Difference between revisions of "Part:BBa K782059"

Revision as of 10:32, 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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 64
21
INCOMPATIBLE WITH RFC[21]
Illegal XhoI site found at 7
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 85
Illegal NgoMIV site found at 820
1000
COMPATIBLE WITH RFC[1000]

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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.
+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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 ===Usage and Biology===