Part:BBa_K323089:Experience

The 2010 iGEM team Slovenia used this part in combination with Part:BBa_K323088 containing a corresponding operator sequence. Both parts were inserted into the low copy vector pSB4C5 (Part:pSB4C5) by tripoint ligation to form a device for in vivo testing of protein-DNA binding.

We have constructed 7 parts for testing of DNA binding proteins HivC (Part:BBa_K323090), Gli1 (Part:BBa_K323092), Zif268 (Part:BBa_K323094), Jazz (Part:BBa_K323096), Blues (Part:BBa_K323098), PBSII (Part:BBa_K323100) and TAL (Part:BBa_K323102).

Binding of the tested proteins was determined with the beta-galactosidase assay (for a detailed description of the method see the 2010 iGEM team Slovenia [http://2010.igem.org/Team:Slovenia/METHODS_and_PARTS wiki]). Beta-galactosidase activity of the culture with no arabinose present should have indicated maximum activity due to minimal expression of the DNA binding protein. Therefore, we normalized the activity of the latter culture to 100 percent, and the beta-galactosidase activity of the culture with 1% arabinose present in the media was compared to the latter. Results of a representative experiment are shown below.

Figure: HivC, Gli1, Zif268, Jazz, Blues, PBSII and TAL bind to their corresponding DNA binding sites. In all constructs tested, the beta-galactosidase activity decreased with addition of arabinose, which induces the transcription of the DNA binding protein. These results show that all of the tested DNA binding proteins bind to their specific operator sequences.

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