Difference between revisions of "Part:BBa K782059"
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Alginate lyases are not present in mammalian cells but have been found in bacteria, algae and marine mollusks. It has already been demonstrated that the addition of alginate lyase degraded alginate-poly(L-lysine)-alginate microcapsules (Breguet et al., 2007). Alginate lyases degrade alginate by the reaction of β-elimination and are classified based on their substrates: some prefer M-rich alginate, whereas others like G-rich more. Therefore we selected an enzyme that could degrade both, G- and M-blocks of alginate. We found the alginate lyase from bacteria Pseudoalteromonas elyakovii to be a promising candidate, which can degrade all types of alginate (Sawabe et al., 2007). We replaced the original bacterial signal peptide with the preprotrypsin leader sequence to ensure the efficient secretion from mammalian cells and attached a Myc tag at the C-terminus to facilitate the detection of secreted enzyme. | Alginate lyases are not present in mammalian cells but have been found in bacteria, algae and marine mollusks. It has already been demonstrated that the addition of alginate lyase degraded alginate-poly(L-lysine)-alginate microcapsules (Breguet et al., 2007). Alginate lyases degrade alginate by the reaction of β-elimination and are classified based on their substrates: some prefer M-rich alginate, whereas others like G-rich more. Therefore we selected an enzyme that could degrade both, G- and M-blocks of alginate. We found the alginate lyase from bacteria Pseudoalteromonas elyakovii to be a promising candidate, which can degrade all types of alginate (Sawabe et al., 2007). We replaced the original bacterial signal peptide with the preprotrypsin leader sequence to ensure the efficient secretion from mammalian cells and attached a Myc tag at the C-terminus to facilitate the detection of secreted enzyme. | ||
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Figure 1: Schematic representation of the BioBrick part for the alginate lyase engineered for secretion from eukaryotic cells. PPT LS denotes preprotrypsin leader sequence, aly is mature alginate lyase coding sequence and Myc indicates Myc peptide tag for detection. E = EcoRI restriction site, X = XbaI restriction site, S = SpeI restriction site, P = PstI restriction site. | Figure 1: Schematic representation of the BioBrick part for the alginate lyase engineered for secretion from eukaryotic cells. PPT LS denotes preprotrypsin leader sequence, aly is mature alginate lyase coding sequence and Myc indicates Myc peptide tag for detection. E = EcoRI restriction site, X = XbaI restriction site, S = SpeI restriction site, P = PstI restriction site. | ||
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<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
===Usage and Biology=== | ===Usage and Biology=== | ||
Revision as of 10:33, 24 September 2012
Alginate lyase
Alginate lyases are not present in mammalian cells but have been found in bacteria, algae and marine mollusks. It has already been demonstrated that the addition of alginate lyase degraded alginate-poly(L-lysine)-alginate microcapsules (Breguet et al., 2007). Alginate lyases degrade alginate by the reaction of β-elimination and are classified based on their substrates: some prefer M-rich alginate, whereas others like G-rich more. Therefore we selected an enzyme that could degrade both, G- and M-blocks of alginate. We found the alginate lyase from bacteria Pseudoalteromonas elyakovii to be a promising candidate, which can degrade all types of alginate (Sawabe et al., 2007). We replaced the original bacterial signal peptide with the preprotrypsin leader sequence to ensure the efficient secretion from mammalian cells and attached a Myc tag at the C-terminus to facilitate the detection of secreted enzyme.
Figure 1: Schematic representation of the BioBrick part for the alginate lyase engineered for secretion from eukaryotic cells. PPT LS denotes preprotrypsin leader sequence, aly is mature alginate lyase coding sequence and Myc indicates Myc peptide tag for detection. E = EcoRI restriction site, X = XbaI restriction site, S = SpeI restriction site, P = PstI restriction site.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 64
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 7
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 85
Illegal NgoMIV site found at 820 - 1000COMPATIBLE WITH RFC[1000]