Difference between revisions of "Part:BBa K3739115"
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K525998-Aly01-his-3SpyTag-lecA-B0010 | K525998-Aly01-his-3SpyTag-lecA-B0010 | ||
| + | ===Biology=== | ||
| + | =====SpyTag===== | ||
| + | SpyTag (a 13-residue peptide) and SpyCatcher (a 116-residue domain) are engineered split fragments of the second immunoglobulin-like collagen adhesin domain (CnaB2) of the fibronectin-binding protein (FbaB) of ''Streptococcus pyogenes''. IGEM team Peking 2016 has modified the peptide and domain into triple SpyTag and triple SpyCatcher, which can form bonds with each other and create Spy Crosslinking Network, enabling the formation of hyper-branched products and polymer network at low concentrations, and gels with a high degree of cross-linkage at high concentrations.<sup>1</sup> | ||
| + | |||
| + | =====LecA===== | ||
| + | LecA is a soluble galactose-binding lectin from bacterium ''Pseudomonas aeruginosa''. The lectin is specific for α-galactose.<sup>2</sup> | ||
| + | |||
| + | =====Aly01===== | ||
| + | Aly01 is alginate lyase from ''Vibrio natriegens'' SK42.001, which is secreted out and able to digest alginate to unsaturated alginate oligosaccharide. Its signal peptide (named Aly01 in our parts), which is fused with heterogenous protein, is performed well in ''E. coli''. It is implied that the heterogenous protein fused with Aly01 signal peptide may be also secreted efficiently. | ||
| + | |||
| + | ===Usage=== | ||
| + | In order to get secreted 3SpyTag-LecA and further purify it, we added the signal peptide Aly01, followed by a his-tag (6*His) at the N-terminal of 3SpyTag-LecA. We used both <partinfo>BBa_K081005</partinfo> and <partinfo>BBa_K525998</partinfo> to construct the expression system and obtained the composite <partinfo>BBa_K3739035</partinfo> and BBa_K3739115, which are assembled on the expression vector pET-28a(+) by standard assembly. The constructed plasmids were transformed into ''Vibrio natriegens'', then the positive transformants were selected by kanamycin and confirmed by colony PCR and sequencing. | ||
| + | |||
| + | ===Characterization=== | ||
| + | =====1. Agarose Gel Electrophoresis===== | ||
| + | When we were transferring the plasmid into ''Vibrio natriegens'', colony PCR was used to certify the plasmid was correct. We got the target (1154bp). | ||
| + | |||
| + | [[File:T--XMU-China--aly01-his-spytag-lecA.png|200px|alt text]] | ||
| + | |||
| + | '''Fig.1''' The result of colony PCR. Plasmid pET-28a (+). | ||
| + | |||
| + | ===Reference=== | ||
| + | 1. Yang, X.; Wei, J.; Wang, Y.; Yang, C.; Zhao, S.; Li, C.; Dong, Y.; Bai, K.; Li, Y.; Teng, H.; Wang, D.; Lyu, N.; Li, J.; Chang, X.; Ning, X.; Ouyang, Q.; Zhang, Y.; Qian, L., A Genetically Encoded Protein Polymer for Uranyl Binding and Extraction Based on the SpyTag-SpyCatcher Chemistry. ''ACS Synth Biol'' '''2018''', 7 (10), 2331-2339.<br/> | ||
| + | 2. Kuhaudomlarp, S.; Gillon, E.; Varrot, A.; Imberty, A., LecA (PA-IL): A Galactose-Binding Lectin from Pseudomonas aeruginosa. ''Methods Mol Biol'' '''2020''', 2132, 257-266. | ||
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Revision as of 00:49, 22 October 2021
K525998-Aly01-his-3SpyTag-lecA-B0010
K525998-Aly01-his-3SpyTag-lecA-B0010
Biology
SpyTag
SpyTag (a 13-residue peptide) and SpyCatcher (a 116-residue domain) are engineered split fragments of the second immunoglobulin-like collagen adhesin domain (CnaB2) of the fibronectin-binding protein (FbaB) of Streptococcus pyogenes. IGEM team Peking 2016 has modified the peptide and domain into triple SpyTag and triple SpyCatcher, which can form bonds with each other and create Spy Crosslinking Network, enabling the formation of hyper-branched products and polymer network at low concentrations, and gels with a high degree of cross-linkage at high concentrations.1
LecA
LecA is a soluble galactose-binding lectin from bacterium Pseudomonas aeruginosa. The lectin is specific for α-galactose.2
Aly01
Aly01 is alginate lyase from Vibrio natriegens SK42.001, which is secreted out and able to digest alginate to unsaturated alginate oligosaccharide. Its signal peptide (named Aly01 in our parts), which is fused with heterogenous protein, is performed well in E. coli. It is implied that the heterogenous protein fused with Aly01 signal peptide may be also secreted efficiently.
Usage
In order to get secreted 3SpyTag-LecA and further purify it, we added the signal peptide Aly01, followed by a his-tag (6*His) at the N-terminal of 3SpyTag-LecA. We used both BBa_K081005 and BBa_K525998 to construct the expression system and obtained the composite BBa_K3739035 and BBa_K3739115, which are assembled on the expression vector pET-28a(+) by standard assembly. The constructed plasmids were transformed into Vibrio natriegens, then the positive transformants were selected by kanamycin and confirmed by colony PCR and sequencing.
Characterization
1. Agarose Gel Electrophoresis
When we were transferring the plasmid into Vibrio natriegens, colony PCR was used to certify the plasmid was correct. We got the target (1154bp).
Fig.1 The result of colony PCR. Plasmid pET-28a (+).
Reference
1. Yang, X.; Wei, J.; Wang, Y.; Yang, C.; Zhao, S.; Li, C.; Dong, Y.; Bai, K.; Li, Y.; Teng, H.; Wang, D.; Lyu, N.; Li, J.; Chang, X.; Ning, X.; Ouyang, Q.; Zhang, Y.; Qian, L., A Genetically Encoded Protein Polymer for Uranyl Binding and Extraction Based on the SpyTag-SpyCatcher Chemistry. ACS Synth Biol 2018, 7 (10), 2331-2339.
2. Kuhaudomlarp, S.; Gillon, E.; Varrot, A.; Imberty, A., LecA (PA-IL): A Galactose-Binding Lectin from Pseudomonas aeruginosa. Methods Mol Biol 2020, 2132, 257-266.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]