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 Ze 5 cna 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

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

Transformation with Antifreeze Proteins

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

Team:WPI Worcester/Results