Part:BBa_K5033006
OncoBiotica: mFadA[B]_GSLinker_eGFP
If you are interested in an overview of the parts designed by the iGEM Team Aachen 2024, visit our Parts page.
This part, developed by iGEM Aachen 2024, is a reporter variant of the basic part BBa_K5033000. The codA cytosine deaminase (CDA) domain has been exchanged with an enhanced Green Fluorescent Protein (eGFP) coding domain.
Its main use is to evaluate the binding of the mFadA B-domain to specific types of fusobacteria.
This part encodes a fusion protein designed to combine two functionalities. Binding specific bacteria and having a reporter function. This part is to be cloned into a vector based on an inducable expression system. iGEM Aachen 2024 used a pET21b(+) vector.
iGEM Aachen 2024 did assays that indicate a binding to fusobacteria.
This part is only used as an additional tool to analyze the binding domain, which has already been been well described by the iGEM23_CPU-CHINA team.
See: mFadA B-Domain (BBa_K4990002)
Contents
Part Composition
As previously discussed, the first protein domain is derived from the part BBa_K4990002 but it has been codon optimized for expression in E. coli. It is the mFadA B-domain, found in various Fusobacterium strains. This domain should be able to bind to FadA pili on Fusobacterium nucleatum and its former subspecies Fusobacterium nucleatum nucleatum, F. polymorphum, F. vincentii, F. animalis via self assembly.
The second functional protein domain is linked to the mFadA B-domain by a synthetic flexible linker consisting of Glycin and Serine in alternating order. This linker is 12 amino acids long.
This second functional domain of the fusionprotein is eGFP which originally is native to Aequorea victoria (water jellyfish). The eGFP sequence was aqcuired using PCR methods on a template provided by the lab our team was working in.
The fusionprotein encoded by this part also contains a downstream hexa-histidine tag for protein purification.
Protein Modeling
Biochemical Properties
The fundamental biochemical properties like molecular mass and extinction coefficient are important for a lot of SynBio work done with proteins. To see an overview of these properties, have a look at figure 2.
Protein Structure Prediction
Cloning of the Plasmid
To build the plasmid containing the gene for our reporter variant, we used the plasmid we already had for our WT-Fusionprotein (BBa_K5033000; pET21b(+)_mFad[A]_GSLinker_CDA[WT]). The gene sequence for this part contains a BamHI restriction site between the linker and the enzyme. The backbone contains a XhoI restriction site at the end of the gene insert.
After deciding on eGFP as an reporter we started cloning the plasmid using restriction digestion and ligation.
The backbone was prepared using the BamHI and XhoI restriction enzymes. After digestion, the cut backbone was cleaned up using an agarose gel and a gel extraction kit. The same was done for the Insert.
After gel cleanup the cut backbone and insert were ligated using the T4 Ligase.
To enhance the efficiency of the plasmid transformation into E. coli BL21 (DE3) the plasmid was first propagated via transformation in E. coli DH5α.
The propagated pET21b(+)_mFadA[B]_GSLinker_eGFP plasmid could then be purified with a plasmid miniprep kit and used for transformation into the production organism E. coli BL21 (DE3).
If you are interested in reviewing the cloning process a bit more detailed, visit our Experiments and Results pages.
Producing the Fusionprotein
After successful transformation of the pET21b(+)_mFadA[B]_GSLinker_eGFP plasmid into the production organism E. coli BL21 (DE3) the protein could be expressed and purified. The pET21b(+) backbone has a lac operon (including the lacI repressor) which can be induced with IPTG (IUPAC: Propan-2-yl 1-thio-β-D-galactopyranoside).
Expression and Purification of the Fusionprotein
The fusionprotein was expressed by adding IPTG to the medium to a final concentration of 1mM.
The His-tagged protein was purified using a Protino Ni-IDA 2000 packed column by Macherey & Nagel®.
This gel shows that the E50 fraction contains the highest concentration of our fusionprotein, while containing nearly no impurities. That is why this fraction has been selected for desalting (removing the imidazole) and storing in 50mM TRIS buffer for usage in assays.
Binding Assays
To investigate the binding of the bacterial adhesin mFadA[B] to the different Fusobacterium strains, two specific binding assays, flow cytometry and a photometric assay, were performed. The aim of these tests was to quantify and evaluate the interaction between the eGFP (enhanced green fluorescent protein) tagged variant of our fusion protein and the Fusobacterium strains. This approach will help us to understand the binding specificity of the protein in the tumor microenvironment.
For deeper insights into the methods visit our Experiments page.
Cultivation of Fusobacteria
Before conducting binding assays it is necessary to cultivate the strains of Fusobacterium that shall be tested. The cultivation of fusobacteria is not without its pitfalls, as they are obligate anaerobic bacteria.
In order to detect the binding of the bacterial adhesin mFadA to various Fusobacterium strains, the different strains were first cultivated. The growth dynamics of Fusobacterium nucleatum, Fusobacterium animalis, Fusobacterium vincentii and Fusobacterium polymorphum were measured to ensure that the bacteria were in their exponential growth phase for the subsequent binding assay. This is important as we expect the bacterial surface structure and adhesion capabilities to be enhanced in this phase.
In this experiment, each of the strains were inoculated in two different liquid media and growth curves were generated by measuring the optical density over a period of 28 hours. Figure 5 shows the comparison of the four Fusobacterium strains in BHI medium and figure 6 the comparison in Columbia medium. Due to limited access to the laboratory, the data for the overnight growth measurements remained incomplete. Despite this, a clear increase in growth during the first hours, without a significant lag phase, is recognizable in both curves. This leads us to the suggestion that the strains adapted rapidly to the culture conditions. Based on this, we assume that the exponential phase started early in the cultivation, even though a full growth curve was not captured.
