In this project, we aim to provide a test kit that detect beta-2-Transferrin, which is the specific biomarker that only present in Cerebrospinal fluid (CSF). However, sialo Transferrin (sTF) is a major component that present in both serum and CSF. The minor differences in the Transferrin (TF) based glycol chains make it difficult to distinguish from sTF, which may lead to sTF binding with anti-TF antibody to create a false positive result.

Figure 1. Schematic diagram of the structure of two transferrin proteins

(Sialo Transferrin has a Sialo group and beta-2-Transferrin doesn’t)

Therefore, for removing the sTF in the nasal sample, we design the Deletion Chamber (DC), which contains nickel beards immobilized with Siglec-1, a lectin that binds specific to the sialic acid. We use BL21 E.coli to produce lectin connecting with double 6 His tag.

Figure 2. Schematic diagram of Siglec-1 protein binding to nickel ions

(Function of His tag)

When the nasal sample is collected, it is sent to the Deletion Chamber to remove the sTF. The deletion of sTF highly reduces the risk of false positive result in the detection of cerebrospinal fluid.

Figure 3. Process of deletion in Deletion Chamber

After the deletion, the sample is sent to the chamber that contains AuNP-anti Transferrin antibody.

The conjugated sample is then added on the Nitrocellulose test strip immobilized with anti-Transferrin antibody. The red line indicates the positive result.

Figure 4. Function of the AuNP and the Test Strip

Our Engineering Design Cycle involves four cycles.

1. In the first cycle, we created a bacterium that produce Siglec-1 connecting with double 6 His tags. We then construct Deletion Chambers using Nickel beads and Siglec-1. We use DNA sequencing to verify the result and test the function of deletion chamber via ELISA.

2. In the 2-4 cycles, we constructed a bacteria synthesis beta-2-Transferrin (bTF), mCheery (mC) and beta-2 Transferrin-mCheery (bTF-mC). We conjugated the sTF and bTF samples with AuNP and assemble simple test strips to simulate the practical situation. The bTF-mC complex is verified by protein electrophoresis. The conjugated sample is dripped on the test strip for testing, and the bTF-mC is used on the test strip to trace the path via spectrometer.

DBTL 1: pET28a-Siglec-1 (BBa_K4847010)

Design 1:

Aims: Creates the bacteria that can translate 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.

pET28a was chosen as the vector of the experiment. It contains a sequence of DNA that for resistance to the antibiotic Kanamycin, which plays an important role in selection of bacteria that contains pET28a. The Lac l operator on the plasmid is used to control expression of the protein. When the inducer IPTG is added to the E.coli culture medium, it inactivates Lac I to produce repressor protein and allows RNA polymerase to bind on the promotor. The translation of the desirable protein thus started.

The double 6 His tags ensures the protein can be purified in the following Nickel column extraction and immobilized on the Ni-beads for constructing the Deletion Chamber. The Siglec-1-His tag complex have a lower cost and easier to produced compared to the biotin conjugated Siglec-1 lectin. Meanwhile, the use of Siglec-1-His tag complex reduces the steps need for conjugation, which increases the efficiency of the experiment.

-Plasmid: pET28a

-Bacterium: BL21 E.coli

-Key gene: Siglec-1

-Enzymes: protease, ligase, primase

Figure 5. Plasmid map of pET28a-Siglec-1

Build 1:

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

PCR was used to amplify Siglec-1. By using corresponding Primers R and F, the new synthesized stands are elongated, which allows specific restriction enzymes to cut sticky ends in the following processes. Using gel electrophoresis to test the results (Figure 6A), we found that the gene length was over 5000bp (in fact 5130bp). Next, we connected to the digested plasmids by T4 DNA ligase, and we transferred plasmids to the component cell BL21. The bacteria were then cultured on the culture medium containing Kanamycin. Only the bacteria with successfully transformed plasmids would grow normally, due to the antibiotic resistance on the plasmids (Figure 6B).

Test 1:

1. SDS-PAGE

Products of the Ni-extracted are placed in holes of sodium dodecyl sulfate gel and electrophorized. The result is shown below.

