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| − | This part contains the coding sequence of a single chain variable fragment (scFv) that binds specifically to dihydroxyacid dehydratase DHAD ([https://parts.igem.org/Part:BBa_K1621006 Bba_K1621006]) of ''Salmonella'' Typhimurium.
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| − | Meyer ''et al.'' (2012) showed that [https://parts.igem.org/Part:BBa_K1621006 DHAD] acts as a specific antigen for ''S.'' Typhimurium. This subtype of the ''Salmonella enterica'' subspecies ''enterica'' causes more than 90% of all ''Salmonella'' infections. scFvs binding specifically against DHAD were identified by phage display using the human naïve antibody library HAL7/8. For the scFv that is encoded by this part (TM228.2.3-D9) an EC[50] value of 50 nM was determined by titration ELISA and the binding to DHAD was verified by immunoblotting.
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| − | After cloning the part into an expression vector, the scFv can be efficiently overexpressed in ''Escherichia coli''. Figure 1 shows the vector that was used for overexpression and the conditions for growth of the bacteria and induction of the expression are summarized in table 1.
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| − | The harvested cells were lyzed by sonification and proteins were separated from cell debris by ultracentrifugation. Afterwards, the scFv was purified by affinity chromatography. The His-tag that is C-terminally fused to the protein specifically binds to Ni-NTA agarose beads and is eluted with 500 mM imidazol. The same method was used to overexpress and purify the corresponding antigen [https://parts.igem.org/Part:BBa_K1621006 DHAD].
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| − | The protein solutions before and after affinity purification were analyzed by SDS PAGE. Figure 2 shows that the scFv (~30 kDa) as well as [https://parts.igem.org/Part:BBa_K1621006 DHAD] (~63 kDa) were efficiently enriched and successfully purified from the whole cell lysate.
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| − | Both purified proteins were used to perform Western Blot analysis to show the specific binding properties. This is visualized in figure 3.
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| − | The part was shipped to the registry in standard pSB1C3, starting with a start codon (ATG). It was inserted into the shipping backbone by Gibson Assembly and the sequence was verified afterwards.
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