Team:WPI Worcester/Results
Results
Biofilm Quantification
Antifreeze Proteins against Biofilm Formation
HIStag Purification
HIStag purification was conducted to purify antifreeze proteins (IA and Ze) and GFP from BL21 strains of E. coli. These strains produce a larger quantity of proteins. They were activated using IPTG to produce more protein. The antifreeze proteins and GFP contain histadine, and therefore bind to components of the purification, allowing other parts of the E. coli cells to be removed.
Protein Concentration Assay
Samples from the Eluate of the protein purification were compared with BSA on a protein assay to quantify the concentration of protein recovered from purification. The graph shows that the purification yielded between 0.42 and 0.54 mg/ml of protein.
SDS Page Gel
Purified GFP can be found in well 4 of the first gel. It is around the expected size of GFP, which is 27 kDa. Therefore, it is likely that GFP was purified in this sample. Purified IA4 is seen in well 8 of the first gel, while IA5 can be seen in well 4 of the second gel. Both eluate samples of IA are around 24 kDa, when compared to the ladders in the samples. This is the expected size when compared to literature. Lastly, purified Ze4 can be seen in lane 8 of the second gel and Ze5 can be seen in lane 5 of the third gel. These are the smallest, around 14 kDa, and fall toward the bottom of the gel. Something to note is that there is a band that is present in all gels. It is a non-specific band that seems to not have been purified out using HisTag purification. It is possible that this could contribute to future results of our assays. Future work would require a second round of purification to eliminate this band of proteins before running our assays.
Biofilm Assay Result
The first graph shows the amount of biofilm produced in EMG, DH5𝛼, NCTC, S. Aureus, and S. epidermidis with IA4, IA5, Ze1, Ze4, or GFP coating the well plate. When comparing the red no protein bar to every other bar, most proteins seem to show a reduction in biofilm formation in these trials. This occurs in every strain except for EMG. It is possible that EMG felt threatened by the addition of the AFPs and produced more biofilm as a protection method.
The second graph shows the amount of biofilm produced in EMG, DH5𝛼, NCTC, S. Aureus, and S. epidermidis with IA4, IA5, Ze1, Ze4, or GFP mixed into the bacteria that was then added to the well plate. When comparing the red no protein bar to every other bar, most proteins seem to show a reduction in biofilm formation in these trials. This occurs in every strain except for EMG. It is possible that EMG felt threatened by the addition of the AFPs and produced more biofilm as a protection method.
The addition of AFPs or GFP seem to have an impact on the formation of biofilm in all strains that were tested. It is possible that any trends seen in this data could be due to the nonspecific band present during the protein purification protocol.
Curcumin Assays
As can be seen, the EMG strain had the most pronounced biofilm development. While DH5α had an apparent drop in biofilm development, it like S. epidermidis and S. aureus showed an increase in biofilm growth. NCTC similarly shows an initial drop but an uptick in measured biofilm after 20 µM curcumin solution was applied. However, the overlap between error bars in these four aforementioned strains among ratios means overall measurements may be suspect.
Discussion and Future Work
Curcumin’s antibacterial ability against different strains of bacteria was assessed. To see if it had a blanket response among different bacterial species was an objective of this study. Observing the data, particularly the mismatch in drops and increases in biofilm measurement a few reasons for this occurrence arises. One concerns the very development of biofilm initially, wherein, when placed under taxing, harsh conditions nonideal for development, some bacteria form biofilm (Kostakioti, 2013). Curcumin, posited in the literature to kill bacteria, may have proven so harsh for bacteria with membrane deconstructing mechanism that cells went into biofilm development to endure, explaining the increase in biofilm detected in S. aureus for example. Another reason may concern the measurement technique used. While great care was taken to be consistent among all assayed plates in all trials, there was no standardization in the initial wash step of the plates to apply the crystal violet stain, which, on those occasions the water was applied for longer than needed, removing the biofilm, thus skewing data.
Future extensions of this project could involve running this experiment once more with the aforementioned issues addressed and remedied. Moreover, discerning a scalable method conducive for industrial production of curcumin from synthetic bacteria (like what is executed currently with insulin) may prove insightful and provide for a case study for a new industry of standardized herbal biologics. If desired, the genes comprising the mechanism of curcumin could be spliced into plant genomes to have the plants produce curcumin themselves.
Lettuce Colony Counts
Gene Gun Transformation Results
The team has built a low-cost gene gun that costs only $416.35, and the gun was proven to be functional with GFP reporter system. For more information, please see the hardware page.
Transformation with Antifreeze Proteins
Biofilm assay plates made from transformed cultures of IaAFP, ZeAFP, GFP, Empty psB1C3 plasmid in EMG competent cells. The goal was to induce the various AFPs in bacteria that are known to form biofilms. This was tested on biofilm assay plates containing a sample of each protein with and without arabinose. The results from the biofilm assay plates show that the samples induced with arabinose produced less biofilm when compared to the same protein sample not induced with arabinose. This is because the arabinose binds to the AraC protein and turns on the operon that triggers the expression of the AFPs.
Image of a protein gel that confirms the expression of the proteins. The protein gel confirms expression of AFPs. The red boxes outline the IaAFP with arabinose, ZeAFP with arabinose and GFP with arabinose all contain bands that show protein expression. The samples of IaAFP, ZeAFP and GFP without arabinose do not show the band that confirms expression of the proteins. The last two wells contained samples of empty psB1C3 plasmid and therefore do not have the bands of protein.
Image of GFP cultures. The two tubes on the right that contain arabinose and the two tubes on the left do not. It can be seen that the tubes containing arabinose are glowing and therefore confirm the expression of the GFPs.
Project Achievements
We achieved a lot over the course of our project, both positive and negative results. Below are our lists of successes and failures.
Successes of ICEberg
- Successful Purification of Antifreeze proteins in BL21 strains of E. coli
- Reduction of Biofilm formation with any application of
- Transformation of EMG cells to express Antifreeze protein
- Production of a functional and inexpensive Gene Gun
- Transformed lettuce leaves to express GFP using the team Gene Gun
Failures of ICEberg
- Inability to make competent NCTC cells with multiple protocols
- IPTG inducible operon did not turn on
References
Kostakioti, M., Hadjifrangiskou, M., & Hultgren, S. J. (2013). Bacterial Biofilms: Development, Dispersal, and Therapeutic Strategies in the Dawn of the Postantibiotic Era. Cold Spring Harbor Perspectives in Medicine, 3(4). doi:10.1101/cshperspect.a010306