Team:KAIST Korea/Project Background

1. Promoter : BBa_J23119
2. RBS : BBa_B0034
3. att sites : Recognition site for BBa_K907000(Mycobacteriophage Bxb1 integrase).

The FlipFlop, initially, promotes the transcription and translation of down-stream gene, Protein A in the Figure 2, due to its promoter orientation.
When BBa_K907000(Mycobacteriophage Bxb1 integrase, Bxb1_Int) recognizes and inverts the sequence flanked with attB and attP sequences, promoter orientation is reversed, leaving recombined attL and attR sequences.

Then this device promotes the transcription and translation of upstream gene(must be designed in reverse orientation at construction step), or Protein B(Signal 1) in Figure 4.


Also, the signal can be restored to its initial state when BBa_K907000/ BBa_K907001 (Mycobacterio-phage Bxb1 excisionase, Bxb1_Xis) complex recognizes and inverts the sequence flanked with attL and attR sequences back to original state.

Using the FlipFlop device, we can generate biological machine that operating the metabolism as we programmed. We decided to call the system as Auto-regulating FlipFlop. To demonstrate the idea, we applied the device to control bio-indigo production, which is catalyzed by bacterial flavin-containing monooxygenase(bFMO).

What is bFMO?

Bacterial flavin-containing monooxygenase(bFMO) convert indole, which is produced from primary metabolite of organisms, into isatin, sequentially catalyzed into indigoid compounds that express indigo color.
Because the enzyme utilizing the primary metabolite, we can easily notice the enzyme is working well or not. For the following experiments, engineered bFMO gene from Methylophaga sp. Strain SK1 is kindly provided by Duhee Bang from Yonsei University, Republic of Korea.

At its initial state (while signal = 0), downstream gene of promoter, LuxI, produces AHL(N-Acyl homoserine lactone). Separated from FlipFlop device, LuxR gene is transcribed under the control of constitutive promoter. Generated LuxR molecules and AHLs form complex to initiate the transcription of Bxb1 integrase, positioned at the downstream of pLuxR promoter. Then, Bxb1 integrases specifically bind to attB and attP sequences to generate signal 1.

At its inverted state(while signal = 1), bFMO gene of upstream sequence can be transcribed and translated. So that bio-indigo starts to be produced. Upper genes of bFMO, AHL-Lactonase and Bxb1 Excisionase are expressed in order, degrading AHL-LuxR complexes and forming Bxb1 integrase-excisionase complexes, respectively. Finally, Int-Xis complexes bind to attL and attR sequences and invert the promoter into its original orientation, regenerating attB and attP sequences.