OspA and Salp12 Protein Expression
The Lyme AID project aims were worked on concurrently, dependent upon recombinant protein expression of Salp12, OspA, and INP-OspA.
Design - Salp12 is a protein found in the salivary glands and midgut of the Ixodes scapularis tick, the main host of the Borrelia burgdorferi spirochete and causative agent of Lyme Disease. The protein has been found to function as a chemoattractant for B. burgdorferi, playing a role in the acquisition of the spirochete into new tick hosts from infected prey. The amino acid sequence of Salp12 was found in the supplemental data for the 2019 paper published by Murfin et al. and reverse translated, codon optimized for E. coli.
OspA is expressed on the surface of B. burgdorferi and functions as a signal transducer or as a receptor (Li, 1997). The OspA sequence was found at the National Center for Biotechnology Information database (NCBI, 2016). The ice-nucleating protein (INP) is a natural surface expressing protein, and we hypothesized that by linking OspA to the INP, OspA would be expressed on the surface of the cell. The INP sequence was found at the National Center for Biotechnology Information database.
Build - The Salp12 pET-28a(+) plasmid was designed on Benchling and synthesized by Twist Bioscience, as shown in Figure 1.
The OspA pET-28a(+) plasmid and INP-OspA pET-28a(+) plasmid were designed in Benchling and synthesized by Twist Bioscience, as shown in Figure 1.
Figure 1: Salp12, OspA, INP-OspA Plasmids. The plasmids we created in Benchling had a pET-28 backbone. On the left is our Salp12 plasmid, in the center is our OspA plasmid, and on the right is our INP-OspA plasmid
Test - The pET-28a(+) plasmids containing OspA, INP-OspA, and Salp12 were transformed into E. coli BL21/DE3. Transformed cells were induced using 1mM IPTG, and extracted proteins were analyzed using 10% SDS PAGE. SDS PAGE results displayed no difference compared to wild type BL21/DE3 cells, as seen in Figure 2.
Figure 2: Initial SDS PAGE Analysis. The initial SDS PAGE analysis showed no changes in the band size of our Salp12, OspA, or INP-OspA. After running the SDS Page, we decided to remove OspC from our design and focus our efforts on OspA. OspC will be investigated further in future steps
Learn - No OspA, INP-OspA, and Salp12 proteins were expressed, as indicated by the negative results of SDS PAGE. We decided to use Colony PCR to determine the presence of successfully transformed cells.
Colony PCR
Test - Colony PCR was used to screen for positive colonies with the OspA, INP-OspA, and Salp12 genes present. Gel electrophoresis results displayed that only 20% of the cells were successfully transformed. In Figure 3, bands at ~822 bp and ~348 bp were consistent with anticipated sizes of OspA and Salp12, respectively.
Figure 3: Colony PCR for OspA and Salp12. We can see a band in both PCRs where we anticipated them to be. At ~822bp for OspA and at ~348bp for Salp12
The colony PCR-verified cells were then used to undergo induction. 10% SDS PAGE analysis, as shown in Figure 4, at varying dilutions revealed protein expression for these conditions consistent with INP-OspA via a band at 60 kDa, but did not show Salp12 expression. A western blot and IFA-immunofluorescence assay also did not reveal successful Salp12 expression by E. coli.
Figure 4: INP-OspA SDS Page Results. By using SDS PAGE, we confirmed that out INP-OspA was sucessfully expressed
Learn - INP-OspA was successfully transformed as confirmed by Colony PCR. INP-OspA was successfully expressed as confirmed by SDS PAGE results. We decided to use nickel affinity chromatography to purify our protein products. Meanwhile, we similarly learned through colony PCR that Salp12 transformation was also only successful in 20% of the cells. However, many modes of protein analysis of successfully transformed cells did not yield protein expression.
Nickel Affinity Chromatography
Test - The INP-OspA proteins are attached to a His6 affinity tag, and nickel affinity chromatography was utilized to purify protein products. The initial wash samples had the INP-OspA target protein and others present in the lysate, but with increasing washes, other proteins were “washed out” and only the target protein remained in the final elution. Shown in Figure 5,in the case of INP-OspA, we have it at the anticipated MW of 60 kDa, as well as a smaller, secondary band which is consistent with a protein degradation product.
Figure 5: Nickel Affinity Purification of INP-OspA. In the INP-OspA Ni Affinity gel, we have the anticipated MW of 60 kDa, as well as a smaller, secondary band which is consistent with a protein degradation product
Learn - We successfully purified our target proteins, INP-OspA, through nickel affinity chromatography.
Immunocytochemistry/Fluorescence
Test - Immunocytochemistry was used to confirm that OspA is expressed on the surface of the cell. The primary antibody (1AB), Anti-OspA Rabbit, was specifically designed to bind to OspA and the secondary antibody (2AB), AlexaFluor 488 Goat Anti-Rabbit, tags onto the primary, adding a fluorescent signal. For wild-type E. coli, the primary and secondary antibody will not bind, and therefore we do not expect to see fluorescence associated with these cells, consistent with Figure 6. Fluorescence was seen in the INP-OspA, as shown in Figure 7, indicating its surface expression, while wild-type E. coli displayed no significant fluorescence.
