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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Protein data table for BioBrick BBa_ automatically created by the BioBrick-AutoAnnotator version 1.0
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) 1  101  MAGATSTKKLHKEPATLIKAIDGDTVKLMYKGQPMTFRLLLVDTPETKHPKKGVEKYGPEASAFTKKMVENAKKIEVEFDKGQRTDKYGRGLAYIYADGK MVNEALVRQGLAKVAYVYKPNNTHEQHLRKSEAQAKKEKLNIWSEDNADSGQTG*
Sequence features: (with their position in the amino acid sequence, see the list of supported features) None of the supported features appeared in the sequence
Amino acid composition: Ala (A) 15 (9.7%) Arg (R) 5 (3.2%) Asn (N) 6 (3.9%) Asp (D) 8 (5.2%) Cys (C) 0 (0.0%) Gln (Q) 6 (3.9%) Glu (E) 12 (7.8%) Gly (G) 12 (7.8%) His (H) 4 (2.6%) Ile (I) 5 (3.2%) Leu (L) 11 (7.1%) Lys (K) 23 (14.9%) Met (M) 5 (3.2%) Phe (F) 3 (1.9%) Pro (P) 6 (3.9%) Ser (S) 5 (3.2%) Thr (T) 11 (7.1%) Trp (W) 1 (0.6%) Tyr (Y) 7 (4.5%) Val (V) 9 (5.8%)
Amino acid counting Total number: 154 Positively charged (Arg+Lys): 28 (18.2%) Negatively charged (Asp+Glu): 20 (13.0%) Aromatic (Phe+His+Try+Tyr): 15 (9.7%)	Biochemical parameters Atomic composition: C763H1231N213O230S5 Molecular mass [Da]: 17228.8 Theoretical pI: 9.48 Extinction coefficient at 280 nm [M-1 cm-1]: 15930 / 15930 (all Cys red/ox)
Plot for hydrophobicity, charge, predicted secondary structure, solvent accessability, transmembrane helices and disulfid bridges
Codon usage Organism: E. coli B. subtilis S. cerevisiae A. thaliana P. patens Mammals Codon quality (CAI): good (0.79) excellent (0.83) excellent (0.82) excellent (0.81) good (0.72) acceptable (0.60)
Alignments (obtained from PredictProtein.org) SwissProt: P00644 (99% identity on 150 AAs), Q5HHM4 (98% identity on 150 AAs), Q6GB41 (98% identity on 150 AAs), Q6GIK1 (98% identity on 150 AAs), Q7A6P2 (98% identity on 150 AAs), Q8NXI6 (98% identity on 150 AAs), Q99VJ0 (98% identity on 150 AAs) TrEML: A3QSR2 (98% identity on 140 AAs), A3QSR3 (98% identity on 140 AAs), A3QSR6 (98% identity on 140 AAs), A5A513 (98% identity on 148 AAs), A5A520 (98% identity on 148 AAs), A5A523 (98% identity on 148 AAs), A5IQZ5 (98% identity on 150 AAs), A6QFA0 (98% identity on 150 AAs), A6TZS0 (98% identity on 150 AAs), A7WZZ3 (98% identity on 150 AAs) PDB: 1ey0 (100% identity on 136 AAs), 1eyd (100% identity on 136 AAs), 1nsn (100% identity on 138 AAs), 1snc (100% identity on 135 AAs), 1sta (100% identity on 135 AAs), 1stb (100% identity on 136 AAs), 1stg (100% identity on 136 AAs), 1sth (100% identity on 136 AAs), 1stn (100% identity on 136 AAs), 1sty (100% identity on 136 AAs)
Predictions (obtained from PredictProtein.org)
Subcellular Localization (reliability in brackets) Archaea: cytosol (100%) Bacteria: secreted (55%) Eukarya: cytosol (24%)	Gene Ontology (reliability in brackets) Molecular Function Ontology: - Biological Process Ontology: GO:0030541 (28%)
Predicted features: Disulfid bridges:  - Transmembrane helices:  -
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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.

Figure 1: Construct schematic of part assembled with a Gal1 promoter upstream to trigger galactose dependent endonuclease activity

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).

Figure 2: CFU assay results from pGNucA-H(BY4741) and pGNLSNucA-H(BY4741), ratio of colonies counted from plates with the nuclease induced to the colonies counted from the plates with nuclease uninduced. Nuclease with the NLS attached reduces the total number of live colonies on the plate.

Additional Information Please visit the part registry page for our improved part BBa_K3927015 for more information on the part improvement.