Part:BBa_K1071014
RFP-MTS
RFP (BBa_E1010) with a C-terminal membrane translocation sequence (MTS). The origin is the protein YlxH from the bacterium Geobacillus thermodenitrificans. The MTS has a hydrophobic site and an affinity for membranes.
Usage and Biology
An important aspect in synthetic biology is the cellular compartmentalization of complex enzyme reactions. Therefore, it would be great to establish simple and autonomous parts that allow the recruitment of synthetic components to cellular membranes – eventually with specificity to certain lipid environments (e.g. anionic phospholipid clusters).
Therefore, we decided to establish membrane scaffolds, which are known as membrane targeting sequences (MTS). These sequences form amphipathic helices, which autonomously bind to membranes often with specificity to anionic phosphor lipids (compare to: Parlitz et al., 2007, JBC and Szeto et al., 2003, JBC). To challenge that idea, we fused the reporter RFP (red fluorescent protein) with a C-terminal membrane targeting sequence (MTS). The origin is the protein YlxH from the bacterium Geobacillus thermodenitrificans. To show the functionality of the MTS, we fused the targeting sequence to the C-terminus of reporter RFP to gain RFP-MTS. The construct was transformed into Escherichia coli and an IPTG inducible promoter controlled expression of the RFP-MTS fusion protein (Figure).
RFP-MTS localized at the membrane with a slight preference for the poles, which in E. coli are enriched in anionic phospholipids. Taken together, we were able to create an additional BioBrick for our toolbox and to improve the in the registry existing RFP.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 676
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 555
Illegal AgeI site found at 667 - 1000COMPATIBLE WITH RFC[1000]
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