Part:BBa_K3746009
T7-LacO-NSP4-tvLac-6His
Description
This is a part design for the Phase I project (2021 iGEM) of Team Hong Kong JSS.
The IPTG inducible secretory expression construct of laccase from Trametes versicolor (tvLac) (BBa_K3746002). It is the secretory version of expression construct (BBa_K3746006).
The coding sequence of tvLac is driven by T7 promoter (BBa_I719005), a lac operon (BBa_K3076802), strong RBS (BBa_K3076801), a signal peptide for protein secretion (NSP4 (BBa_K3746004)) and a T7 terminator (BBa_K395601).
The laccase protein is tagged with a 6-His Tag (CATCATCATCATCATCAT) for protein purification.
The NSP4 is a newly discovered signal peptide used in E. coli for protein secretion by signal recognition particle (SRP) pathway. This signal peptide was developed by Han, S.K., et al. (2017) based on the sequence of DsbAss signal peptide. They reported that this Novel Signal Peptide 4 has about 4 times higher secretory efficiency than the conventional DsbAss signal peptide. [1]
Reference
[1] Han, S. J., Machhi, S., Berge, M., Xi, G., Linke, T., & Schoner, R. (2017). Novel signal peptides improve the secretion of recombinant Staphylococcus aureus alpha toxinH35L in Escherichia coli. AMB Express, 7(1). https://doi.org/10.1186/s13568-017-0394-1
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 138
Illegal PstI site found at 1248
Illegal PstI site found at 1291 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 138
Illegal PstI site found at 1248
Illegal PstI site found at 1291 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1646
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 138
Illegal PstI site found at 1248
Illegal PstI site found at 1291 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 138
Illegal PstI site found at 1248
Illegal PstI site found at 1291
Illegal NgoMIV site found at 119
Illegal AgeI site found at 230
Illegal AgeI site found at 1043
Illegal AgeI site found at 1473 - 1000COMPATIBLE WITH RFC[1000]
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