CSF-Rhinorrhea is a dangerous disease due to its resemblance to rhinorrhea, yet it has the potential for rapid deterioration to lethal diseases, such as meningitis. Therefore, we propose a cheap and easily accessible means of detection of CSF-rhinorrhea and fulfill the current absence of POC (point of care) diagnosis.

After thorough research into the subject matter, we recognized the obstructions to the diagnosis test kit (which is sialo transferrin, as elaborated in the previous sections). We attempt to counter this issue by synthesizing lectin (Siglec-1), and conjugating it at the deletion chamber, which is an approach to remove sTF from the sample fluid proposed by Oh^[1].

Table 1. Part Contribution

1. Add new experimental data to an existing part: mCherry BBa_K4335004

mCherry (BBa_K4335004) is a red fluorescent protein, which is one of the reports for developing a universal gene expression method that uses 200 random nucleotides as regulatory sequences to drive the expression of coding sequences^[2].

In our project, we introduced mCherry to link our protein in order for the better visualization. We used mCherry to construct a fusion protein as a reporter and constructed plasmid pET28a-mCherry (BBa_K4847004) and pET28a-bTF-mCherry (BBa_K4847005). Next, we expressed the mCherry protein (about 30kDa) and fusion protein bTF-mCherry (about 107kDa) per the SDS-PAGE gels.

Figure 1 electrophoresis gel of mCherry PCR and SDS-PAGE

Under the spectrometer, we can observe the color of mCherry present in the red band, which is attached to bTF protein.

Figure 2. bTF-mCherry protein is observed under spectrometer

2. Create new parts BBa_K4847000 and BBa_K4847005

bTF ( BBa_K4847000) is a peptide protein produced by the activation of neuraminidase in the brain, only present in cerebrospinal fluid and perilymphatic fluid, making it an important marker for specific identification of cerebrospinal fluid. So we We obtained the plasmid pET28a-bTF(BBa_K4847003) and pET28a-bTF-mCherry (BBa_K4847005) through genetic engineering methods. As shown in figure 3, we can confirm that we successfully constructed the plasmid pET28a-bTF and pET28a-bTF-mCherry.

Figure 3. Construction results

We managed to co-express mCherry and bTF to obtain the protien and analyze the diffusion of bTF-mC on NC membrane.

As shown in figure 4, we successfully expressed the beta-2-Transferrin (79 kD), mCherry(30 kD) and beta-2-Transferrin-mCherry(107 kD).

Figure 4. SDS-PAGE results of protein expression and purification

(P represents “Precipitation”, S represents “Supernatant)

As we can see in figure 5, there are antigen bands on the test strip, which proves that antibodies and colloidal gold can be used for visualization of the detection of bTF. Furthermore, the spectrometer experiment also proves that the Deletion Chamber barely affects the result for bTF detection. Under the spectrometer, the color intensity of mCherry presented in both of the bands are visibly similar, which proves that the appearing red line is caused by bTF, and the Deletion Chamber has a minor effect on the depletion of bTF.

Figure 5: bTF-mC sample, bTF-mC sample with deletion chamber processed (bTF-mC+DC)

3. Create new parts BBa_K4847009 and BBa_K4847010

In order to obtain the complex of Siglec-1 and double 6 His tag, which provide scaffold for later researches of deleting sTF using the Nickel beards immobilized with the Siglec-1 (BBa_K4847009), we designed to construct pET28a-Siglec-1 (BBa_K4847010). As shown in Figure 6, the Siglec-1 fragments were found over 5000bp (5130 bp) as expected.

Figure 6. Plasmid construction results of pET28a-Siglec-1

Products of the Ni-extracted are placed in holes of sodium dodecyl sulfate gel and electrophorized. The result is shown in figure 7 where the Siglec-1 protein was found at 183 KDa.

Figure 7. Results of pET28a-Siglec-1 protein expression

(S represents “Supernatant”, T represents “Flow through”, E represents “Eluent”)

[1] Oh, Jusung, et al. “Determination of Cerebrospinal Fluid Leakage by Selective Deletion of Transferrin Glycoform Using an Immunochromatographic Assay.” Theranostics, vol. 9, no. 14, 1 Jan. 2019, pp. 4182–4191, www.ncbi.nlm.nih.gov/pmc/articles/PMC6592183/, https://doi.org/10.7150/thno.34411. Accessed 7 Oct. 2023.

[2] Lale R., Tietze L., Nesje, J., Onsager I., Engelhardt K., Rückert C., et al. A universal method for gene expression engineering.