Team:Baltimore BioCrew/Design

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DESIGN

• Design Philosophy






• MAFF Design






• Killswitch Design

Design Philosophy

There are a number of probiotics advertised to improve gut health, from yogurt to pills. However, we are confident that using these B. theta genes to produce MAFFs is a far better alternative to these common products.

MAFFs are indigenous to the human microbiome, which means they’re safe: to increase MAFF content in the microbiome wouldn’t run the same risks as medicating patients with a non-native protein. E. Coli, which serve as the MAFF producers and delivery system, are also native to the microbiome. Any chance of pathogenesis is nullified by the xylose-dependent killswitch that limits their lifespan and reproduction once the supplement runs out.

Furthermore, MAFFs amplify what’s already there, rather than introduce new bacterial species to the gut. Because every microbiome is unique, MAFFs give a boost that’s tailored to each patient without the need for any extra testing. Our project combines simplicity with safety to create a powerful probiotic that’s perfect for everyone.

MAFF Design

The backbone of these composite parts are the BT2268 and BT2269 genes, found in Bacteroides thetaiotaomicron and indigenous to the human gut. The signal peptide was removed to prevent the proteins from remaining bound to the cell membrane, thus making them easier to purify from the cells. A 6xHis tag was added for protein collection, and they were codon-optimized for transformation into E. Coli.

A significant concern when designing the basic parts were the signal peptides and transmembrane domains. Membrane proteins are difficult to purify from cells, complicating verification of our results. The signal peptide was easily located and double-checked by a variety of online tools, so it was removed from both sequences. However, the transmembrane domains of both genes were impossible to identify, so they were left as-is.

Both composite parts use a strong T7 inducible promoter. This enabled the BioCrew to culture the bacteria without producing significant quantities of MAFFs, but also allowed production to be induced as needed. It provided flexibility for the researchers and stability for the bacteria.

The composite parts use different terminator sequences; this is because IDT was repeatedly unable to synthesize the BT2269 composite part due to complexity within the terminator sequence. For unknown reasons, this issue did not affect the BT2268 composite part, so only the BT2269 composite part was altered.

Killswitch Design

We, of course, are well aware of the dangers of inserting a foreign organism into the body. One mistake or mutation could prove fatal. So, in order to guarantee safety, we implemented a killswitch into the system. This killswitch has two different promoters: one, a low-efficiency constitutive promoter, promotes ydcE, which encodes the EndoA toxin. The second promoter, a xylose inducible promoter, promotes ydcD to produce the EndoAI antitoxin. Our strategy was to have the MAFF supplement taken in a xylose solution. Until the xylose is metabolized, the bacteria will produce MAFFs. However, once the xylose is metabolized, the inducible promoter will stop, and the toxin will inhibit further growth. This design was based on the HKUST iGEM team own killswitch, part BBa_K733012. Their killswitch was the inverse of ours, with the presence of xylose stopping antitoxin production, rather than its absence. However, we felt that this left room for error in the system. There are a myriad of different factors that could affect the xylose transportation. As it would have to be administered after the MAFF bacteria had time to express, we could not guarantee cell death. So, with a flipped system, we are able to ensure that, even if the xylose misses its mark, the system will still die. On top of this change, we used a new terminator, part BBa_B1002, a well characterized terminator. The original terminator that HKUST used was far too strong than was needed, and added needless complexity to the system, making it less cost effective.