Team:UIUC Illinois/Project/Design


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Take up
The take up of L-carnitine is amplified by the addition of Pseudomonas aeruginosa's native L-carnitine uptake system. We desired to amplify this uptake system to allow for our bacteria to outcompete our gut bacteria. This system consisted of two genes cbcVW and caiX. cbcVW is the membrane bound protein that allows for L-carnitine to go through the bacteria's membrane. caiX is a free protein that directs the L-carnitine to the membrane bound transporter. We have introduced both of these genes into Nissle and tested the uptake of L-carnitine.

After each gene was isolated, we ligated them into two different plasmids. cbcVW was added into pET 26b and caiX was added into pACYC. These vectors were selected because of their excellent expression capabilities and the inducable T7 promoter.

Break down
The break down is achieved by cdhCAB, also originating from the Pseudomonas aeruginosa genome. This enzyme breaks down L-carnitine into 3-dehydrocarnitine. This process allows, as we mentioned on the Background page, for the alternative breakdown of L-carnitine, thus diverting TMAO production.
When designing primers for this biobrick, we modified the gene for cdhCAB. The gene originally began with a GTG sequence, but was modified to ATG to improve the transcription factor's recognition of the start codon. After gene isolation and digestion, it was ligated into pCDF, another excellent expression vector with an inducable T7 promoter.

Nissle 1917
Nissle 1917 is a strain of E.coli that is safe for digestion because it is currently used on the market today. We transformed and tested all of our parts into Nissle to ensure that our project is feasible as an actual pill on the market. See Safety page for more info.

Delivery system
In order for Cardiobiotics to be successful, it must reach the final destination: the small intestine. It is in the small intestine that L-carnitine promote the growth of carnitine loving gut bacteria and where L-carnitine is converted into the toxic TMA. It is difficult for Cardiobiotics to reach the small intestine due to the stomach acid's low pH. We sought to develop a protective, nutritious medium for the Cardiobiotics to survive: alginate gel. The gel was then encapsulated in gel capsules and sprayed with an enteric coating to prevent degradation at low pH.