a colony from the D. Aquaticus plate

2.     Place in the shaking incubator for 48 hours

3.     Autoclave a flask of 200 ml TGY with 1.5% agar to pour plates

4.     Reheat the agar on a hot plate and pour

5.     Let the plates sit overnight to solidify

6.     Autoclave a four flasks of 10 ml TGY with 0.5% agar to pour atop the hard agar

7.     Reheat the agar on a hot plate, remove TGY, add tetracycline, and vortex for six seconds

a.     0 ug/ml: remove 0 ml TGY, add 0 ml Tet.

b.     10 ug/ml: remove 40 ul TGY, add 40 ul Tet. 

c.     100 ug/ml: remove 400 ul TGY, add 400 ul Tet.

d.     1000 ug/ml: remove 4 ml TGY, add 4 ml Tet.

8.     Before the soft agar solidifies pipette 5 ml from one flask into the plate labeled with the corresponding concentration

a.     There will be two plates for each concentration

9.     Pipette 100 ul of D. Aquaticus into the plate

10.  Sterilize an inoculation loop with the flame of a Bunsen burn, when cooled streak the bacteria on the plate 

11.  Repeate step 9 and ten for all plates

12. Place the plates in the 30 degrees Celsius incubator for 48 hours

The picture below shows the growth of bacteria after 48 hours. The only plate with growth was the one will about any antibiotic. The antibiotic killed or inhibited the bacteria from growing at 10 ug/ml, 100 ug/ml, and 1000 ug/ml. This does not support the hypothesis that D. Aquaticus would have a resistance to tetracycline because of the efflux pump. The reason for this might be that the tetracycline prevented the gene for tetR from being read. Therefore, it would have never released the repression for the tetA. The next steps are to test the susceptibility of Deinococcus Canei, a strain closely related to D. Aquaticus. 





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