Team:UIUC Illinois/Results

Results

"I have had my results for a long time: but I do not yet know how I am to arrive at them."

Karl Friedrich Gauss

Feeding Assay

Experimental analysis began with verifying biobrick BBa_K734000. To verify growth from solely caffeine or theobromine, the organisms were sent to us from UT Austin with the guaB gene knocked out. Guanine comes from xanthine, which is a necessary factor for microbial growth. Without the ability to produce guanine, xanthine degradation is imperative for guanine biosynthesis therefore addicting the microorganisms to caffeine. We set out experimentally to show that theobromine degradation could also be used for xanthine degradation using the same N-Demethylation pathway. Our results are as follow:

Figure 1. All organisms were grown on M9 Media with specified amounts of Theobromine & Caffeine. Refer to lab notebook.[reduce font size]
By verifiying that our ΔguaB cells could only grow in media with caffeine or theobromine present, we verified that part BBa_K734000 was functional. Subsequent experiments involved monitoring growth through an arrary of theobromine amounts. HPLC results indiciating linear degradation of theobromine have yet to arrive!

Reconstruction of pdCAF

To qualify with iGEM standards, all illegal cutsites were made to be removed.

Figure 2. Cut sites were removed using standard PCR procedure.[reduce font size]
To remove cutsites, we had designed primers upstream of each illegal cutsite and attempted to ampify out and reassemble using golden gate assembly methods. Unfortunately, we were not able to remove the cutsites in time. Additional primers have been ordered and we are currently in the process of modifiying the part further. Our PCR reactions were performed using Q5 Polymerase however did not yield our anticipated results.

Lactobacillus

To create our probiotic, it was mandatory that we transform our gene into a strain of Lactobacillus. With the help of the Miller lab at UIUC, we were able to culture our cells anaerobically. Using shuttle vector pnZ8048, the initiative was to induce Chloramphenicol resistance into the cells. When trying to transform with our frozen stocks of electrocompetent cells with our shuttle vector, we realized that Plantarum, and many Lactobacillus species have to be cultured fresh almost every day. The advent of this realization had been temporally placed conveniently near part submission, ergo results are to come.