Introduction
NanoLuciferase is an enzyme that uses furimazine as a substrate to produce light, and GfasPurple and TsPurple are chromoproteins, which are vibrant proteins. We aimed to express anti-Cys C nanobody-nanoluciferase, anti-Cys C NanoLuciferase-NanoLuciferase, anti-Cys C nanobody-TsPurple, and anti-Cys C nanobody-GfasPurple. In designing these constructs, we wished to create protein conjugates that could both bind Cys C and help detect Cys C.
Cloning
Using Golden Gate assembly, we removed the lac operon from pSB1C3 (BBa_K2842666) plasmids by using Type IIS restriction enzymes (BsaI). gBlocks were also digested with BsaI to generate complimentary overhangs. Then, T4 DNA ligase ligated gBlocks into plasmids through these complementary overhangs.
The way we expressed our proteins broadly follows this outline, with some deviations (as detailed in the registry):
- The plasmids were transformed into E. coli DH5-alpha cells and cultured on agar media.
- The cells were cultured on chloramphenicol to kill those that were not transformed, then the colonies were left overnight to grow.
- Blue/white screening was performed using IPTG and X-gal to identify cells containing the modified plasmids with gBlocks (white) and the unmodified plasmids not containing the gBlock (blue).
- Selected white colonies were subjected to miniprep to isolate the plasmids.
- A sample of the isolated plasmids was sent to FullCircle labs for sequencing to confirm correct transformation.
gBlocks
Each gBlock either encodes a control reporter protein or a fusion protein which contains a nanobody (NB) linked via a GS linker to another one or two reporter proteins that are vibrant or give out light. The nanobody is complimentary to Cys C, and the other protein is either one NanoLuciferase (NL), two tandem NLs or a chromoprotein. All protein units are linked together via GS linkers. All gBlocks were synthesised by GenScript (NB-NL, NL, NB-GfasPurple, NB-TsPurple gBlocks, NB-NL-NL within a plasmid) or IDT (NB, reverse primer).
All gBlocks were codon-optimised for expression in E. coli BL21. Forbidden restriction sites within the gBlocks were removed before ordering the gBlocks and replaced with synonymous codons to ensure type IIS (RFC1000)/RFC 10 compatibility. All transcriptional units contain a T7 promoter, an RBS, the coding sequence attached to a linker-His tag, and a T7TE terminator. The PelB signal sequence was used to target proteins to the periplasmic space, which is a reducing environment. This enabled correct disulphide bond formation. The PelB sequence is cleaved off after having targeted the protein to the periplasm. All sequences within plasmids are flanked by EcoRI and XbaI prefixes and SpeI and PstI suffixes. The composite sequences we designed are as follows:
All gBlocks were codon-optimised for expression in E. coli BL21. Forbidden restriction sites within the gBlocks were removed before ordering the gBlocks and replaced with synonymous codons to ensure type IIS (RFC1000)/RFC 10 compatibility. All transcriptional units contain a T7 promoter, an RBS, the coding sequence attached to a linker-His tag, and a T7TE terminator. The PelB signal sequence was used to target proteins to the periplasmic space, which is a reducing environment. This enabled correct disulphide bond formation. The PelB sequence is cleaved off after having targeted the protein to the periplasm. All sequences within plasmids are flanked by EcoRI and XbaI prefixes and SpeI and PstI suffixes. The composite sequences we designed are as follows:
A. NB-gfaspurple: The anti Cys-C NB conjugated via a GS linker to Gfaspurple
B. NB-NL: the anti-Cys C NB conjugated via a GS linker to NL
C. NB-NL-NL: the anti-Cys C NB conjugated via a GS linker to NL, which is conjugated to another NL via a GS linker. The reason for having 2 NL’s is to see if this will enhance the limit of detection (LOD) of Cys C.
D. NB-TsPurple: The anti-Cys C NB conjugated via a GS linker to TsPurple.
E. NL: Used as a control to compare to the enzymatic activity of NB-NL, seeing if the linker-NB significantly affects enzymatic activity of NL.
F. NL-NL: used as a control to compare to the activity of NB-NL-NL, seeing if the linker-NB significantly affects enzymatic activity of NL.
G. Gfaspurple: Used as a control to compare to NB-GfasPurple, to see whether the NB contributes significantly to binding to Cys C.
No control TsPurple composite part was designed as this was provided by Jack Jeffries, one of our supervisors.
Parts Table
|
Code |
Name |
Category |
Type |
|
E. coli Ribosome Binding Site (RBS) |
Basic |
RBS |
|
|
T7 Promoter |
Basic |
Promoter |
|
|
6 X His Tag |
Basic |
Coding |
|
|
Pectate Lyase B (PelB) Signal Sequence |
Basic |
Coding |
|
|
(GGGGS)3 Linker |
Basic |
Coding |
|
|
GGSG Linker |
Basic |
Coding |
|
|
T7Te Terminator |
Basic |
Terminator |
|
|
Anti-Cystatin C Nanobody |
Basic |
Coding |
|
|
TsPurple Chromoprotein |
Basic |
Coding |
|
|
GfasPurple Chromoprotein |
Basic |
Coding |
|
|
PelB-GfasPurple-GGSG Linker-6xHis Tag Coding Sequence |
Composite |
Coding |
|
|
NanoLuciferase |
Basic |
Coding |
|
|
PelB-NanoLuciferase-GGSG linker-6xHis Tag Coding Sequence |
Composite |
Coding |
|
|
PelB-anti-Cystatin C Nanobody-(GGGGS)3 linker-NanoLuciferase-GGSG linker-6xHis tag Coding Sequence |
Composite |
Coding |
|
|
PelB-NanoLuciferase-(GGGGS)3 linker-NanoLuciferase-GGSG linker-His tag |
Composite |
Coding |
|
|
PelB-anti-Cystatin C nanobody-(GGGGS)3 linker-GfasPurple-GGSG linker-6xHis tag |
Composite |
Coding |
|
|
PelB-anti-Cystatin C nanobody-(GGGGS)3 linker-TsPurple-GGSG linker-His tag |
Composite |
Coding |
|
|
PelB-anti-Cys C nanobody-(GGGGS)3 linker-NanoLuciferase-(GGGGS)3 linker-NanoLuciferase-GGSG linker-6xHis tag |
Composite |
Coding |
|
|
Reverse primer for PelB-NanoLuciferase-(GGGGS)3 Linker-NanoLuciferase-GGSG linker-His tag assembly |
Basic |
Primer |
|
|
(GGGGS)3 linker-NanoLuciferase-His tag-T7 terminator |
Basic |
Coding |