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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?

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.
2. Aliquot specified amounts of purified enzyme into specified wells. Invert tubes
3. Cover plates and let sit for specified times at room temperature.
4. 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?

1. 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.
2. After 72 hours, aliquot specified amounts of purified enzyme into specified wells. Invert tubes.
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. 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.

Amylase and DispersinB Biofilm Assay Version 2 (modified from 2014 BYU iGem Team Biofilm Assay on 13 Oct 2014)

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 biofilm breakdown" or "biofilm breakdown". 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 triplicate for each instance of the test. Leave at room temperature and do not shake. Use 1.5 mL Eppendorf test tubes. Check for biofilm breakdown and dispersion by inverted each tube and tapping it twice on a hard surface. To determine when enough time has passed for proper biofilm adherence to test tubes, fill 7 test tubes with 50 uL of biofilm sample and use one each day to determine by tap test whether or not enough time has passed for the biofilm to have properly set up.

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: Will enzyme break down pre-existing biofilm?

1. Add approximately 50 uL of biofilm into each of the tubes as outlined in the table below. 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. Be sure to fill extra tubes with same amount of biofilm which will act as test tubes to determine when enough time has passed for the biofilm to properly set up in the tubes.
2. Aliquot amounts specified of solution and enzyme into tubes as specified on table below.
3. Once biofilm adherence verified using before-mentioned test tubes, invert each tube and tap on hard surface twice. Record whether biofilm remained adhered to tube or whether any biofilm was dislodged by the tap test.

Results (17 Oct 2014)

For this test we did not use all the tubes as were highlighted due to scarcity of purified protein.

While the results were not as clear as last time, we were still able to see a marked difference between those biofilm samples with and without amylase. This difference between last time and this time is likely due to only letting the assay run for 5 days instead of 7 days as was previously done.

Observations - Alpha Amylase: Despite the biofilm not being set up quite as well as last time, a marked difference was able to be seen in those tubes with biofilm and Alpha Amylase protein. In the tests with higher concentrations of Alpha Amylase, more fragments of biofilm were discharged when the tubes were tapped on the table, whereas those with just biofilm or those with biofilm and control solution did not see this change. There was one case, however, where a chunk of biofilm was discharged in one of the biofilm + solution tubes, but the corresponding tubes with amylase saw discharges of smaller fragments. From this we can infer that the biofilm was actively degrading the biofilm and causing the consistently smaller fragments of biofilm the protein was in contact with.

Observations - DispersinB: As far as the tests run with DispersinB go, only one showed a difference in pre- and post- tap where the presence of the protein showed a marked breakdown of the biofilm as with the Amylase. The other two tests saw no difference pre- and post- tap. And while some biofilm was discharged in the solution control tube, when compared to the tube with 25 uL of DispersinB there was a significant increase in biofilm discharge. This test will need to be duplicated to proffer any definitive data.