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

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

2. Define the concentration of the sTF used in the Deletion Chamber.

Based on the actual situation, different concentration of sTF are prepared. The freeze-drying sample is diluted using sample diluent. By pipette concentrated sample to the EP tube with sample diluent for several times, a sample with gradient is being prepared. These solutions are split charing and used in the later ELISA experiment.

Figure 8. sTF concentration before (x-axis) and after (y-axis) deletion chamber

Meanwhile, 10 ng/mL bTF solution and 100 ng/mL sTF solution is also prepared.

-Immobilization of Siglec-1 with Nickel beads

After adding PBS solution, the Nickel beads is centrifuged and the supernatant is discarded and the PBS solution is added again. This process is repeated for 3 times. Siglec-1 in the Cycle 1 is added to the solution containing Ni-beads. Due to the double 6 his Tags connecting on the Siglec-1, the Siglec-1 with immobilized on the Nickel beads.

-Assemble the deletion chamber and sTF testing

The mixture of lectin and Nickel Beads is incubated overnight and centrifuge. After adding PBS again, the incubated Nickel Beads solution is split charged into different EP tubes. The different concentration sTF solution is added in the Nickel beads separately. After 30 minutes waiting, the sample is added to the EP tubes and Elisa Test for sTF solutions before and after the Deletion is applied.

3. Elisa testing

The micro-plate is prepared and 100μL of different sTf and bTf solutions to the well in the order. The plate is then covered with the adhesive strip and incubated at 37°C for 60 minutes. After incubation, the liquid is removed and 200μL of wash buffer is added. For 2 minutes waiting, the buffer is then removed and this process is repeated for 3 times. After that, we invert the ELISA plate on the filter paper, tap the plate gently to remove the remaining liquid. HRP-conjugate (1x) is then applied to each well and the washing step is repeated for 5 times. Next, TMB (the chromogenic substrate adopted in the experiment) is added and the plate is incubate at 37°C for 20 minutes in a darkroom. Stop solution to each well to terminate the reaction. Finally, within 5 minutes, we measure the solution at a wavelength of 450nm.

Based on the standard curve, we calculated the concentration of each well, and the results are shown below.

Figure 9.concentration of sTF and bTF before and after (sTF+DC, bTF+DC), respectively

Learn 1:

The double 6 his Tags connecting with Siglec-1 allows the conjugation of Siglec-1 with Nickel beads. Meanwhile, it enables us to extract sufficient Siglec-1 in the latter process of assembling the Deletion Chamber.

Based on the successful extraction of Siglec-1, we start our 2^nd DBTL cycle, the test of Deletion Chamber. The obvious decrease concentration for sTF before and after Deletion Chamber indicated the effectiveness of deletion by Siglec-1. The increasing trend of concentration after Deletion Chamber shows that the sTF is correctly measured. However, we can see there is an increase in some of the samples with low concentrations. That is partially due to measure errors on ELISA itself.

DBTL 2: pET28a-Transferrin (BBa_K4847003)

Design 2:

Figure 10. Plasmid map of pET28a-Transferrin

We used the T7 promoter as the strong promoter in our plasmid, which can efficiently initiate gene expression. We chose pET28a as the plasmid vector and used restriction endonucleases NheI and BamHI to cut Transferrin gene fragments and plasmid, then connected them with DNA ligase, we will get the recombinant plasmid pET28a-Transferrin (Figure 10). Then we transformed them into Escherichia coli E.coli BL21(DE3) in the heat shock reaction. Finally, we will use IPTG to induce our target protein Transferrin expression.

Build 2:

Firstly, we amplified Transferrin gene fragments by PCR, as shown in Figure 11A and then we used restriction endonucleases NheI and BamHI to cut plasmid pET28a (Figure 11B). After obtaining the recombinant plasmid, we transformed it into Escherichia coli BL21(DE3), successfully obtained the transformant (Figure 11C). Gene sequencing the recombinant we found out that there was no mutations and matched to our envisions (Figure 11D).

