Difference between revisions of "Part:BBa K1362160"

Revision as of 00:24, 22 October 2019

RBS + Gp41-1 N-intein RFC[105] assembly construct (with His6)

Sequence and Features

Improvement:Freiburg 2019

Group: Freiburg 2019
Author: Fabian Bäzner
Summary: We improved BBa_K1362160 by designing BBa_K3009016:a ready-to-use version of the gp41.1 N-Intein with maltose-binding protein 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_K1362160 by replacing the cassette for expression of the red fluorescent protein (RFP) with maltose-binding protein (MBP) 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_K3009015.

Characterization

To demonstrate the advantage of our part over BBa_K1362160 we tested the expression of both constructs. Therefore, we had two 4 mL cultures of BL21(DE3) containing BBa_K1362160 or BBa_K3009016 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_K3009016 has been expressed in high amounts compared to the original part and can be used together with BBa_K3009015 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 by adding the maltose-binding protein 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.

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