This experiment has two parts: one tested the effectiveness of chitinase breakdown using our synthesized strain of ChiA and the other tested the ability of the strain to be mutated. We also used mathematical models to predict whether or not enzyme evolution via error-prone PCR is possible.
In proving the ability of GST-ChiA-FLAG (our synthesized construct) to secrete chitinase, we used two methods. In the solution assay, there was significant chitinolytic activity and the levels of degradation were measured using colorimetric methods and absorbance measurements. (see Figure 1)
|Figure 1: The levels of chitinolytic activity were measured using a serial dilution of chitinase to test to see that an increase in chitinase concentration is correlated with increased chitin degradation. As the concentration of chitinase decrease from left to right, there is a decrease in absorbance/color showing that there is, in fact, a direct correlation.|
Chitinase secretion also is also expressed in the plate assays were rings of chitin degradation are observable by the naked eye on chitin agar plates. (see Figure 2)
|Figure 2: The plate on the right was plated with colonies of bacteria that was transformed with our GST-ChiA-FLAG construct and the plate on the left was plated with bacteria transformed with a negative control (pBAD-D4). There are visible rings and more colonies on the right as there is actual ChiA sequences in those bacteria and the vector used for this construct had ampicillin resistance. The negative control didn’t have a sequence for ChiA but did have ampicillin resistance although there does seem to be a lower survival rate in the negative control colonies regardless.|
This proves that chitinase is effective in degrading chitin when transformed into e.coli and that it is, in fact, the GST-ChiA-FLAG that is degrading the chitin (this notion is supported by the negative control which had no chitin degradation). After we proved that the enzyme is effective we tried to purify the protein and produce on a small scale by utilizing the GST sequence. We purified the protein using GST purification protocol outline by (insert source here) and used western blot to determine the concentration of arabinose to maximize protein expression and also to check protein expression. The arabinose check was successful; however, the western blot was not. When we reattempted the protein purification and then did western blot, we modified the protocol to increase induction efficiency, and also did the Bradford Assay to check the concentration of protein. (see figure 3)
|Figure 3: These are the measured absorbance from the Bradford assay which we did in triplicate. The cells highlighted in blue was the known controls (1000, to 500, 250, 125, 62.5, and 0 ng/µl from top to bottom) while the cells highlighted in pink are the samples of protein. Wells 1A-C are samples from frozen bacteria and wells 2A-C are samples from fresh bacteria and we performed a 1:10 dilution. Based on these results, there is a very small concentration of protein but when we did western blot later there was no protein that appeared on the blot.|
The Bradford assay showed a very small concentration of protein that was purified while the western blot showed none at all (it is important to note that these results were after the purification protocol was modified) however this is not due to induction inefficiency or lack of protein (we proved both in the arabinose check and the plate and solution assay). Therefore, the only error most likely has to do with protein that was lost during the purification process and then dissolved during SDS-PAGE, transfer, or blotting (as the concentration of protein was already very low it would be easier for some of it to be lost during the blotting process). These errors could be due to problems with the protocol or incorrect lab techniques but we were able to extract some results: protein exists, can be purified to some extent, and the concentration of arabinose to efficiently induce protein expression.
The second part of our experiment is trying to prove the ability of GST-ChiA-FLAG to be mutated to encourage future research on the enzyme evolution of the chitinase strain via error-prone PCR. This part of our experiment was inspired by a mathematical model known as Monod’s equation to predict where a mutation will occur and the growth rate. Then we proved the ability of Serratia Marcescens chitinase to be mutated experimentally through our construct (GST-ChiA-FLAG). (see figure 4)
|Figure 4: Based on this graph, there is clearly several sites of mutation between the negative control and the positive control (positive and negative for Mn+ which the mutation agent). Clearly showing that mutagenesis is possible.|
We mutated our strain of GST-ChiA-FLAG construct following a protocol for error-prone PCR, as described by Wilson_et_al 2001. By using both the mathematical model and the experiment, we proved that mutagenesis was possible and that error-prone PCR could be used to mutate Serratia Marcescens Chitinase. We will expand on this project either in future competitions or in future research by further evolving the enzyme using this method to improve the chitinolytic efficiency and increase the activity.
After the synthesis and test of chitinolytic activity of our GST-ChiA-FLAG construct, it has been proven that GST-ChiA-FLAG produces chitinase to some degree and that there is a way to mutate ChiA from Serratia Marcescens. Secretion of the chitinase in both the solution and plate assays is evident in showing that the transformation efficiency of the construct into competent E.coli cells is high enough where degradation is significant. Secretion of chitinase was observed in the solution assay by observing the reaction between chitinase and horseradish peroxidase producing hydrogen peroxide as a product. The solution was visually pink, but the absorbance measurement also holds true to this observation, there is clear chitin degradation in solution. On the agar plate, there were visible halos around colonies transformed with the GST-ChiA-FLAG tag plasmid and plates with bacteria transformed with a negative control (known as pBAD-D4) not only showed a lower colony survival rate but also zero chitin degradation. We were able to successfully design a bacterial strain capable of secreting Serratia Marcescens chitinase to break down cell walls in fungi which cause diseases such as Fusarium oxysporum, Rhizoctonia solani, Bipolaris sp, Alternaria raphani, and Alternaria brassicicola. Our strain has the potential to deconstruct fungi which affect a wide range of common agricultural products like bananas and potatoes, allowing for increased food supply. By talking to a local farmer and nursery manager, we learned that they were both vulnerable to crop loss due to fungi, and are looking for organic remedies. Our discussion with Dr. Schmelz helped us develop the idea that many variations of our strain of chitinase would be beneficial in treating this problem since the plants with positive results will reproduce. This lead to the integration of error-prone PCR in our experiment. Our project has economic and commercial implications since current treatment involves fumigation which is expensive and harmful to crops. Utilizing the chitinase strain would make this unnecessary, cutting the costs of fumigation, as well as revenue loss in crop sales. Additionally, the use of our strain can potentially combat world hunger, benefitting health since malnutrition will be reduced. Mycotoxins have also been found to accumulate in poorly stored food crops so the risk of cancer will also be diminished. Overall, the lives of those regularly affected by fungi-susceptible foodstuffs would drastically improve. We found the optimal conditions for arabinose induction, western blot, and the chitinase assay which were all used to successfully check gene expression. The GST-ChiA-FLAG construct was proven to secrete the enzyme when plated on chitin agar plates where chitin degradation was present, indicated by small halos. In order to maximize chitin degradation, error-prone PCR was used, proving that our strain of chitinase can be mutated. We began the PCR process, as shown in the figure, where each color represents a base pair. The top row and bottom row represent regular PCR and error-prone PCR where MnCl2 was added, respectively. The frequency of each base clearly varies between the two methods. The small second curve on the error-prone PCR graph indicates that a different base was present. Future research could entail further evolving our chitinase strain by using error-prone PCR until we have found the most efficient one. This is done by selecting the mutants with beneficial properties, or which degrade the most chitin and continually performing PCR. Using this method it is possible to simulate the process of natural selection to evolve the enzyme and increase chitinolytic activity. This connects to the model that we used that was based on the derivative of Monod’s equation that was derived using calculus. This model that inspired our Error-Prone PCR method for mutations can depict the rate and type of mutation that occurs based on a set of predetermined features. This model combined with the experimentally proven fact that GST-ChiA can be mutated proves that controlled evolution of Serratia Marcescens ChiA is possible.