The focus of our summer has been engineering control over bacterial adhesion. At NEGEM 1.0, we were excited to learn that our friends at Worcester Polytechnic Institute were working on a novel approach to controlling biofilm formation using antifreeze proteins. These proteins were able to disrupt biofilm formation under certain circumstances, and during our exchanges at the NEGEM meetup we became inspired to collaborate. We were interested in the possibility of using antifreeze proteins as a trigger for releasing pilli attachment. The first step in pursuing this potential control “switch” was asking WPI to help us characterize how our modified Pili would behave in the team’s quantitative biofilm formation assay. If they found a significant difference between biofilm formation in our Bactogrip strains and their controls, it might indicate a relationship between Type 1 Pili and the adhesion mechanisms disrupted by their antifreeze proteins. In return, we validated WPI’s biofilm formation assay using unique biofilm forming strains worked on in our host lab. This showed reproducibility of results and that the biofilm formation assay protocol is robust in various experimental conditions. We feel like we have only just begun to scratch the surface of this collaboration’s potential and look forward to a fruitful dialogue with WPI long into the future!
1 round-bottom 96-well plate
1 flat-bottom 96-well plate
Appropriate antibiotic stock
15 mL conical tubes or glass test tubes for growing liquid cultures
0.1% crystal violet
30% acetic acid
A large beaker
A tray or box that is slightly larger than a 96-well plate
A plate reader
1. Transform desired plasmids into appropriate organism.
2. Prepare 5mL LB liquid cultures (supplemented with appropriate antibiotics and inducers) from stock or a colony from the transformation plate of the desired strains. Allow the cultures to grow for 18-20 hours at 37 degrees Celsius in a shaking incubator.
3. Prepare 1:100 dilutions, with a total volume of 1 mL, with desired media for each liquid culture.
4. Prepare 1:100 dilutions, with a total volume of 1 mL, with desired media for each liquid culture.
5. Plate 100 μL of each dilution in sets of 4 wells in a round-bottom 96 well plate.
6. Shake the plate out over a tray to remove all planktonic bacteria.
7. Rinse the 96-well plate in a large beaker of water and shake the water out over the tray.
8. Lay a paper towel out on the bench top. Hit the 96-well plate against the covered bench top until no liquid remains in the wells.
9. Stain all wells used in the assay with 125 μL of 0.1% crystal violet for 10 minutes.
10. Shake the 96-well plate over the tray again and rinse out the crystal violet in a large beaker of water.
11. Cover the bench top with more paper towels and hit the plate against the bench top until all wells are free of liquid crystal violet. Note: Make sure that the only crystal violet remaining is bound to a biofilm at the bottom of a well. Rings of crystal violet around a well are not indicative of biofilm formation and should be rinsed again, as excess stain will skew the results of the assay.
12. Leave the plate face up on the bench top overnight to dry.
13. Add 200 μL of 30% acetic acid to all wells that were stained to solubilize the crystal violet. Allow the acetic acid to sit for 10 minutes.
14. Pipette up and down the mix the acetic acid and crystal violet in the wells.
15. Transfer 125 μL of the acetic acid/crystal violet solution from each well into a well in a flat-bottom 96-well plate.
