Team:UIUC Illinois/Project/Design

Take up
The take up of L-carnitine is being 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 out compete our gutt 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 in the Nissle and tested the uptake of L-carnitine.

Break down
The break down is achieved by cdhCAB, also originating from the Pseudomonas aeruginosa's 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 diverting TMAO production.

"Take up" system
After each gene was issolcated 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 its excellent expression capabilities and with an inducable T7 promoter.

"Break down" system
When designing primers for this biobrick, we modified the gene. The gene originally began with a GTG sequence but was modified to ATG to improve the transcription factors recognition of the start codon. After gene isolation and digestion it was ligated into the pCDF which is another excellent expression vector and with an inducable T7 promoter.

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

"Deliver" system
In order for the probiotic to be successful, it must first reach the final destination: the small intestine. It is in the small intestine that L-carnitine is broken down into the toxic TMA by gut bacteria. It is difficult for bacteria to reach the small intestine due to the stomach acid's low pH. We sought to develop a protective, nutritious medium for the cells to survive: alginate gel. The gel was then encapsulated in gel capsules and sprayed with an enteric coating to prevent degradation at low pH.