Results
On this page, we describe all our results in detail
In order to achieve the purification of the target proteins, a total of five plasmids were constructed. These plasmids were named as follows: pET28a-6×His-HMGB1_FL, pET28a-6×His-HMGB1_AB, pET28a-6×His-HMGB1_A, pET28a-6×His-HMGB1_B, and pET28a-6×His-PslG. Upon completion of the sequencing process, it was ascertained that the plasmid had been accurately sequenced and was deemed suitable for utilization.
The plasmids exhibiting accurate sequencing outcomes were subsequently introduced into Escherichia coli BL21 for the purpose of inducing expression. Following this, Escherichia coli cells were disrupted, and the desired protein was isolated by affinity chromatography and molecular sieve techniques, resulting in the acquisition of a very pure form of the target protein.
Crystal violet is a viable option for the quantitative analysis of biofilm quantities. Through the application of several proteins, we have successfully proved their capacity to mitigate the formation of biofilms. One notable observation is that the presence of these proteins in the surrounding environment has the capacity to impede the formation of biofilms. Moreover, with respect to a fully developed biofilm, these proteins possess the capability to degrade its architecture within a specific temporal duration.
In the experiment involving the disassembly of biofilms, the treatment group using PslG demonstrated a reduction in the absorbance of OD550, which corresponds to the biomass of the biofilm, from approximately 1.6 to approximately 1.0 within a one-hour timeframe. On the other hand, among the four HMGB1 modified proteins, HMGB1_FL and HMGB1_B box exhibited a stronger ability to disassemble the biofilm. Based on these findings, we have decided to select PslG and HMGB1_FL as our functional proteins.
In the inhibition of biofilm growth experiment, it was observed that the group treated with PslG efficiently suppressed the growth of biofilm in comparison to the control group. Additionally, four proteins modified with HMGB1 exhibited similar efficacy in inhibiting biofilm growth.
In our experimental approaches, we employed confocal microscopy to enable the visual depiction of changes occurring within the biofilm. Through the utilization of this imaging method, we have successfully proved the complete elimination of fully grown biofilms through the action exerted by these proteins. The use of these proteins led to a significant reduction in biofilm biomass as compared to the control group. The absence of the DNA and polysaccharide network is apparent, yet the persistent presence of fluorescent dots, which serve as representations of P. aeruginosa, confirms the effectiveness of our strategy in degrading the biofilm.
The comparative study of the two sets of trials allows for the observation of changes in biofilm thickness before and after the treatment. The experimental group demonstrated a statistically significant drop in extracellular DNA (eDNA) and polysaccharide content, together with a reduction in biofilm thickness, in comparison to the control group. These findings provide more evidence to substantiate the influence of the target protein.
Given the expected release of these proteins by genetically engineered bacteria in the human gastrointestinal tract, it is crucial to establish that these proteins do not induce any adverse effects on human cells. The experimental results suggest that the proteins being studied exhibit low levels of toxicity, as they only cause cell death at very higher concentrations. Moreover, it is important to acknowledge that these proteins do not induce a significant inflammatory response.
The quorum sensing system was selected as the sensor, and afterwards, our system's ability to detect the presence of pathogenic bacteria was demonstrated. Initially, we conducted an investigation to determine the ideal concentration range for the functionality of the quorum-sensing system. Subsequently, we effectively illustrated that the system is capable of detecting the presence of pathogenic bacteria (namely, Pseudomonas aeruginosa) with high efficiency.
In this study, green fluorescent protein (GFP) was employed as a surrogate for the downstream target protein. The fluorescence intensity was quantified as a measure of the relative expression level of the target protein. Initially, the concentration of a quorum-sensing effector molecule (3OC12HSL) surrounding P. aeruginosa was quantified, yielding a value of approximately 0.1μM. We proceeded to forward the process. The experiment was initially conducted on two gradients. There are two factors to consider in this context: the concentration of quorum-sensing effector molecules and the duration of the induction period. Ultimately, it was determined that our system achieved optimal expression efficiency at approximately 0.1μM over a duration of 30 minutes, so showing the effectiveness of the system. The average rate of GFP production per minute per cell was subsequently computed over a period of 3 hours. This calculation was performed under conditions where the concentrations of quorum-sensing effector molecules were approximately 0.1μM, representing the peak potency of the expression system.
In the end, the proteins can be liberated by the modified bacteria following their stimulation via the presence of arabinose. In the beginning, we established that the introduction of arabinose resulted in the successful activation of Lysis E7, hence leading to a notable decrease in the survival rate of genetically engineered bacteria. Following this, an experimental verification was performed in order to demonstrate that the reason for this decline was associated with bacterial lysis generated by Lysis E7.
In the first phase of our study, we conducted an analysis of the protein expression levels of the downstream targets controlled by the arabinose promoter under different levels of arabinose induction. Upon the introduction of arabinose, successful expression of Lysis E7 was attained, leading to the observed phenomenon of a decline in optical density (OD) and eventual lysis of Escherichia coli as the duration of induction increased.
Furthermore, we conducted a comparative analysis of the DNA content in the lysate of Escherichia coli that was lysed using Lysis E7, in comparison to the bacterial solution and the lysate obtained using physical fragmentation. Ultimately, it was determined that the DNA composition of Lysis E7 lysate closely resembled that of physical Lysis lysate, although exhibited notable dissimilarity from that of the bacterial solution. This observation suggests that Lysis E7 possesses a commendable capacity for lysing.
Following the completion of the aforementioned investigations, we devised the following future plan:
For repeating our experiments, we strongly recommend that future iGEM teams refer to our experiment page, which details what should be noted in each part of the experiment.