Part:BBa_K4847010
Siglec-1-pET28a
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pET28a-Siglec-1 (BBa_K4847010)
Plasmid Design
We engineered the bacteria that can translate the complex of Siglec-1 (BBa_K4847009) and double 6 His tags, which provide a scaffold for later research of deleting sTF using the Nickel beads immobilized with the Siglec-1. pET28a (BBa_K3521004) was chosen as the vector of the experiment. It contains a sequence of DNA that is resistant to the antibiotic Kanamycin, which plays an important role in the selection of bacteria that contain pET28a. The Lac I operator on the plasmid is used to control the 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 to the promoter. The translation of the desirable protein thus starts.
The double 6 His tags ensure 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 has a lower cost and is easier to produce compared to the biotin-conjugated Siglec-1 lectin. Meanwhile, the use of the Siglec-1-His tag complex reduces the steps needed for conjugation, which increases the efficiency of the experiment.
Engineering Principle
Siglec-1 (Sialoadhesin, CD169) is a monocyte/macrophage-restricted adhesion molecule that can bind to granulocytes, red blood cells, B cells, and to CD43 T cells [1,2]. Siglec-1 is a type I transmembrane protein belonging to the linoleic acid binding immunoglobulin-like lectins family[3]. Siglec-1 hybridizes with various sialic acid molecules[4]. The molecular basis of carbohydrate binding for lectin receptors has been studied through site-specific mutagenesis, crystallography, and nuclear magnetic resonance analysis. Within the critical V-set domain of Siglec-1, arginine 106 and tryptophan at positions 97 and 2 were identified as key residues for interaction with sialic acid[5].
Construction and Cultivation
PCR was used to amplify Siglec-1. By using corresponding Primers R and F, the newly synthesized strands are elongated, allowing specific restriction enzymes to cut sticky ends in the following processes. Using gel electrophoresis to test the results (Figure 2A), we found that the gene length was over 5000bp (in fact 5130bp). Next, we connected the digested plasmids by T4 DNA ligase, and we transferred the 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 2B).
Characterization/Measurement
SDS-PAGE
IPTG-induced protein was purified by the Ni-extracted and placed in holes of sodium dodecyl sulfate gel and electrophorized. The result is shown below.
Based on the actual situation, different concentrations of sTF are prepared. The freeze-drying sample is diluted using sample diluent. By pipetting concentrated sample to the EP tube with sample diluent for several times, a sample with a gradient is being prepared. These solutions are split charging and used in the later ELISA experiment.
Meanwhile, 10 ng/mL bTF solution and 100 ng/mL sTF solution are also prepared.
Immobilization of Siglec-1 with Nickel beads
After adding a PBS solution, the Nickel beads are centrifuged, and the supernatant is discarded and the PBS solution is added again. This process is repeated for 3 times. Siglec-1 in 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 centrifuged. After adding PBS again, the incubated Nickel Beads solution is split charged into different EP tubes. The different concentration sTF solution is added to 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.
ELISA
The micro-plate is prepared, and 100μL of different sTf and bTf solutions are added to the well in 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. After 2 minutes of 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 incubated at 37°C for 20 minutes in a darkroom. Stop solution is added 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.
Learn
The 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 2nd DBTL cycle, the test of the Deletion Chamber. The obvious decrease in concentration for sTF before and after the Deletion Chamber indicated the effectiveness of deletion by Siglec-1. The increasing trend of concentration after the 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.
References
[1] Crocker PR, Hartnell A, Munday J, Nath D. The potential role of sialoadhesin as a macrophage recognition molecule in health and disease. Glycoconj J 1997;14:601–9.
[2] van den Berg TK, Nath D, Ziltener HJ, et al. Cutting edge: CD43 functions as a T cell counterreceptor for the macrophage adhesion receptor sialoadhesin (Siglec-1). J Immunol 2001;166:3637–40.
[3] Crocker P.R., Paulson J.C., Varki A. Siglecs and their roles in the immune system. Nat. Rev. Immunol. 2007;7:255–266.
[4] Hartnell A. Characterization of human sialoadhesin, a sialic acid binding receptor expressed by resident and inflammatory macrophage populations. Blood. 2001;97, 288–296
[5] Erikson E, Wratil PR, Frank M, et al. Mouse Siglec-1 Mediates trans-Infection of Surface-bound Murine Leukemia Virus in a Sialic Acid N-Acyl Side Chain-dependent Manner. J Biol Chem. 2015;290(45):27345-27359.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 4672
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 4402
Illegal BglII site found at 5858
Illegal BamHI site found at 5059
Illegal XhoI site found at 4872 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 2622
Illegal NgoMIV site found at 2782
Illegal NgoMIV site found at 4370
Illegal NgoMIV site found at 6129 - 1000COMPATIBLE WITH RFC[1000]
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