Difference between revisions of "Part:BBa K4390026"
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'''This part is not compatible with BioBrick RFC10 assembly but is compatible with the iGEM Type IIS Part standard [[Help:Standards/Assembly/Type_IIS|which is also accepted by iGEM.]]''' | '''This part is not compatible with BioBrick RFC10 assembly but is compatible with the iGEM Type IIS Part standard [[Help:Standards/Assembly/Type_IIS|which is also accepted by iGEM.]]''' | ||
| + | |||
| + | '''This is a level 1 part formed by assembly of the following level 0 parts:''' | ||
| + | {| class="wikitable" style="margin:auto" | ||
| + | |- | ||
| + | | Promoter || [[part:BBa_J23100|J23100]] | ||
| + | |- | ||
| + | | RBS || [[part:BBa_B0034|B0034]] | ||
| + | |- | ||
| + | | N-O part || [[part:BBa_K4390025|K4390025]] | ||
| + | |- | ||
| + | | C part || [[part:BBa_K4390009|K4390009]] | ||
| + | |- | ||
| + | | Terminator || [[part:BBa_K4390001|K4390001]] | ||
| + | |} | ||
==Usage and Biology== | ==Usage and Biology== | ||
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SB7 is a 7-amino-acid silica-binding motif from the spore coat protein CotB1 of Bacillus cereus. According to studies, its three arginine residues are important for binding to silica surfaces. We tagged this sequence to the N-terminus of the metallothionein of Mytilus edulis. Therefore, when the lysate from a cell culture expressing SB7-tagged metallothionein is mixed with silica beads followed by washing off the supernatant, we would be left with metallothioneins bound to the silica beads. This entire construct could then be applied as an ultrafiltration device for removing heavy metals from the wastewater. This is one of the three types of silica-binding tags we explored for immobilising proteins to silica beads. | SB7 is a 7-amino-acid silica-binding motif from the spore coat protein CotB1 of Bacillus cereus. According to studies, its three arginine residues are important for binding to silica surfaces. We tagged this sequence to the N-terminus of the metallothionein of Mytilus edulis. Therefore, when the lysate from a cell culture expressing SB7-tagged metallothionein is mixed with silica beads followed by washing off the supernatant, we would be left with metallothioneins bound to the silica beads. This entire construct could then be applied as an ultrafiltration device for removing heavy metals from the wastewater. This is one of the three types of silica-binding tags we explored for immobilising proteins to silica beads. | ||
| + | |||
| + | ==Characterization== | ||
| + | After protein expression in the BL21(DE3) cell cultures, the cultures were lysed by sonication, and the lysates were run on an SDS-PAGE gel to confirm the presence of our CBD fused proteins (Figure 1). This SDS-PAGE step also serves as a solubility test to confirm that all of our desired proteins are in the soluble fraction and can be properly incorporated into protein immobilisation methods. | ||
| + | |||
| + | [[File:SDS_MT_CBD.png|600px|centre|frameless|link=]] | ||
| + | ''Figure 2. SDS-PAGE gel of the lysates containing our expressed constructs. Lane 0 represents the negative control, which is the BL21(DE3) strain containing only the pJUMP29 LacZ acceptor plasmid without any insert. The red lines indicate the bands representing our constructs. The ladder we used was Prestained Protein Marker, Broad Range (7-175 kDa) (NEB #P7708S).'' | ||
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Latest revision as of 23:28, 13 October 2022
SB7-tagged M. edulis Metallothionein
This part is not compatible with BioBrick RFC10 assembly but is compatible with the iGEM Type IIS Part standard which is also accepted by iGEM.
This is a level 1 part formed by assembly of the following level 0 parts:
| Promoter | J23100 |
| RBS | B0034 |
| N-O part | K4390025 |
| C part | K4390009 |
| Terminator | K4390001 |
Usage and Biology
Metallothionein (MT) is a small protein (around 6-7 kDa) which is rich in cysteine. These thiol group in cysteines provide ability to chelate almost all heavy metal ions including Cd2+, Hg2+, Pb2+ and As3+, but had been shown that has higher binding affinity with Hg2+ (Manceau, A. et al., 2019). The ability of chelating heavy metals provides the metal tolerance for its hosts. This MT sequence was obtained from Mytilus edulis, a blue mussel which originally live in aqueous environment (MACKAY E. A. et al.,1993).
SB7 is a 7-amino-acid silica-binding motif from the spore coat protein CotB1 of Bacillus cereus. According to studies, its three arginine residues are important for binding to silica surfaces. We tagged this sequence to the N-terminus of the metallothionein of Mytilus edulis. Therefore, when the lysate from a cell culture expressing SB7-tagged metallothionein is mixed with silica beads followed by washing off the supernatant, we would be left with metallothioneins bound to the silica beads. This entire construct could then be applied as an ultrafiltration device for removing heavy metals from the wastewater. This is one of the three types of silica-binding tags we explored for immobilising proteins to silica beads.
Characterization
After protein expression in the BL21(DE3) cell cultures, the cultures were lysed by sonication, and the lysates were run on an SDS-PAGE gel to confirm the presence of our CBD fused proteins (Figure 1). This SDS-PAGE step also serves as a solubility test to confirm that all of our desired proteins are in the soluble fraction and can be properly incorporated into protein immobilisation methods.
Figure 2. SDS-PAGE gel of the lysates containing our expressed constructs. Lane 0 represents the negative control, which is the BL21(DE3) strain containing only the pJUMP29 LacZ acceptor plasmid without any insert. The red lines indicate the bands representing our constructs. The ladder we used was Prestained Protein Marker, Broad Range (7-175 kDa) (NEB #P7708S).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 11
Illegal NheI site found at 34
Illegal NotI site found at 73 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References
MACKAY, E. A. et al. (1993) Complete amino acid sequences of five dimeric and four monomeric forms of metallothionein from the edible mussel Mytilus edulis. European journal of biochemistry. 218 (1), 183–194.
Manceau, A. et al. (2019) Mercury(II) Binding to Metallothionein in Mytilus edulis revealed by High Energy‐Resolution XANES Spectroscopy. Chemistry : a European journal. 25 (4), 997–1009.