Part:BBa_K3009015
RBS + gp41.1 C-intein with TRX (+His6)
Freiburg 2019
Group: Freiburg 2019
Author: Fabian Bäzner
Summary: This part is an improved version of BBa_K1362161.It is a ready-to-use version of the gp41.1 C-Intein with thioredoxin added as extein, for direct testing and establishing of protein splicing conditions
Usage and Biology
Inteins are naturally occuring proteins that autocatalytically splice out of a precursor protein and attach the flanking regions together by forming a new peptide bond1. Inteins can be contiguous or split. In this case, two separate parts, the N- and C-terminal intein fragments, need to bind together to reconstitute a full intein and perform the protein splicing reaction. Split inteins are very powerful tools for several applications, like post-translational protein modifications or the easy in vitro synthesis of fusion proteins2. We decided to improve BBa_K1362161 by replacing the cassette for expression of the red fluorescent protein (RFP) with thioredoxin (TRX) and thereby implement a functioning ORF. Like this, the construct is ready for use and future iGEM teams can easily establish the optimal splicing conditions for their application without the need of any cloning. This construct should be use in combination with BBa_K3009016.
Characterization
To demonstrate the advantage of our part over BBa_K1362161 we tested the expression of both constructs. Therefore, we had two 4 mL cultures of BL21(DE3) containing BBa_K1362161 or BBa_K3009015 and induced them, following an OD600 of 0.5, with 1 mM IPTG, then incubated them at 37°C, 200 rpm. After 3 h the cultures were pelleted and lysed. Denatured samples of the lysates were loaded to a SDS-PAGE gel (100 V, 1h 10 min) followed by Coomassie staining of the gel (Figure 1).
Expression system:
Backbone: pET302
Promoter: T7 promoter
Terminator: T7 terminator
RBS: T7 RBS (BBa_K1362090)
E.coli strain: BL21(DE3)
The SDS-PAGE shows that the improved BBa_K3009015 has been expressed in high amounts compared to the original part and can be used together with BBa_K3009016 for analyzing different splicing conditions. To demonstrate that the optimized parts are qualified for this application, we tested the splicing reaction. We cultured BL21(DE3) cells with gp41.1 C-Int + TRX and gp41.1 N-Int + MBP, induced them at an OD600 of 0.5 with 1 mM IPTG, then incubated them at 37°C, 200 rpm. After 3 h, the cultures were pelleted and lysed. The lysates were centrifuged at 21000 xg, 4°C and the supernatants mixed und incubated at 42°C, with and without 4 mM Dithiothreitol (DTT). Samples were taken at different time points and a Western Blot assay was performed with them (Figure 2).
The WB analysis shows that our constructs conduct the splicing reaction rapidly and under different reducing conditions. Overall, we managed to improve BBa_K1362161 by adding the thioredoxin to it and thereby enabling strong expression, protein splicing at desired levels and facile WB detection. This makes BBa_K3009015 and BBa_K3009016 ideal parts for establishing the optimal splicing conditions for every iGEM team that wants to work with the gp41.1 split inteins.
References
[1] Hirata, R. et al., Molecular structure of a gene, VMA1, encoding the catalytic subunit of H+-translocating adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae (1990). J. Biol. Chem. 265, 6726–6733.
[2] Li Y. et al., Split-inteins and their bioapplications (2015). Biotechnology Lett. 37 (11), 2121-2137
Sequence and Features:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 93
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