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Team:BYU Provo/Modeling
From 2014.igem.org
BYU 2014 Team Modeling |
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Amylase and DispersinB Biofilm Assay (adapted from the 2013 BYU iGem Team Biofilm Assay)
This test will act as a qualitative check as to whether or not the biofilm is broken down/inhibited by the presence of amylase. Each well should be classified after the specified time limit with either no, low, medium, or high levels of biofilm. As more of the enzyme is used, we should see an inverse relationship occurring with the amount of biofilm on the wells when compared to the control wells that should see no decrease in the levels of biofilm. If this inverse relationship does occur between amount of enzyme and amount of biofilm, then we can conclude that the enzyme is causing the wastewater biofilm to be broken down/ inhibited.
Run assay in duplicate (one will run for 24 hours and the other for 48 hours). Leave at room temperature and do not shake. May need to use 10 mL glass tubes instead of 96-well plate if there are issues getting the biofilm from the samples to adhere to the walls of the wells.
Along with a control with just biofilm, we will use a control of biofilm with glycerol and protein elution buffer to see if those substances, which the enzymes were stored in, have any effect on the biofilm.
Amounts of enzyme to be aliquoted are given in the table below.
Protocol 1: Will enzyme prevent aggregation of biofilm?
- Aliquot approximately 50 uL of biofilm into each of the 96 wells of a 96-well plate. Cut off the tip of a 1000 uL pipette tip so that as little of the biofilm is broken up when transferring it from the sample tubes into the well plate.
- Aliquot specified amounts of purified enzyme into specified wells. Invert tubes
- Cover plates and let sit for specified times at room temperature.
- For plate one, after 24 hours have passed, invert the plate so that any supernatant will fall out of the plate leaving only biofilm adhering to the wells. For plate two, repeat the procedure, but after 48 hours have passed.
Protocol 2: Will enzyme break down pre-existing biofilm?
- Add approximately 50 uL of biofilm into each of the 96 wells of a 96-well plate. Cut off the tip of a 1000 uL pipette tip so that as little of the biofilm is broken up when transferring it from the sample tubes into the well plate. Allow biofilm to set in wells at room temperature for 72 hours. Cover plates.
- After 72 hours, aliquot specified amounts of purified enzyme into specified wells. Invert tubes.
- For plate one, after 24 hours have passed, invert the plate so that any supernatant will fall out of the plate leaving only biofilm adhering to the wells. For plate two, repeat the procedure, but after 48 hours have passed.
96-well plate 1 2 3 4 5 6 A 0 uL of enzyme + 50 uL biofilm 12.5 uL of control solution + 50 uL biofilm 12.5 uL of enzyme + 50 uL biofilm 0 uL of enzyme + 50 uL biofilm 12.5 uL of control solution + 50 uL biofilm 12.5 uL of enzyme + 50 uL biofilm B C D E F Results (13 October 2014)
The table below shows the initial set-up of tests run. We decided to not run the test where we would let the biofilm sit for 72 hours before introducing the enzyme but instead used those tubes as a check to see whether enough time had passed to allow the biofilm to adhere to sides of the tube. We would check biofilm adherence by inverting the tube and tapping it on the table twice. Once we were sure the biofilm had had enough to set, we tested the rest of the tubes from the actual test (what we had meant to let run for 48 hours but instead allows to run for 7 days). From the tests run in triplicate, we were unable to reject our initial hypothesis - that of the purified amylase enzyme being able to degrade pre-existing biofilm. We saw that the test tubes that had the enzyme would drop clumps of biofilm when tapped on the table, whereas the tubes with no enzyme or solution and those with the glycerol/elution buffer solution did not release any biofilm when tapped on the table. We will be refining the test and duplicating the results.