The growth of the Fusobacterium strains in both BHI and Columbia media is largely similar and comparable. However, small differences in growth rate and final optical density were noted, with slightly faster growth and higher final OD in Columbia compared to BHI. Despite these differences, we chose to use BHI medium for our binding assays. This decision was driven by the fact that Escherichia coli, used as a negative control, as it is not expected to bind to the mFadA[B] binding domain, is known to grow better in BHI.
For deeper insights into the methods visit our Experiments page.
Fluorescence Activated Cell Sorting (FACS)
In this test, the eGFP fusion protein (in the following only described as eGFP) was brought together with several cultivated fusobacteria strains and incubated for one hour. While incubating we aimed for the eGFP to bind to the natural occurring mFadA[B] binding domain on the surface of the bacteria. After the incubation we washed off the unbound eGFP and tested the emitting fluorescence from the cells via a flow cytometer. The results of the first flow cytometriy test are shown in figure 7.
Fig. 7: Dot plots displacing the fluorescence signal detected of the Fusobacterium nucleatum. The X- axis shows the fluorescence intensity of each detected event, while the Y-axis indicates cell size (Forward Scatter). A) F. nucleatum without incubation with the eGFP, to show the autofluorescence of the bacterium. B) F. nucleatum after 1 hour incubation with the eGFP, without washing off the unbound eGFP as a positive control. C) F. nucleatum after 1 hour of incubation with the eGFP, with washing off the unbound eGFP, as the first sample. A Gate was inserted to show the amount of presumably eGFP positive events.
Figure 7A shows the dot plot with mainly one population of events and nearly no fluorescence detected if no eGFP was added. In figure 7B a clear trend of the same population with an increased fluorescence signal is noticeable when the eGFP is added but not washed off. The population also shifts to the right indicating a higher fluorescence signal due to the addition of eGFP which could not be washed off and is therefore bound to the surface of the bacteria. With these results we can say the eGFP bound in a certain amount to the surface of the bacteria, but it is unclear if the mechanism used is specific to the fusobacteria due to the mFadA[B] binding domain. Therefore, we repeated the test with several more bacterial strains known to be found in the intestinal tract, like four different fusobacteria and E. coli.
If you are interested in taking a look at the dot plots from this assay, visit our Results page. In this context they may be overwhelming, since there are 15 of them.
Unexpectedly the results are not as conclusive as before. Nearly no plot shows a significant increased fluorescence signal representing eGFP positive events. The visualization of the positive control, in which the eGFP was not washed off the bacteria, also shows no significantly increased fluorescence signal. As the signals are not as previously observed, these data cannot be reliably interpreted by analyzing the dot plots.
Instead, the number of events with an increased fluorescence was counted. No value exceeds 2500 events, which in comparison to the number of events detected by the flow cytometer (50.000) is very low. We can conclude that there does not appear to be an eGFP signal in any of the samples detected, still there is a trend at almost every of the strains. The positive control has a higher event count than the other samples and the negative control has a lower count in events. This trend was best seen in Figure 10 and appears to apply to all samples. Because of that, we assume there might be a specific interaction between the eGFP fusion protein and the mFadA[B] on the surface of the bacteria, but this method might not be specific enough to show a significant result.
This could be due to the fact that the autofluorescence of the bacteria is too high to detect a difference between a bacterium with and without single eGFP fusion proteins bound to the surface of the cells. This could also explain the lack of differentiable cell populations in the dot plot with and without the eGFP fusion protein bound to the surface.
Another explanation for the inconsistency of the results between the first and second test with the flow cytometer might be the difference in effectiveness of the eGFP fusion protein. In the second test, the positive control also showed no increase in fluorescence. Further tests may be required to confirm the specificity of the binding of the fusion protein. For this reason, we have developed the photometric binding assay.
Photometric Assay
The plate reader results (figure 8) show a comparison of all mentioned Fusobacterium strains and E. coli, tested both with and without the eGFP fusion protein to assess the binding of the fusion protein to the bacteria. The analysis of the pure fusion protein showed the highest fluorescence intensity, indicating that the eGFP can generate a high fluorescence signal intensity. The results of the bacteria show that the binding affinity with the eGFP fusion protein generally tends to be higher in the bacteria than without it. Unexpectedly E. coli had the highest measured fluorescence intensity together with F. polymorphum. This could be due to the general adhesive properties of E. coli or unspecific binding of the eGFP fusion protein.
Conclusion
All in all, the results indicate that only a small amount of our protein binds to the cells, making it challenging to measure this interaction accurately. The assays performed may not have been sensitive enough to detect this binding affinity. While our binding test results suggest a tendency for the mFadA[B] fusion protein to bind, this interaction does not appear to be particularly specific, as the binding to E. coli in the photometric assay shows us.
Additionally, the results of the flow cytometry analysis were not significant, suggesting that further testing is required to characterize the binding properties in more detail. Our findings suggest that although there is a trend for a binding affinity for our protein, the assays used may not be optimal. One potential problem could be that the eGFP fusion protein might be too big, which leads to those unspecific binding properties.
In the future, it may be promising to change the eGFP reporter domain of our fusion protein and use more sensitive detection methods for detecting the bindingt. For example, the binding to mFadA[B] could be further investigated using alternative protein-protein-interaction assays, such as ELISA (enzyme-linked immunosorbent assay) tests. Another promising approach would be to identify a new binding domain of the fusobacteria for our fusion protein. More specific binding should significantly improve the therapeutic efficiency of our approach in the tumor environment and enable the development of more targeted treatment strategies.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 115
Illegal XhoI site found at 835
Illegal XhoI site found at 842 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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