Part:BBa_K4888005
IPTG/Theophylline induced Lpp-OmpA D1/D2 SpaC
This composite part expresses the D1/D2 domains of SpaC on the surface of E. coli, under the constraint of a double induction system: IPTG and Theophylline dependant.
Please, note that another composite with the D2 domain only exists BBa_K4888006. Our results show that D1/D2 domains were more effective in binding to the mucus. However, the D2 domain only is smaller.
This composite part has been designed to express the protein at the surface and stop the production when the bacterium is no more exposed to both inducers. It was crucial to have an expression system that wasn't leaky to provide additional security.
To see the double induction system BBa_K4888001
The protein has to be expressed on the bacterial surface. The well-known Lpp-OmpA display system is used to deliver our protein to the membrane.
To see transport system BBa_K1694002
SpaC is a mucus-binding protein. Since the original protein is quite heavy and the D1/D2 domains are sufficient to bind to the mucus, it has been truncated to preserve only the relevant domains.
The original SpaC protein BBa_K4888002.
The truncated D1/D2 domains BBa_K4888004
Composition and Functionnement of the part
Expression of the protein - western blot
Method
First, the induction system and the protein expression were assessed with a western blot, as our construct contained a FLAG tag.
If our protein is indeed expressed, its detection is achievable through the use of chemiluminescence.
For an illustration of the operational concept of the western blot, please look below :
Western blot functionnement
Result
In our experiments, the negative control consisted of transformed bacteria that had not been exposed to a specific molecule. We conducted incubation experiments using IPTG only, theophylline only, and a combination of both.
Western experiment - expression of truncated SPAC using the double expression control system
Western Blot analysis of boiled bacteria hosting the D1D2 construct, induced with Theophylline (Th), IPTG, both, or neither.
D1D2 show the highest protein expression levels when induced with both Theophylline and IPTG than with other conditions. The observed bands corresponded to the expected sizes of approximately 80 kDa for D1D2.
Notably, IPTG or Theophylline alone still led to a modest level of protein expression, indicating that both expression systems have some degree of leakiness. However, when both inducers were absent D1D2 expression was minimal but still present.
We have confirmed that the proposed expression system is effective in minimizing the leakiness of both promoters when they are combined and that the protein is expressed correctly.
Surface expression - Immunofluorescence assay
Method
Secondly, an immunofluorescence assay has been conducted to verify that the protein is correctly transported to the surface with Lpp-OmPA
The protocol is based on Park, Seongjin, et al.3 . [1]
If we observe a significantly higher level of fluorescence in the bacteria expressing D1/D2 SpaC compared to the negative control, this indicates the presence of the protein on the surface of the bacteria.
The schematic of this experiment can be seen below :
Schematic of the working system of Immunofluorescence on SpaC
Result
Paraformaldehyde was used to fix the bacteria, and a permeabilization condition was induced by Tween 20, a membrane-disrupting detergent.
Strong fluorescence for anti-Flag antibody detection was exhibited by bacteria hosting the D1D2-construct that had been induced, aligning precisely with the positions of HOECHST-stained bacteria. In contrast, uninduced bacteria (negative control) showed no fluorescent signal for the His-tag.
left: Paraformaldehyde-fixed D1D2-construct expressing bacteria. right: Paraformaldehyde-fixed uninduced D1D2-construct hosting bacteria as negative control.
HOECHST stain in blue for DNA visualization, and Anti-Flag antibody stain in red.
The fluorescence signal intensity for 20 individual bacteria in each condition was quantified and summarized in a boxplot. Statistical significance, indicating differences in means, was determined using a two-tailed unequal variance Student's t-test. The obtained p-values were very low, indicating statistical significance.
Boxplots illustrating the intensity measures of the Immunofluorescence (IF) signals for various conditions. 'Surface D1D2' represents D1D2 construct-expressing bacteria on the surface, 'Negative control' refers to uninduced D1D2 construct-hosting bacteria, and 'Permeabilized D1D2' indicates induced D1D2 construct-carrying bacteria that have been permeabilized. Statistical significance was determined using a two-tailed unequal variance Student's t-test (***: p-value<0.001).
Remarkably, the signal intensity of permeabilized bacteria was found to be significantly lower than that of induced bacteria. This discrepancy could be attributed to potential protein losses from the bacterial surface during Tween 20 treatment or the relatively shorter incubation time with the primary antibody, which was due to the permeabilization step that took approximately 1.5 hours in the permeabilized samples, despite being overnight.
This shows that the protein is correctly expressed on the surface.
Half-life assessment
Method
D1/D2 SpaC's half-life was determined through a series of Western Blot analyses. This involved conducting regular Western Blots at different time intervals after the cessation of SpaC induction.
Initially, assessments were made every 6 hours, followed by subsequent evaluations every 12 hours, and finally, every 24 hours. The remaining protein levels were inferred from the signal intensity observed in the Western blot results.
Result
Loading control (Ponceau S staining) on the left and corresponding Western Blot on the right, showing D1D2 construct-expressing bacteria at various time points after induction.
