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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.

Start both methods at the same time and run in duplicate (one will run for 24 hours and the other for 72 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.

Amounts of enzyme to be aliquoted are given in the table below.

Method 1: Will enzyme prevent aggregation of biofilm?

1. 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. Allow biofilm to set in wells at room temperature for 24 hours. Cover plates.
2. After 24 hours, aliquot specified amounts of purified enzyme into specified wells.
3. 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. Fill wells with carbohydrate-specific dye and invert plate again so that stain falls out. For plate two, repeat the procedure, but after 72 hours have passed.

Method 2: Will enzyme break down pre-existing biofilm?

1. Aliquot specified amounts of purified enzyme into specified wells and allow to set for 24 hours. Cover the plate.
2. 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.
3. 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. Fill wells with carbohydrate-specific dye and invert plate again so that stain falls out. For plate two, repeat the procedure, but after 72 hours have passed.

96-well plate	1	2	3	4	5	6	7	8
A	0 uL of enzyme + 50 uL biofilm (method 1)	1.5 uL of enzyme + 50 uL biofilm (method 1)	12.5 uL of enzyme + 50 uL biofilm (method 1)	25 uL of enzyme + 50 uL biofilm (method 1)	0 uL of enzyme + 50 uL biofilm (method 2)	1.5 uL of enzyme + 50 uL biofilm (method 2)	12.5 uL of enzyme + 50 uL biofilm (method 2)	25 uL of enzyme + 50 uL biofilm (method 2)
B
C
D
E
F