Results
Below are the results of the characterization of the three mechanisms that make up our system: sensing, motility, and killing.
Sensing
Accessory gene regulator (agr) Sensing
Purpose:
We constructed a new BioBrick for the agr system, BBa_K3191101, that includes a sequence modification of an existing agr BioBrick: BBa_K1022100. We first conducted experiments to find if BBa_K3191101 function is improved when compared to BBa_K1022100. Two experimental protocols were followed: the first, designated as pre-permeabilized, followed the procedure used in the original characterization of BBa_K1022100. The second, designated as permeabilized, used a Tris-EDTA solution to permeabilize the outer membrane of the E. coli cells.
The native agr system contains SarA, which is a transcriptional regulator believed to activate P2, increasing transcription in the agr system. To determine if SarA improves P2 transcription, we conducted a second set of experiments adding SarA to BBa_K3191101. The new construct, BBa_K3191102, contains the same sequence as BBa_K3191101 with the addition of the SarA gene.
Results:
Figure 1: Total fluorescence of pre-permeabilized cells with and without AIP induction.
Figure 2: Total fluorescence of permeabilized cells at 0 and 3 hours post-induction with AIP.
Figure 3: Change in fluorescence between permeabilized cells with and without AIP induction.
Figure 4: P-values of fluorescence values for BBa_K206000, BBa_K1022100, BBa_K3191101, and BBa_K3191102 using the modified sensing characterization protocol.
Discussion:
Our characterization data displays the difference in fluorescence values between BBa_K206000, BBa_K1022100, BBa_K3191101, and BBa_K3191102. This data was analyzed with a two-tail t-test of unequal variances with a significance level of 0.05 to draw conclusions of significance. Based on these values, fluorescence for BBa_K1022100 and BBa_K3191101 tend to exceed the control. This demonstrates that BBa_K1022100 and BBa_K3191101 both function properly.
Figure 1 demonstrates fluorescence values under the pre-permeabilization protocol. These data indicate that AIP induction caused minimal difference in fluorescence intensity. Figures 2 and 3 show fluorescence intensity and change in fluorescence, respectively, of cells after the permeabilization protocol. When comparing total fluorescence, BBa_K1022100 tends to yield significantly greater fluorescence values than BBa_K3191101 at hour 8, as supported by a p-values of 0.002 for the AIP trial. This suggests the modified sequences, while more complete, do not successfully increase GFP production under the original characterization procedures. The addition of SarA also does not appear to provide a significantly greater fluorescence under the influence of AIP. This is seen in a comparison of BBa_K3191101 and BBa_K3191102 at hour 3 in figure 4 (p-value = 0.902).
Trends in the data suggest that the introduction of AIP appears to have a greater effect on BBa_K3191101 and BBa_K3191102 than on BBa_K1022100 under the permeabilization characterization conditions. The fluorescence values for these conditions are seen in figures 2 and 3, and the corresponding p-values are listed in figure 4. In these conditions, a Tris-EDTA solution is used to attempt to increase AIP intake by the cells. BBa_K1022100 does not experience significantly greater fluorescence than the control at hour 3 with AIP induction (p-value = 0.111). In comparison, BBa_K3191101 and BBa_K3191102 have significantly greater fluorescence at hour 3 with AIP induction. This is demonstrated with p-values of 0.010 for BBa_K3191101 and 0.002 for BBa_K3191102. This suggests that AIP induction does not significantly increase fluorescence in BBa_K1022100, while it does increase in BBa_K3191101 and BBa_K3191102.
The increase in fluorescence may be explained by the modified experimental procedure. If the presence of Tris-EDTA successfully increases AIP uptake, then an increase in AIP uptake only improves fluorescence in BBa_K3191101 and BBa_K3191102. This suggests that our parts are truly induced by AIP, while BBa_K1022100 is not.
CheZ Motility
Purpose: CheZ directed motility allows for the controlled movement of cells in response to a chemical stimulus. Overexpression of CheZ has been noted to constrict overall movement, therefore our system utilizes a degron sequence, YbaQ, to control this phenomenon. Degron sequences tag a protein for degradation, preventing CheZ overexpression in our system. The CheZ-YbaQ sequence is placed under the control of a P2 promoter controlled by the agr system as a means to encourage movement towards AIP.
After characterization, we concluded our degron was successful, however overall motility failed.
The strain UU2685 E. coli cells without the transformed CheZ system exhibited the expected lack of motility. The cells transformed with our CheZ parts, pBAD+CheZ and pBAD+CheZ+YbaQ displayed significant motility, even without the induction of arabinose.
The pBAD+CheZ showed consistent diffusion motility for both 0% and 0.02% induction. A two-tail t-test of unequal variances with a significance level of 0.05 displayed a p-value of 0.092, indicating a lack of significance in the area of diffusion. This contradicts the hypothesis of pBAD+CheZ relying on arabinose induction for motility. The reasoning behind this lack of significance is inconclusive, but may be a result of arabinose contamination from an unaccounted source such as the cell media. An additional possibility is our overestimation of promoter regulation. The pBAD+CheZ induced with 0.2% arabinose displayed motility significantly different (p-value of 0.005) from the combined motility of uninduced pBAD+CheZ and pBAD+CheZ induced with 0.02% arabinose. It was concluded this was likely a result of the greater concentration of arabinose providing the cells with more sugar to consume over time, permitting a greater displacement.