Figure 6: Wild-Type E. coli with INP-OspA. For wild-type E. coli, the primary and secondary antibody will not bind, and therefore we do not expect to see fluorescence associated with these cells. Figure made with Biorender
Figure 7: E. coli with INP-OspA (left) with primary AB and secondary AB, exhibiting green fluorescence of AlexaFluor 488. WT E. coli (right) treated with primary AB and secondary AB, exhibiting no fluorescence. We sucessfully were able to express INP-OspA on the surface
Learn - We learned that INP-OspA is displayed on the surface of the cell. Our next goal is to show that the aptamers associate with the surface-displayed OspA.
Aptamers
The final aspect of our B. burgdorferi detection system uses OspA aptamers and gold nanoparticles.
Gold nanoparticles (AuNP) have a unique ability to colorimetrically change when shifting from a separated state to an aggregated state (Alsager et al., 2015). The deaggregated state of AuNP appears as a transparent pinkish hue and the aggregated state of AuNP appears as a transparent bluish-grey color, as shown in Figure 8. More details on our implementation of aptamers can be found on our Description page.
While designing our aptamer solution, we optimized the buffer by altering its salinity to ensure the colorimetric change occurred in a noticeable manner.
Buffer Optimization
Figure 8: Model of Aptamer Binding Sequence with OspA on Surface of B. burgdorferi. Binding of aptamer to B. burgdorferi results in colorimetric change due to AuNP aggregation. Figure made with Biorender
Initially, the iGEM team focused on creating buffer solutions to test the efficiency of aptamers. The goal of this focus was to determine optimal conditions for OspA detection. In the first trial, the aptamer group created buffer solutions containing the following salts: NaCl, KCl, Na Phosphate, and MgCl2. The results show all but one solution changing color from the normal reddish-pink hue of the solution to the blueish-grey hue of the gold nanoparticles. Therrefore, the buffer solution was determined to be too strong.
We learned from the results from this first round of gold nanoparticle testing that the nanoparticles required a weaker buffer for aggregation. We altered the buffer solution in the following trial.
Figure 9: Results from 2nd Trial. Shows gradual colorimetric change aligned with gradual buffer concentration increase
In the second trial, we created buffer solutions containing only NaCl. We predicted that because this buffer solution was weaker, a gradient of hues would be seen rather than an immediate color change in the presence of AuNP, as indicated in Figure 9.. From the most dilute buffer solutions to the most concentrated ones, the hues show a gradual color change from the reddish-pink to the blueish-grey hue with various intermediate shades in between and we had successfully optimized the buffer solution for ideal AuNP aggregation.
From the results of this second round of gold nanoparticle aggregation verification, we learned that lower concentrations of salts present in a buffer can produce desired results with aptamers. Since the solutions formed a visible color gradient, the exact measurements and specific components can be replicated in the future to produce optimal effects with the AuNP.
Determining Concentration Requirements
After creating the optimal buffer solution, we determined the concentration of ssDNA in an OspA aptamer sample. The team added DI water to several vials of aptamer samples possessing various lengths of sequences. After diluting the ssDNA, the absorbance values were measured. By measuring the absorbance values, the original concentration of the aptamer itself was calculated via the Biers Law equation.
Considering the concentration values of the OspA aptamers are now calculated, the iGEM team can determine the exact concentration of AuNP needed for a 3:1 aptamer to AuNP ratio. This ratio has been proven by past experiments by AFRL to be an option for optimal amounts of each component in order to produce the most efficient detection of molecules. We will achieve this 3:1 ratio with OspA, the target of Lyme AID.
Next Steps
We plan to implement a mechanism to use aptamers in the detection of OspA from Lyme Disease (Tabb et al., 2022).
Desiging our protocol to match Dr. Alsager’s work in colorimetric detection of 17β-estradiol, we will test the colorimetric signaling mechanism of our Lyme disease detection system (Alsager et al., 2015).
Using aptamers designed over the 2023 summer at Air Force Research Laboratory, we intend to combine our efforts in designing a BSL1-safe model organism of B. burgdorferi with OspA on E. coli. We will first test our AuNP-aptamer detection system against a purified OspA and eventually move to testing the detection system against our model organism for Lyme disease.
References
Alsager, O. A., Kumar, S., Zhu, B., Travas-Sejdic, J., McNatty, K. P., & Hodgkiss, J. M. (2015). Ultrasensitive colorimetric detection of 17β-estradiol: the effect of shortening DNA aptamer sequences. Analytical chemistry, 87(8), 4201–4209. https://doi.org/10.1021/acs.analchem.5b00335
Li, H., Dunn, J. J., Luft, B. J., & Lawson, C. L. (1997). Crystal structure of lyme disease antigen outer surface protein a complexed with an fab. Proceedings of the National Academy of Sciences of the United States of America, 94(8), 3584–3589. https://doi.org/10.1073/pnas.94.8.3584
Murfin, K. E., Kleinbard, R., Aydin, M., Salazar, S. A., & Fikrig, E. (2019). Borrelia burgdorferi chemotaxis toward tick protein Salp12 contributes to acquisition. Ticks and Tick-Borne Diseases, 10(5), 1124–1134. https://doi.org/10.1016/j.ttbdis.2019.06.002
NCBI. (2016). Outer Surface Protein A (ospa) (plasmid) [Borreliella burgdorferi Bol26] - protein – NCBI. National Center for Biotechnology Information. https://www.ncbi.nlm.nih.gov/protein/226202344
Tabb, J. S., Rapoport, E., Han, I., Lombardi, J., & Green, O. (2022). An antigen-targeting assay for Lyme disease: Combining aptamers and SERS to detect the OspA protein. Nanomedicine: Nanotechnology, Biology and Medicine, 41, 102528. https://doi.org/10.1016/j.nano.2022.102528