Difference between revisions of "Part:BBa K3927015"

Revision as of 13:38, 18 October 2021

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

Usage

Design

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.

@@ Line 27: / Line 27: @@
 [[File:T--NUS_Singapore--NLSNucA_schematic.png |800px|thumb|center|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.
+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).
 [[File:T--NUS_Singapore--NLSNucA.png |800px|thumb|center|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.
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