Difference between revisions of "Part:BBa K4847005"

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pET28a-Transferrin-mCherry (pET28a-bTF-mCherry)

pET28a-Transferrin-mCherry

pET28a-Transferrin-mCherry (pET28a-bTF-mCherry)

Engineering Principle

bTF (BBa_K4847000) is a peptide protein produced by the activation of neuraminidase in the brain, which is only present in cerebrospinal fluid and perilymphatic fluid, making it an important marker for specific identification of cerebrospinal fluid[1]. The mCherry (BBa_K4335004) is a red fluorescent protein that provides fluorescent labeling for bTF, allowing for tracking within the NC membrane[2].

Construction Design

Figure 1. Plasmid map of pET28a-Transferrin-mCherry

pET28a-bTF-mCherry (BBa_K4847005) is mainly composed of three basic parts: the vector pET28a, bTF (BBa_K4847000) and mCherry (BBa_K4335004). We amplified the mCherry and bTF gene fragments, and then connected to the vector pET28a for plasmid construction. Finally we transformed into E. coli BL21 to express protein in vivo.

Construction Approach

Figure 2. Plasmid construction results of pET28a-Transferrin-mCherry

Firstly, we obtained a single gene fragment Transferrin and mCherry through PCR, and then connected the two fragments together to form a long fragment Transferrin-mCherry (2835bp) through overlap-PCR, as shown in the Figure 2A. After the fragments was connected to plasmid pET28a, it was transformed into Escherichia coli and a monoclonal was successfully grown, as shown in the Figure 2C. Then we found that 9 out of 16 samples were found to be positive in E.coli (DH5α) and only 1 positive sample in E.coli (BL21) by PCR identification (Figure 2B). Finally, through gene sequencing (Figure 2D), the results also proved that we successfully constructed the recombinant plasmid pET28a-Transferrin-mCherry.

Characterization

1. SDS-PAGE

Upon IPTG inducing expression, proteins purified by Ni-extracted are placed in holes of sodium dodecyl sulfate gel and electrophorized. The band locations match the molecular weight of beta-transferrin (79 kDa), mCherry protein (30 kDa) and beta-transferrin-mCherry (107kD).

Figure 3. Results of pET28a-Transferrin-mCherry protein expression
(P represents “Precipitation”, S represents “Supernatant”)

2. AuNP testing

sTF, bTF, sTF/bTF mixture before and after Deletion Chamber is added on the test strips, and the color change is shown below (Figure 4). sTF will be detected with the same antigen band as bTF which is a false positive result. After DC, sTF is removed while the bTF can be still detected by presenting a positive antigen band, therefore, we can conclude that the deletion chamber can successfully eliminate the effect from sTF in the real context. Under the spectrometer, we can see there is no light of sTF while there is obvious light in bTF-mC samples which the presence of the red line on the strip is not induced by the AuNP itself but the color of bTF-mC.

Figure 4. Results of sTF, bTF, and sTF/BTF mixture test strips before and after Deletion Chamber

Experiment notes

Due to the use of a pipette, the immobilizing of the antibody on the test strip is hard to achieve because the antibody solution will diffuse on the NC membrane, and the color change for AuNP is not as distinctive as the normal test strip.Meanwhile, the camera used to record also makes the red color dimer compared the observation with bare eyes.However, the color change on the key test strip can also be observed. By using the dispenser to immobilize the antibody on the NC membrane, the result of the test strip might become more obvious.

Conclusion

The DNA sequencing and the characterization prove the successful construction and function of pET28a-Transferrin-mCherry. In addition, the tracing experiment proves that the presence of the red line on the test strip is caused by the Anti-transferrin antibodies on the NC membrane that traps the bTF-AuNP complex. Therefore, this method can be used for the visualization of our bTF detection kit.

References:

1. Haft GF, Mendoza SA, Weinstein SL, Nyunoya T, Smoker W. Use of beta-2-transferrin to diagnose CSF leakage following spinal surgery: a case report. Iowa Orthop J. 2004;24:115-8.
2. Fages-Lartaud, M., Tietze, L., Elie, F., Lale, R., & Hohmann-Marriott, M. F. (2022). mCherry contains a fluorescent protein isoform that interferes with its reporter function. Frontiers in bioengineering and biotechnology, 10, 892138

Sequence and Features