Fig1. (pBAD+CheZ 0.2%)
Fig2. (pBAD+CheZ 0.02%)
Fig3. (pBAD+CheZ 0%)p>
The pBAD+CheZ+YbaQ induced with 0.02% arabinose produced an area of diffusion comparable to that of pBAD+CheZ+YbaQ when induced with 0% arabinose. A two-tail t-test of unequal variances with a significance level of 0.05 displayed a p-value of 0.978, indicating a lack of significant difference. The lack of significance can be attributed to similar conditions discussed in the pBAD+CheZ analysis when the same trend was observed. The pBAD+CheZ+YbaQ induced with 0.2% arabinose was significantly more motile than the motilities of pBAD+CheZ+YbaQ with no induction and 0.02% induction combined. This was maintained by a p-value of 0.0039 generated by a t-test similar to the one mentioned above.
Fig4. (pBAD+CheZ+YbaQ 0.2%)
Fig5. (pBAD+CheZ+YbaQ 0.02%)
Fig6. (pBAD+CheZ+YbaQ 0%)
The motility observed for the pBAD+CheZ construct with no arabinose induction was registered as a false positive; the lack of arabinose should have yielded no motility. This implies the presence of background CheZ. However, the pBAD+CheZ+YbaQ displayed significantly less motility (two-tail t-test p-value of 0.02) indicating a decrease in the CheZ protein with the presence of the degron. We have concluded this indicates the degron is successful in degrading excess CheZ to allow for the efficient function of chemotaxis. The inhibition of excess CheZ is imperative to the success of the motility mechanism because it ensures that MRSA is precisely targeted as intended. Under optimal conditions (an induction of 0.2% arabinose), pBAD+CheZ showed no significant difference (p-value of 0.766) in motility compared to pBAD+CheZ+YbaQ.
While there was significant diffusion of the CheZ parts as detailed above, there were no indicators of successful directionality. The cells proliferated in a circular region that contested any form of directed motility.
P2+CheZ+YbaQ was never successfully sequenced and therefore never characterized. It remains unknown how the P2 promoter would have reacted in the context of our sensing system.
Fig7. Motility Assays
Killing
Garvicin KS
Purpose:
The small antimicrobial peptide (AMPS) Garvicin is the final step in our 3-modular system. Although newly discovered, this multi-peptular bacteriocin has been noted to be more potent than many other bacteriocins. Once the motility function has carried the system to the desired Staph. Aureus, the payload , garvicin KS, will be released into the infested site. As E. coli is a Gram-negative bacterium, additional steps must be taken to ensure that the garvicin KS peptides can be secreted from the cell. Using a novel signal peptide (NSP4) this peptide tags proteins for secretion by the Sec pathway allowing for greater production of Garvicin into the system.
We are testing the effectiveness of our garvicin part through Bradford analysis and comparing its concentration to a negative control, garvicin alone and garvicin with the NSP4 secretion tag. It is our prediction that garvicin with the NSP4 should be secreted at a higher concentration.
The results of the Mini-Protean Tris-Tricine Gel, which separates proteins by size similar to SDS-PAGE, revealed that all three samples might contain some level of protein contamination. This will interfere with the identification of the target protein. While there are bands present at the size that corresponds to the Garvicin KS bacteriocin, the blurr associated with possible contamination of the sample interferes with the identification. Nevertheless, Mass Spectrometry analysis will be conducted to further identify the proteins that are present in the band associated with the molecular weight Garvicin KS.
Figure 1: Protein gel analysis of BBa_K3191301 and BBa_K3191302 against the empty vector pSB3K3
We used known concentration of Bovine serum albumin (BSA) to compare concentration to absorbance as standard for our BBa_K3191301, BBa_K3191302, and pSB3K3 parts.
Bovine Serum Albumin Concentrations
Figure 2: Concentration vs. Absorbance of BSA Standard.
| Curve Name | Curve Formula | A | B | R2 |
| StdCurve | Y = A*X + B | 0.801 | 0 | 0.993 |
Figure 3: Concentrations of Undilute and 1:1 dilutions of BBa_K3191301, BBa_K3191302, and pSB3K3
Discussion:
The dilution of all 3 parts showed mostly inconclusive results. The pSB3K3 vector and the Garvicin + NSP4 had concentration below a significant case of standard. We did this dilution in case our undiluted samples were too concentrated for the analysis.
For the undiluted samples an increasing trend was made clear. The negative control, pSB3K3, had the lowest noted concentration of 0.335 mg/mL when compared to the garvicin and garvicin + NSP4. Although the negative should demonstrate a value closer to 0 this distinction between the parts shows to justify the existence and significance of the garvicin part and its modified form. In the comparison of the garvicin and the garvicin + NSP4 concentrations, the value of the NSP4 secretion tag is made quantitatively clear. The garvicin alone produced a concentration of 0.528 mg/mL while the garvicin + NSP4 with a concentration of >1.044 doubled the untagged part.
The increase in concentration suggest that our addition of the NSP4 secretion tag is a more optimal inclusion than utilizing garvicin alone. This helps to ensure that once our E. coli is able to sense and reach the target Methicillin Resistant Staph. Aureus it will be in high enough concentration to achieve its goal.
The group had plans to include the regulatory promoter, P2, in placement of the pBAD promoter utilized in each garvicin part. The intentions of the group was that P2 would provide greater activation than the pBAD itself, however time constraints disallowed this inclusion.