16. Read the OD595nm of each well in the flat-bottom plate with a plate reader.
17. Subtract the average of the blank wells from the OD of each well that contained a sample.
18. Calculate the average of the sets of wells containing the same sample.
19. Normalize the averages to the average of the control wells.
The WPI team completed the biofilm assay described above with relevant BactoGrip strains. The strains they tested were JW4283 (a fim H knockout and the negative control for biofilm formation) and C2M+C5 and C9-3+C5 which express modified pili when induced by arabinose and rhamnose. They reported that receiving our agar stabs, they streaked plates and incubated them at 37 degrees Celsius for 24 hours. Afterwards, they placed the plates in the refrigerator until they received the arabinose and rhamnose needed to induce pili formation in the experimental strains. Once they had all necessary materials, they grew 5mL liquid cultures of each of the 3 strains in LB in accordance with the protocol. The JW4283 culture was not supplemented with antibiotics or inducers, but the C2M+C5 and C9-3+C5 cultures were supplemented with 5 μL of chloramphenicol, 5 μL of ampicillin, 0.01% arabinose, and 0.5% rhamnose. They prepared the 1:100 dilutions in LB broth and M9 minimal media, which they found to support biofilm formation while designing their biofilm assay. The remainder of the protocol was left unchanged. Below are the results of the assay.
A photo of the biofilm plate after it was stained with crystal violet on 9/3/15. Although there appears to be some remaining crystal violet, biofilm formation was not robust in any of the strains.
A table of the OD595nm for each well with the blank subtracted. Average ODs for each strain in both LB and M9 are also included.
A graph of average OD595nm for C2M+C5 and C9-3+C5 in M9 minimal media standardized to average OD595nm for JW4283 in M9 minimal media (indicated by the line at 1). Error bars = +/- standard error for the 4 ODs measured for the two strains in M9.
According to the results we collected, the Harvard strains with modified pili do not form biofilms under the conditions we subjected them to. These strains may be able to form biofilms under different conditions. Further investigation would have to be done to determine if different if different media types, incubation periods, or transformation into different strains of E. coli could induce biofilm formation. Determining optimal biofilm formation conditions could allow the Harvard team to use the biofilm assay to gather quantitative results for their various binding assays. However, it is possible that the modified pili interfere with biofilm formation. In that case, the biofilm assay would not be a valid method of quantification.
Following the above protocol, we grew up our delta FimB strain (negative control), wild type CsgA curli bacteria (positive control) obtained from the Joshi Lab, the curli bacteria transformed with the BBa_J23117 plasmid and two separate strains with the curli bacteria transformed with two separate antifreeze protein plasmids developed by WPI (TiAFP and TiAFP-Beta). However, in executing the protocol we made a few changes which we felt were valid to strive for the success of the assay. Namely, round-bottom plates at the Wyss were not sanitary so we used a flat-bottom plate for that part of the assay instead. In addition, the curli strain obtained from the Joshi Lab was grown in 1 mL pure YESCA media (100 mg Casamino acids, 10 mg yeast extract dissolved in ddH2O per 1 mL solution) instead of LB and M9 (we were told this would be optimal for cell growth). Furthermore, the liquid cultures were also supplemented with 5 μL of carbomycin (since the curli strain itself was grown on a chloramphenicol + carbomycin resistance plate). Finally, having been informed by the Joshi Lab that the bacteria would almost certainly perish if left in a 96-well plate for 48 hours at 37 degrees Celsius, we followed their advice and incubated the plate for 48 hours on a room-temperature shaker (25 degrees Celsius) instead. The plate reader results can be seen below:
Our results indicated that on average there was more biofilm formation on the positive control (WT CsgA in row B) than on the curli bacteria strains that had been transformed with the antifreeze protein plasmids developed by WPI (rows D and E), while there was also greater average biofilm formation in the positive control than in the negative control (delta FimB in row A), both of which we expected to see. Repetitions of the assay will have to be done in the future to further verify our results.
In order to better develop our research ideas and have a chance to work with and learn from other iGEM teams in the area, we attended the New England iGEM (NEGEM) conferences, the first in June and the second in September! At the two meetups, we had the chance to listen to others' project presentations and give feedback, as well as receive insight on our own presentation and ideas. In addition, we had a good opportunity to begin to talk about collaboration opportunities (eventually leading to our own wet lab collaboration with WPI!) The opportunity to meet other like-minded young, eager researchers and to truly explore others' ideas gave us invaluable insight into the iGEM community beyond our laboratory, and also allowed us to learn more about the diverse possible applications of genetic engineering.