Part:BBa_K1761000:Experience

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Application TU Eindhoven 2015

Implementing the amber stop codon TAG for building in the an azide-functionalized amino acid. The protein is expanded with a BamHI linker and mNeonGreen and with a BsoBI linker and NanoLuc. The sequence for these two proteins was inserted in a pETDuet-1 vector. This vector, together with the pEVOL-pAzF (coding for tRNA + tRNA synthethase) were inserted in BL21(DE3) competent cells by double transformation. The proteins were expressed and the DBCO-PEG4-TAMRA, Fluorescence and bioluminescence confirmation were successful (see BBa_K1761001 [1] and BBa_K1761002 [2]).

Additionally, two different oligos with functionalized DBCO groups were clicked on membrane proteins. Fluorescently labeled complementary strands were incubated with the bacteria as an extra confirmation of the protein expression and click reaction (see Figure 1).

Figure 1: To verify whether the click reaction has occured with DBCO-modified DNA, we incubate the cells with DBCO-functionalized complementary strands. If the DBCO-modified DNA clicks, the fluorescently labeled DBCO-functionalized complementary strands will anneal with the clicked strands. In that case, the cells will remain fluorescent after a few washing steps.

By using strand displacement (see Figure 2), the two membrane proteins are brought in close proximity (see Figure 3).

Figure 2: Schematic overview of DNA strand displacement. DNA Strand displacement is initiated when the input binds to the toehold region of a partially hybridized DNA strand. After the initiation, branch migration takes place and the input fully hybridizes with its perfect match, yielding the output.

Figure 3: The membrane proteins can be brought in close proximity by clicking oligonucleotides on the loops and adding a long strand complementary to both oligonucleotides. It is expected that this results in a measurable signal. The longer strand is functionalized with a toehold region. Upon addition of a strand perfectly complementary to the longer strand, the system disassembles and the signal will fade out.

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