Part:BBa_K3927015

NLS-NucA

Gene encoding for Staphylococcus aureus endonuclease nucA with a nuclear localisation sequence. Improved part based on part BBa_K1159105.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 25
  Illegal AgeI site found at 478
  
• 1000
  COMPATIBLE WITH RFC[1000]

Description

Previous characterization work of BBa_K1159105 demonstrated poor functionality as a inducible endonuclease kill switch. 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.

Usage

This part does not contain a promoter or terminator, and requires both to function. Characterization work performed to verify the functionality of this part was done using a Gal1 inducible promoter.

Design

A NLS was added to the N-terminus of BBa_K1159105 to aid in the translocation of NucA from the cytosol into the nucleus in order to improve the endonuclease self-destruction activity.

Characterization

Characterization of original part BBa_K1159105

Sequence for BBa_K1159105 was ordered from IDT, and using Gibson Assembly it was inserted into the pGmFaHBD-H plasmid to form pGNucA-H.

pGNucA-H (BY4741) was cultured in YPD-HygB for 24 hours, and a CFU assay was carried out 5 serial dilutions to a cell count of 10-5 and then plated on either YPD-HygB agar as a negative control, as well as YPGR-H to induce the production of NucA. Culture that was induced showed only a 20% decrease in cell mortality, and it was decided that the part needed to be improved upon.

Characterization of this part (Improved version)

Figure 2: Construct schematic diagram of new and improved NucA with NLS added at N-terminus.

Gibson Assembly was used to insert a 7 amino acid NLS sequence at the N-terminus of pGNucA-H forming the plasmid pGNLSNucA-H. pGNLSNucA-H was transfomed into BY4741 forming the strain pGNLSNucA-H (BY4741). pGNLSNucA-H (BY4741) was in YPD-HygB for 24 hours, and a CFU assay was carried out 5 serial dilutions to a cell count of 10-5 and then plated on either YPD-HygB agar as a negative control, as well as YPGR-H to induce the production of NucA (figure 3).

Figure 3: 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.

By adding the NLS, the overall mortality rate of the cells with nuclease induced increased as assessed from the CFU assay, indicated by a greater decrease in the colonies observed growing from the induced to uninduced cultures.