1/22-1/28 Week 3: Tetracycline Efflux Pump with D. Aquaticus

 Introduction

    What are efflux pumps? According to the National Institutes of Health, efflux pumps are basically "bacterial transport proteins which are involved in extrusion of substrates from the cellular interior to the external enviroment" (Sharma, 2019. p 4). This week in lab, my group and I did a quick and simple experiment with tetracycline and D.aquaticus. Basically, D. aquaticus contains both tetA, which codes for the efflux pump, and tetR, which codes for the regulator of the pump. As for tetracycline, we know that in order for tetracycline to work with bacteria, it has interfere with protein synthesis to destroy the bacterial cells' membrane and stop the bacterial growth. Our question is, if D.aquaticus contain both tetA and tetR that can possibly pump a certain amount of tetracycline out of the cell's membrane, then does increasing the dosage allow tetracycline to reach the protein easier? 


Procedure 

    In order for us to start, we must prep our materials. In week 2, my group and I have made 40 ml of TGY soft agar and aliquot 10 ml of it into 4 125ml flasks and autoclaved it. In another 125ml flask, we grew aquaticus straight from freezeback plates into 20ml of TGY. This will ensure that our sample will be fresh and clean since it was streaked from freezebacks and the plates were not contaminated. We let the aquaticus grow for exactly 2 days. We also have made 8 TGY agar plates (normal agar) as pre-made plates for the protocol; we are planning to do duplicates. Finally, we obtained 10 ml of tetracycline at a concentration of 2.5 mg/ml. 

    On the day of the protocol, we remembered to gram stained our grown aquaticus to check for any contamination and also checked the OD value to ensure good growth (we usually accept OD values between 0.8-1.0). Below is our written protocol, the concentration was given to us by Dr. Tuohy. 

1. Label each of the 4 125ml TGY soft agar flasks and pre-made plates to their corresponding concentration. 

   ---> Flask #1 and Plate #1-2: 0 ug/ml 
  ---> Flask #2 and Plate #3-4: 10 ug/ml 
   ---> Flask #3 and Plate #5-6: 100 ug/ml 
   ---> Flask #4 and Plate #7-8: 1000 ug/ml 

2. Start to heat up each of the flasks so it is broth-like and while it is still hot, take out a certain amount of TGY corresponding to what is needed to be added to the media later on using tetracycline. This is to ensure that our flasks will have the exact volume at the end. If no media was taken out before putting in the tetracycline, it will throw off our total volume. 

    ---> Flask #1 (0 ug/ml): -0 ug/ml of media
    ---> Flask #2 (10 ug/ml): -40 ug/ml of media 
    ---> Flask #3 (100 ug/ml): -400 ug/ml of media
    ---> Flask #4 (1000 ug/ml): -4000 ug/ml of media 

3. If the flasks are still hot after this, let it cool (to luke-warm) but do not let it solidify again. This is so the tetracycline will not "denature" due to the extreme heat but will also allow the tetracycline to easily vortex with the media when it is liquid. 

4. Using the same amount that was taken out of the media from each flasks, put in the tetracycline and vortex.

    ---> Flask #1 (0ug/ml): +0 ug/ml of 2.5 mg/ml tetracycline 
    ---> Flask #2 (10 ug/ml): +40 ug/ml of 2.5 mg/ml tetracycline 
    ---> Flask #3 (100 ug/ml): +400 ug/ml of 2.5 mg/ml tetracycline 
    ---> Flask #4 (1000 ug/ml): +4000 ug/ml of 2.5 mg/ml tetracycline

5. Once each flasks are mixed, quickly lay out the pre-made TGY agar plates and quickly pipette 5 ml from each flasks to their corresponding plates from step 1. For example: contents of Flask #1 will go into both Plates #1 and #2 because they are being evenly aliquoted into the plates. 

6. Once all plates are aliquoted, use a sterilized pipette to pipette out exactly 100 ul of our grown aquaticus and using a sterilized loop each time, spread the aquaticus amongst the surface of the plates. Do this for all plates. 

7. Wait for all plates to solidify. 

8. Place all plates in the 30 degree C incubator for results in Week 4. 






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