Team:UIUC Illinois/Results

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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

To determine the functionality of pdCAF3, we designed a feeding assay to observe the growth of E. coli in theobromine and caffeine. The simple assay used M9 minimal media that contained caffeine, theobromine or neither. The pdCAF3 we received included a guaB knockout, which inhibited the strain’s use of guanine unless it came from a methyl-xanthine carbon source such as caffeine or theobromine. This meant that the bacteria containing the pdCAF3 plasmid could not grow without caffeine or theobromine present. We cultured the pdCAF3-containing E. coli in a theobromine M9 solution, caffeine M9 solution and a solution of just M9 media. We additionally cultured wild type E. coli without the pdCAF3 plasmid in the same solutions. The samples incubated in a shaker and we examined them after three days. The wild type grew in all three solutions, which was the expected result because it had no restrictions on its growth. The pdCAF3 strain grew in the caffeine and theobromine solution, but not in the purely M9 solution. This demonstrated the bacteria’s dependence on the guanine of caffeine and theobromine and confirmed the functionality of the pdCAF3 plasmid. 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]
The design of our original Golden Gate reaction had two goals in mind: insert our operon into the standardized backbone pSB1C3 with lactobacillus promoter, and remove all illegal biobrick restriction sites. We designed primers using the type II restriction enzyme BsaI. The properties type II restriction enzymes allowed us to introduce single-base mutations at the sites incompatible with biobrick standard enzymes EcoRI, PstI, SpeI and XbaI. Additionally, we used the primers to introduce and amplify the first and second halves of our promoter into our operon and vector. By including the promoter sequence within the primers, we avoided having to order the physical DNA. We attempted this assembly design multiple times without success. The design was eventually changed to transfer the operon directly to a lactobacillus shuttle vector pnz8048. Unfortunately, this design also failed. We tried enacting the later design in multiple steps, using PCR to put individual fragments into joint fragments, and combing the resultant pieces into our completed part. However, this approach was unsuccessful and failed at different steps each time

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.