Part:BBa_K4390005
pbrR expression construct
BBa_K4390005 is a working transcriptional unit which expresses a PbrR repressor (BBa_K3470001). It is composed of the constitutive promoter J23100 (BBa_J23100), a ribosome binding site (BBa_B0034), the PbrR repressor sequence (BBa_I721002) and the weak synthetic L2U2H09 Terminator (BBa_K4390001). This unit was assembled using JUMP and can be assembled into a JUMP Level 1 vector plasmid for expression in cells.
Usage and Biology
The PbrR repressor is derived from Ralstonia metallidurans. The repressor will bind to the ppbrR promoter in the absence of Pb(II) salts. We used this transcriptional unit alongside the Pb biosensor (BBa_K346002) so that when we use cell lysate which expresses our transcriptional unit there would be pbrR present in the lysate. When this cell-free extract is combined with the Hg biosensor it induces transcriptional repression of the linear biosensor when Pb (II) salts are not present. When Pb (II) salts are present, the pbrR binds to the metal ions and allows transcription of the biosensor, generating a fluorescent output. It should be noted that T7 RNA polymerase, chemical energy (ATP), NTPs and the DFHBI are also required in the cell-free reaction so that fluorescence is observed. The DFHBI binds to the RNA aptamer which the transcribed when the biosensor is no longer repressed.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 11
Illegal NheI site found at 34 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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