Team:Freiburg/Improve
BBa_K1362160 and BBa_K1362161
Summary: We improved BBa_K1362160 and BBa_K1362161 by adding MBP and TRX, creating BBa_K3009016 and BBa_K3009015 for the fast and easy analysis of different splicng conditions to simplify the usage of the gp4.1-Intein.
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_K1362160 and BBa_K1362161 by replacing the cassette for expression of the red fluorescent protein (RFP) with maltose-binding protein (MBP) or thioredoxin (TRX) and thereby implement a functioning ORF. Like this, these constructs are ready for use and future iGEM teams can easily establish the optimal splicing conditions for their application without the need of any cloning.
To demonstrate the advantage of our parts over BBa_K1362160 and BBa_K1362161 we tested the expression of all four constructs. Therefore, we had four 4 mL cultures of BL21(DE3) each containing one original or one improved construct 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)
Fig. 1: Coomassie staining of SDS-PAGE with lysates from original and optimized gp41.1 split intein constructs expressed in BL21(DE3).
The SDS-PAGE shows that the improved BBa_K3009015 and BBa_K3009016 have been expressed in high amounts compared to the original parts and can be used together 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 N-Int + MBP and gp41.1 C-Int + TRX, 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).
Fig. 2: Western Blot of lysates containing gp41.1 C-Int + TRX (BBa_K3009015) and gp41.1 N-Int + MBP (BBa_K3009016), mixed and incubated with and without 4 mM DTT at 42°C, stained against TRX. * spillover from lane 3.
The WB analysis shows that our constructs conduct the splicing reaction rapidly and under different reducing conditions. Overall, we managed to improve BBa_K1362160 and BBa_K1362161 by adding the maltose-binding protein or thioredoxin to it and thereby enabling strong expression, protein splicing at desired levels and facile WB detection. This makes BBa_K3009016 and BBa_K3009015 ideal parts for establishing the optimal splicing conditions for every iGEM team that wants to work with the gp41.1 split inteins.
[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
