Aromatic Wires

Overview

The trans-genic metabolic wire involved transforming the Hbp operon, which encodes the genes for 2-dioxybiphenol degradation, from pseudomonas nitroreducens into the first ‘sender’ E.coli. The second ‘receiver’ E. coli would have the Ben operon from Acinetobacter baylyi transformed into it.

The Hbp operon is regulated by 2-dioxybiphenol, when present the sender E. coli would produce the enzymes required for degradation of 2-dioxybiphenol into benzoic acid. Benzoic acid would then leave the cell as a ‘signal’. This would be detected by the ‘receiver’ E. coil. The Ben operon is regulated by benzoic acid so the signal would allow the genes for benzoic acid to be transcribed. The benzoic acid would then be degraded into catechol, this would quench the signal. The same promoter which regulates the Ben operon would also be coupled to GFP so that you could see that the signal had been picked up by the receiver E.coli.

Fig 1. 2-dioxybiphenol is converted into benzoic acid by the sender E.coli with the Hbp operon inserted into it. The benzoic acid then leaves the cell and is detected and converted into catechol by the receiver E.coli. This has the Ben operon inserted into it.

Sender Construct

Fig 2. The Hbp operon. HbpR encodes the transcription factor this binds to the UAS sequences shown above.. HbpA encodes 2-hydroxybiphenyl-3-monooxygenase, this converts 2-hydroxybiphenyl into 2,3-dihydroxybiphenyl. HbpC encodes 2,3-dihydroxybiphenyl-1,2-dioxygenase, this converts 2,3-dihydroxybiphenyl into 2-hydroxy-6-oxo-6-phenyl-2,4-hexadienoic acid. HbpD encodes 2-hydroxy-6-phenyl-6-oxo-2,4-dienoic acid hydrolase, this converts 2-hydroxy-6-oxo-6-phenyl-2,4-hexadienoic acid into benzoic acid. This construct would be in the standard backbone vector pSB1C3.

There were problems cloning the Hbp operon from p. nitroreducens so after multi failed attempts these genes were ordered for synthesis. They were ordered in 2kb segments to be assembled on arrival due to a much shorter waiting time for delivery.

Receiver Construct

Fig 3. Ben Operon. BenM encodes a transcription factor which binds to the binding site shown above. BenAB encode a dioxygenase complex whilst BenC encodes an electron transfer component. Together BenABC convert benzoic acid into benzoate diol. BenD encodes DHB dehydrogenase which converts it into catechol.

The ben operon cloned successfully but failed to assemble into a biobrick backbone via paperclip (the assembly method we were using). So new primers were ordered to amplify it with restriction enzymes sites at either end to allow assembly. However there were several illegal restriction sites within the genes which would have taken longer to remove than we had. Therefore we decided to try and assemble just the transcription factor with the promoter attached to GFP. This would allow us to detect the signal but not quench it.

Unfortunately we ran out of time to finish these constructs.