Part:BBa_K1159105
Mature Nuclease NucA from Staphylococcus aureus (Thermonuclease) in RFC[25]
Thermonuclease is a endo-exonuclease from Staphylococcus aureus that degrades dsDNA, ssDNA, dsRNA and ssRNA. This part is coding the mature form of the nuclease, also called NucA. This part is flanked by RFC[25] pre- and suffix for further protein fusions.
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]
Protein data table for BioBrick BBa_ automatically created by the BioBrick-AutoAnnotator version 1.0 | ||||||||||||||||||||||||||||||||||||||||||||||
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Nucleotide sequence in RFC 25, so ATGGCCGGC and ACCGGT were added (in italics) to the 5' and 3' ends: (underlined part encodes the protein) ATGGCCGGCGCAACTTCA ... TCAGGTCAAACCGGT ORF from nucleotide position -8 to 453 (excluding stop-codon) | ||||||||||||||||||||||||||||||||||||||||||||||
Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below)
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Sequence features: (with their position in the amino acid sequence, see the list of supported features)
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Amino acid composition:
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Amino acid counting
| Biochemical parameters
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Plot for hydrophobicity, charge, predicted secondary structure, solvent accessability, transmembrane helices and disulfid bridges | ||||||||||||||||||||||||||||||||||||||||||||||
Codon usage
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Alignments (obtained from PredictProtein.org)
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Predictions (obtained from PredictProtein.org) | ||||||||||||||||||||||||||||||||||||||||||||||
Subcellular Localization (reliability in brackets)
| Gene Ontology (reliability in brackets)
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Predicted features:
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Part Contributions
NUS Singapore 2021
Authors:
Tania Santosh Nair, Chew Chin Wei, Linus Tan
Contribution Summary:
NUS Singapore 2021 has successfully cloned part into S. cerevisiae and tested part by placing nuclease downstream of the galactose inducible promoter Gal1 (Figure 1). Kill switch efficacy was not optimal and post-induction of NucA yielded a 82% survival rate in S. cerevisiae.
It was postulated that part of the poor activity was due to a lack of a nuclear localization sequence (NLS), hence the inability for NucA translated in the cytosol from accessing the genomic DNA within the nucleus in S. cerevisiae chassis.
In order to improve the efficacy of the nuclease, a NLS sequence was added to the N-terminus of the part, as this was expected to increase the amount of NucA that is translocated to the nucleus to digest the genome and DNA, which in turn will increase the mortality rate of the cell culture.
Attempted to perform part improvement by adding a nuclear localization signal tag to the 5' end of the part sequence to improve the kill switch efficacy in part BBa_K3927015. Addition of NLS improved reduced overall overall survival rate post induction (figure 2).
Additional Information Please visit the part registry page for our improved part BBa_K3927015 for more information on the part improvement.
//collections/probiotics
//collections/probiotics/biocontainment
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