Bxb1 Invertible Promoter Switch (PB State)
The switch consists of a strong constitutive promoter (BBa_J23119) flanked by Bxb1 attP and attB (attachment) sites. Bxb1 integrase (BBa_K1039003) recombines Bxb1 attP and attB to form Bxb1 attR and attL, flipping the orientation of the promoter and the promoter’s direction of transcription. “Flipping” of the switch by Bxb1 integrase (BBa_K1039003) forms the “RL” state of the switch (BBa_K1039004).
Recombination of att sites affords the switch two potential states, with the promoter driving transcription of different genes depending on the state. The two states are described as “PB state” and “RL state”, in reference to the Bxb1 att sites flanking the promoter in each state.
There is a terminator (BBa_J61048) upstream of the promoter to prevent transcription of genes that correspond to the opposite state of the switch.
In this state (PB state), genes on the prefix side of the switch are expressed. Note that these genes must be inverted relative to the BioBrick convention, i.e. they are transcribed from suffix to prefix, rather than prefix to suffix.
In the RL state (BBa_K1039004), genes on the suffix side of the switch are expressed. These genes are oriented in accordance with the BioBrick convention, i.e. they are transcribed from prefix to suffix.
This state (PB state) is recovered from RL state (BBa_K1039004) in the presence of Bxb1 integrase (BBa_K1039003) and Bxb1 recombination directionality factor (RDF) (Bba_K1039007) through recombination of Bxb1 attR and attL, which forms Bxb1 attP and attB.
In summary, PB state is flipped to RL state in the presence of Bxb1 integrase, and RL state is flipped to PB state in the presence of Bxb1 integrase and RDF.
In the absence of integrase or integrase and RDF, the switch will hold its state. In this way, it is a passive digital memory device.
All aspects of this switch are directly inspired by the switch developed by Ghosh, 2005. Spacer sequences between genetic elements are also taken directly from the work of Bonnet et al.
Note that there is an NheI site on the prefix side of the switch before the attP site. This was placed there as part of an early design of our switch, but does not contribute to functionality.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12Illegal NheI site found at 1
Illegal NheI site found at 201
Illegal NheI site found at 224
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25Illegal NgoMIV site found at 535
- 1000Illegal BsaI site found at 33
Illegal BsaI.rc site found at 515