The signal intensities observed on the Western blots were quantified using Fiji, and standard curves were established in Excel.
left: Quantifying the signal intensities of the different bands from the Western blot of the D1D2 construct using Fiji software; the numerical values represent the areas of the distinct signals.
right: standard curves
By applying the equation derived from the standard curve, half-lives of 113 hours (equivalent to 4.7 days) for D1D2 were determined.
The observed half-lives of approximately 5 days are well-suited for our objectives. In case of sluggish mucus turnover or any unintended binding elsewhere in the body involving SpaC, the natural degradation of the protein will guarantee its clearance within a few days.
Mucus binding experiment
Method
A "gut on a chip" concept, inspired by the research paper by Chin, Wai Hoe, et al.4, has been use to assess the mucus-binding ability of our bacteria in collaboration with SpaC. [2]
Our method involved Caco2 cells on a transwell over several days, inducing mucus production within these cells. Once the mucus was prepared, bacterial strains for testing and control were standardized based on their optical density (OD), diluted to an appropriate concentration, and applied on top of the mucus. Subsequently, the unbound bacteria were washed away, and the remaining bacteria were quantified.
If the number of bacteria expressing SpaC exceeded that of the negative control by a significant margin, we could conclude that SpaC effectively binds to mucus. A schematic illustrating this experiment is presented below :
Schematic of the working system of the mucus binding chip experiment
Result
The mucus binding experiment was repeated with duplicate experiments to enhance robustness. As observed in the Figures below, a significantly higher colony count of SpaC display was maintained when compared to the induced bacteria.
Prior to mucus incubation, bacterial solutions were plated at various dilutions: 1:1, 1:10, and 1:100 for D2 and D1D2 hosting bacteria, and 1:10 and 1:100 for the negative control. Experiment duplicate 1 is shown on the left agar plate, and experiment duplicate 2 on the right plate. The numbers at the bottom represent the colony counts for the respective lanes.
Left : D1D2 expressing bacteria were plated after mucus incubation. Dilutions 1:1, 1:10, and 1:100 were plated. Experiment duplicate 1 was on the left, and duplicate experiment 2 was on the right. The numbers at the bottom represent the colony counts for the respective lanes.
Right : Negative Control (SpaC uninduced) bacteria were plated after mucus incubation. Dilutions 1:1, 1:10, and 1:100 were plated. Experiment duplicate 1 was on the left, and duplicate experiment 2 was on the right. The numbers at the bottom represent the colony counts for the respective lanes.
To assess the binding capacity, the number of colonies from the initial bacterial solution was compared to those harvested after mucus incubation, as presented in the tables below. The post-mucus incubation colony count for the negative control was approximately 1%. Percentages in the range of 24% to 40% were exhibited by D1D2 hosting bacteria. However, it should be noted that at a dilution of 1:100 for the negative control and at a dilution of 1:10 for D1/D2 hosting bacteria, low colony counts were recorded, with only 1 and 2 colonies, respectively, after mucus incubation. This low count resulted in the obtained percentages being less robust, as even slight variations in colony count could lead to significant percentage changes.
The most robust results were derived from the 1:100 control dilution for both dilutions for D1D2 hosting bacteria. To ensure robust and conservative results, the dilution of 1:1 for D1D2 expressing bacteria was exclusively selected. In these cases, the number of colonies post-mucus incubation was approximately 18 times higher for D1D2 than for the negative control and 6.3 times higher for D2-expressing bacteria.
The first duplicate.
Comparison of the number of colonies growing from the initial bacterial solution to the number of colonies after mucus incubation, presented as a percentage. For the negative control, we display dilutions of 1:10 and 1:100. For the induced testing strains, we provide dilutions of 1:1 and 1:10.
The second duplicate.
In the initial mucus binding experiment, an elevated affinity of mucus binding was observed in bacteria expressing D1D2 when compared to the negative control. It is worth noting that the binding capacity was more pronounced for D1D2 than for D2 displayed on the bacterial surface (refer to BBa_K4888006).
This difference in binding affinity can be ascribed to the extended length of D1D2, which might potentially enable it to reach deeper into the mucus. In addition, it could be linked to an improved binding capability stemming from the presence of the D1 domain, which may stabilize the binding domain D2 in a more favorable conformation.
This study stands as a noteworthy accomplishment, showcasing the effective surface presentation of truncated D1/D2 SpaC variants on E. Coli bacteria and showing his effectiveness for mucus binding.
References
[1] Park, Seongjin, et al. An improved method for bacterial immunofluorescence staining to eliminate antibody exclusion from the fixed nucleoid. Biochemistry 58.45 (2019): 4457-4465.
https://pubs.acs.org/doi/full/10.1021/acs.biochem.9b00724
[2] Chin, Wai Hoe, et al. Bacteriophages evolve enhanced persistence to a mucosal surface. Proceedings of the National Academy of Sciences 119.27 (2022): e2116197119
https://www.pnas.org/doi/abs/10.1073/pnas.2116197119
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 1206
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 2503
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 1206
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 1206
Illegal NgoMIV site found at 1703 - 1000COMPATIBLE WITH RFC[1000]
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