Figure 11. Plasmid construction results of pET28a-Transferrin

Test 2:

Figure 12. SDS-PAGE results of pET28a-Transferrin protein expression

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

From the Figure 12, it can be seen that we have successfully expressed the protein Transferrin, with a protein size of about 79kD, but mainly in precipitation, with weak bands in the supernatant.

Learn 2:

The protein content in the supernatant is relatively weak, possibly because the optimal expression conditions were not found during protein purification. In the future, we will explore the optimal conditions for protein expression and increase the protein expression level.

DBTL 3: pET28a-mCherry (BBa_K4847004)

Design 3:

In order to obtain the red fluorescent protein, we constructed the mCherry fragment onto the plasmid pET28a. Following the same idea as the previous round, the plasmid was transformed into Escherichia coli BL21 (DE3) after obtaining the recombinant plasmid. Finally, we used IPTG to induce the expression of target protein mCherry.

Figure 13. Plasmid map of pET28a-mCherry

Build 3:

Figure 14. Plasmid construction results of pET28a-mCherry

Similarly, we used synthesis of mCherry fragment as templates, primer of mCherry-F and primer of mCherry-R amplified gene mCherry (Figure 14A). Compared to the marker, the gene fragment is 711bp and the amplification is correct. Then, we used BamHI and XhoI to cut plasmid pET28a. Compared to the control, the plasmid pET28a was successfully cut into a linear shape (Figure 14B). After obtaining the recombinant plasmid, we transformed it into Escherichia coli BL21(DE3) and successfully grew transformants (Figure 14C). The sequencing results further demonstrate that we have successfully constructed the plasmid without any mutations (Figure 14D).

Test 3:

Figure 15. Results of pET28a-mCherry protein expression

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

From the above figure, it can be seen that we have successfully expressed the protein, which is purple red in color (Figure 15A). SDS-PAGE experiments have also confirmed that the protein size is about 30 kD (Figure 15B).

Learn 3:

We successfully expressed the protein and later constructed it onto the Transferrin gene to construct a fusion protein.

DBTL 4: pET28a-Transferrin-mCherry (BBa_K4847005)

Design 4:

Figure 16. Plasmid map of pET28a-Transferrin-mCherry

Creates the bacteria that can translate the complex of beta-2-Transferrin and mCherry (bTF-mC), which contribute to the following experiment for the tracing on NC membrane. The mCherry connected with beta-2-Transferrin allows us directly to see whether the protein is express via the color of the solution. Meanwhile, the mCherry also gives a fluorescence marker to the beta-2-Transferrin , enabling the tracing in the membrane. We connected Transferrin gene and mCherry together using GS-linker, and then connected the fusion gene fragments to the plasmid pET28a (Figure 16). Finally we transformed into E. coli to express protein in vivo.

Build 4:

Figure 17. 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 17A. 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 17C. 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 17B). Finally, through gene sequencing (Figure 17D), the results also proved that we successfully constructed the recombinant plasmid pET28a-Transferrin-mCherry.

Test 4:

1. SDS-PAGE

Products of the Ni-extracted are placed in holes of sodium dodecyl sulfate gel and electrophorized.

The resulting band locations matches the molecular weight of beta-transferrin (79 kDa), mCherry protein (30 kDa) and beta-transferrin-mCherry (107kD).

Figure 18. 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 (Left).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.

Figure 19. Results of sTF, bTF, and sTF/BTF mixture test strips before and after Deletion Chamber

Learn 4:

The DNA sequencing for the bacteria and the ELISA testing proves the successful construction of the bacteria bTF-mC, which plays an important role in the following tracing experiment.

Due to the use of pipette, the immobilizing 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 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. The sTF sample, Btf sample after Deletion Chamber, and bTF + sTF after deletion chamber test strip have a change of color. The sTF sample after Deletion Chamber does not have a color change.

By using the dispenser to immobilize the antibody on the NC membrane, the result of the test strip might become more obvious.

Meanwhile, the tracing experiment proves that the presents of the red line is not induced by the AuNP itself (the unconjugated AuNP particles). The Anti transferrin antibodies on the NC membrane traps the bTF-AuNP complex, which results in the color change. Therefore, this method can be used for the visualization of our detection kit.