DNA
Si4-CBPa-B

Part:BBa_K2053002

Designed by: Mathieu Hubert   Group: iGEM16_Pasteur_Paris   (2016-10-11)


Fusion Si4-cellulose-binding domain-B domain protein A

It is a fusion protein between the phage displayed silica-binding peptide (Si4) developed by Naik et al (Journal of Nanoscience and Nanotechnology, Volume 2, Number 1, February 2002, pp. 95-100(6)), the Clostridium cellulovorans cellulose-binding domain of cellulose-binding protein A (CBPa) with the B domain of staphylococcus aureus protein A (BpA). This fusion protein is used to bind silica and antibodies to cellulose. We use these functions to functionalize a silica-cellulose composite-based patch into an immunodetection-based system.

Functional characterization Biosilification. We first tested its ability to bio-condense silicic acid into silica. To do that, we activated tetraethyl orthosilicate (TEOS) by using HCl (1M) in order to remove ethyl groups from the molecule, allowing silic acid release. Then, we mixed TEOS with our fusion protein, and incubated it for 2h at room temperature (RT) (Fig. 1A). After incubation, we clearly noted the appearance of a silica-based precipitate (Fig. 1B). In order to quantify the process, we titrated free silicic acid in the supernatant by a molybdate assay (iGEM Minnesota 2011). Compared to negative control (without the fusion protein), free silic acid significantly decreased (84%), validating its transformation into solid silica (biosilification) (Fig. 1C and 1D).

T--iGEM16_Pasteur_Paris--BBa_K2053002_Fig1.png
Fig. 1 Silification of C2

Cellulose-binding. Then, we tested its ability to bind to cellulose. To do that, we mixed cellulose powder (Avicell, Sigma) with an excess of non-specific proteins, bovine serum albumin (BSA), playing the role of non-specific competitor. Into the tests tubes, we added our fusion protein, and incubated the mix for 1h at RT, whilst stirring. Cellulose-protein is centrifuged, and supernatant was removed (S1). Cellulose-based pellet was washed, and the mix was incubated and centrifuged. The washing supernatant was removed (S2). Pellet-bound proteins were released (P). S1, S2, and P samples were denatured and analyzed by SDS-PAGE (Fig. 2A). No fusion protein was found in the supernatants, but in pellet (Fig. 2B). This data suggesting that our fusion protein binds to cellulose.

T--Pasteur_Paris--BBa_K2053002_Fig2.png
Fig. 2 Cellulose Binding

Usage and Biology

The use of the composite biomaterial in the Patch will ensure both detection and degradation.


Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 160
    Illegal AgeI site found at 460
  • 1000
    COMPATIBLE WITH RFC[1000]


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