Sugar logic wires

Background

By definition, metabolic wiring is a signaling system where cells take up metabolites in order to activate substrate specific inducible promoters, and convert them to a second signal/metabolite. However, the system usually uses toxic aromatic compounds as metabolites. While effective signals (as they are capable of diffusing through the membrane), they are not usually found in a bioreactor, and may cause adverse effects to the microbes within them.

Therefore we investigated an alternative version of MW, that takes advantage of E. coli’s hierarchy of carbon metabolism. While the other metabolic wires directly convert one signal to another, the sugar logic system uses two metabolites where the presence of one inhibits the receiver from detecting the other. This maintains the desirable feature of sequential activation of sender and receiver.

In a culture containing arabinose and xylose, the cells will preferentially uptake and consume arabinose, while xylose specific promoters are repressed (Desai and Rao, 2010). If a reporter were attached to these promoters, we could design a two cell system that looks like this:

The cells in this system are placed in a medium containing xylan (a xylose polymer) and arabinose. The first cell (the sender) contains an arabinose induced xylanase (cex). As the cell takes up arabinose, it also secretes xylanase to the medium. This enzyme converts xylan to xylose and releases xylose to the media; xylose concentration is allowed to build up because it is not metabolised by the cells in the presence of arabinose.

The second cell (the receiver) contains a xylose induced reporter. In a complete system, this would be coupled to a population regulator, or another functional circuit. Once the xylose to arabinose ratio reaches threshold, the reporter will be released from repression, and the receiver cells will express GFP.​

Therefore, the sender in this case “sends” to conversion of xylan to xylose, while the receiver “receives” the removal of arabinose from the system.

The carbon hierarchy in these cells is achieved by two transcription factors: araC and xylR. AraC is both an activator and repressor of transcription at pBAD- usually it binds the promoter to keep it switched off, but changes conformation in the presence of arabinose to switch it on. However, arabinose-bound araC also acts as a repressor of pXylF, and prevents the promoter switching on. If xylose is also present in the cell, xylR can act as an activator of pXylF, but only once the araC repression is released.

When both arabinose and xylose are present, xylR and araC compete for pXylF. Their respective levels within the cell will affect the levels of arabinose and xylose at which the receiver will respond. It has been shown (Groff et al. 2012), that this hierarchy can be removed by expressing xylR and araC in a 2:1 ratio, and that high expression of them will kill the cell. Therefore, the sender and receiver strains should be transformed with low copy number plasmids.

Progression

At the beginning of the summer, following a study of the literature, I set about using the Paperclip method to assemble two constructs, shown below:

The constructs were made using parts already found in the registry:

• araC: BBa_I13458
• Constitutive Promoter: BBa_K880005
• pBAD: BBa_K206000
• cex: BBa_K118022
• xylR: BBa_I741005
• ConsP: BBa_K880005
• pXylR: BBa_I741018
• GFP: BBa_E0040

Realising that designing the primers for Paperclip manually was tedious and subject to human error, I decided to use my limited Python coding skills to write a script to automate it. After two days, it worked! With help from Rikki, it was turned into a stand-alone program: sequences go in, primers come out. Given the huge numbers of primers we made over the summer, I’d like to think it saved us some time.

Paperclip assembles constructs from a mixture of multiple plasmids and special, specific primers called clips (more detail here). While preparing the clips was largely successful, amplifying the parts by PCR proved difficult. Many days were spent by the PCR machine, but xylR and cex refused to produce bands on a gel, due to their high GC content. With time pressing on, I decided to get the Receiver construct synthesised. Once this arrived, it should have been straightforward to assemble it into a plasmid, but sadly I needed to amplify it first, as synthesis only gives a small volume of DNA. Unfortunately, I spent 2 weeks trying to amplify the sequence using the wrong primer sequences (but learnt the hard way how important checking is!). The new, correct versions arrived too close to the end of the iGEM competition to be able to produce a finished construct.

References

Desai, T. A., & Rao, C. V. (2010). Regulation of arabinose and xylose metabolism in Escherichia coli. Applied and environmental microbiology, 76(5), 1524-1532.

Groff, D., Benke, P. I., Batth, T. S., Bokinsky, G., Petzold, C. J., Adams, P. D., & Keasling, J. D. (2012). Supplementation of intracellular XylR leads to coutilization of hemicellulose sugars. Applied and environmental microbiology,78(7), 2221-2229.