Part:BBa_K5357018
PelB-NanoLuciferase-(GGGGS)3 linker-NanoLuciferase-GGSG linker-His Tag
Logic of Assembly
This part encodes a PelB-NanoLuciferase-(GGGGS)3 linker-NanoLuciferase-GGSG linker-6xHis tag protein. PelB (see BBa_K5357003) is a secretion signal ensuring the localisation of the protein to the periplasmic space of E. coli, a reducing environment that enables proper disulphide bond formation. PelB is cleaved off in the periplasm.
We used GS linkers because the presence of hydrophobic (G) and hydrophilic (S) amino acids prevents secondary structure formation of the linker, reducing the risk of the linker interfering with the correct folding of the proteins it is connecting. The final product is a correctly folded NanoLuciferase-(GGGGS)3 linker-NanoLuciferase-GGSG linker-6xHis tag, which can be purified by leveraging its 6xHis tag in IMAC (immobilised metal affinity chromatography).
Usage and Biology
NanoLuciferase is an engineered form of luciferase that luminesces with a significantly higher intensity than luciferase when exposed to its synthetic substrate furimazine. It is smaller (19.1kDa) than normal luciferases (62kDa), which decreases its influence on structure when fused to other proteins. It is also more stable in urea than luciferase. These aspects of NanoLucuiferase made us theorise that it would serve as a good reporter for urinary biomarkers.
As part of our project, we decided to test the luminescence and Cystatin C-binding capabilities of an anti-Cystatin C nanobody bound to two tandem NanoLuciferases (NB-NL-NL), to see if we could increase bioluminescence by attaching two NanoLuciferases together. We therefore also wished to express NanoLuciferase-NanoLuciferase (NL) as a control for NB-NL-NL.
Design Notes
Because there were complications assembling the gBlock, we decided to assemble it ourselves from two separate DNA fragments. These fragments were: (1) BBa_K5357015 (T7 promoter-RBS-PelB-NL-His tag-T7Te terminator) and (2)BBa_K5257024 ((GGGGS)3 linker-NanoLuciferase-His tag-T7 terminator).
For fragment (1), we designed a backward primer (BBA_K5357023) which was used in PCR to trim off the His tag, T7Te terminator, and the original reverse BsaI site. This created a PelB-NL amplicon with a reverse BsaI site overhang that is exclusively complimentary to linker-NL (fragment (2)), which has a BsaI overhang. The end Golden Gate product was a coding sequence of NL-NL that could be ligated into a PSB1C3 plasmid.
The linker-NL DNA was ordered from IDT. The coding sequence for NL was taken from a database (see 'Source' section). The DNA's sequence was codon-optimised for expression in E. coli, and in places where there were consecutive identical amino acids like in the His tag, synonymous codons were used to prevent ribosomal stalling. We used Benchling to assemble the coding sequences.
Characterisation
We carried out gel electrophoresis, and this confirmed that the His tag, T7Te terminator, and reverse BsaI site was trimmed off of fragment (1), giving a PelB-NL sequence that is ready to be assembled with linker-NL.
We transformed the plasmid with the insert into DH5 alpha cells, and performed blue/white screening. We got white colonies, indicating the cells had the plasmid inside with a disrupted LacZ operon. This means that we did manage to insert DNA into the plasmids.
We sent the plasmids for sequencing to FullCircle Labs to test whether the insert was the right size. These ended up being 2867bp long, and this length does not match the length of the plasmid with the NL-NL insert (3851bp), but instead matched the length of the plasmid with a single NanoLuciferase inserted.
We therefore repeated the process (PCR, Golden Gate, cloning into DH5 alpha cells). We also performed gel extraction from PCR, to extract the DNA fragments that were the correct size only, corresponding to the size of the NL amplicon. We cultured the transformed DH5 alpha cells, and performed blue/white screening to confirm the gBlock was inserted into the plasmid.
We extracted plasmids from the cells, and we sent 16 samples for sequencing.
All but one set of plasmids had the correct insert, showing that we successfully carried out PCR and Golden Gate cloning to assemble the NL-NL sequence, and successfully inserted it into PSB1C3 (in most cases).
We hope that future iGEM teams will use this part in their projects and help further characterise it.
Source
NanoLuciferase: https://nanolight.com/content/nanoluc-sequence/ T7 promoter, T7Te terminator, and RBS from the PSB1C3 plasmid (BBa_K2842666)
References
Hall MP, Unch J, Binkowski BF, Valley MP, Butler BL, Wood MG, et al. Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem Biol. 2012;7(11):1848-57.
Krasitskaya VV, Efremov MK, Frank LA. Luciferase NLuc Site-Specific Conjugation to Generate Reporters for In Vitro Assays. Bioconjug Chem. 2023;34(7):1282-9.
Li G, Huang Z, Zhang C, Dong BJ, Guo RH, Yue HW, et al. Construction of a linker library with widely controllable flexibility for fusion protein design. Appl Microbiol Biotechnol. 2016;100(1):215-25.
Ceballos-Alcantarilla E, Merkx M. Understanding and applications of Ser/Gly linkers in protein engineering. Methods Enzymol. 2021;647:1-22.
Sockolosky JT, Szoka FC. Periplasmic production via the pET expression system of soluble, bioactive human growth hormone. Protein Expr Purif. 2013;87(2):129-35.
Lei SP, Lin HC, Wang SS, Callaway J, Wilcox G. Characterization of the Erwinia carotovora pelB gene and its product pectate lyase. J Bacteriol. 1987;169(9):4379-83.
Schafer F, Romer U, Emmerlich M, Blumer J, Lubenow H, Steinert K. Automated high-throughput purification of 6xHis-tagged proteins. J Biomol Tech. 2002;13(3):131-42.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 134
- 1000COMPATIBLE WITH RFC[1000]
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