Caffeinated Coli The Caffeinated Coli project has been part of the Microbe Hackers group since before we existed. These bacteria have been modified to be “addicted” to caffeine. If they don’t have caffeine, they can’t grow! However, if you give them caffeine, they’ll grow until they metabolize all of the caffeine present. We can count the final number of cells grown to determine how much caffeine was originally present. This “bioassay” is very accurate; we can use it to calculate the amount of caffeine in a cup of coffee, a can of coke, or an energy drink. Schematic of how Caffeinated Coli works. The introduced genes allow for demethylation of caffeine, also known as 1,3,7-trimethylxanthine. This results in the formation of xanthine, which is readily converted into guanine, allowing the cells to grow. Figure from Quandt, et al. 2013; “Caffeinated Coli 1.0”. The original “Version 1.0” was pioneered by the 2012 UT Austin iGEM team with Professor Barrick, published in 2013, and used in Austin by the 2014 iGEM students. Our “Version 2.0” (initiated in 2015 and published in 2019) can identify specific methylxanthines: not only caffeine, but also theophylline, theobromine, etc. This bioassay is as good as standard chemical methods, such as HPLC. Current Microbe Hackers are investigating different aspects of the Caffeinated Coli system or developing a educational kit for using Caffeinated Coli in high schools. Our active subprojects: Caffeinated Coli Educational Kit. We have created a kit with reagents and materials for a modified, accessible version of our Caffeinated Coli bioassay, along with a five-week synthetic biology module, for high school students to learn about synthetic biology and perform their own bioassay experiment. This kit was implemented for the first time in Austin high schools in Fall 2023. Learn more about the Caffeinated Coli Educational Kit here! Long-Term Evolution Experiment (LTEE). Caffeinated Coli are reliant on methylxanthines due to the deletion of the guaB gene, which is required for de novo guanine synthesis; without this gene, the bacteria must obtain guanine in some other way (such as through the “xanthine salvage pathway,” which converts xanthines into guanine). We are investigating mutations that might arise when ΔguaB E. coli are grown under different selective pressures. LTEE Pipeline: The guaB gene was extracted from the E. Coli genome, using a P1 phage. The LTEE experiment is being conducted on the ΔguaB E. Coli, and a competitive fitness assay is run using CFP plasmid. Figure by Roopa Bindingnavele. OD600 Absorbance of ancestral ΔguaB E. coli, seen in brown and mutated strains, seen in blue and orange.Figure by Roopa Bindingnavele. PurR Project. We have previously found that our ΔguaB E. coli have a mutation in the purR gene. The PurR repressor protein normally regulates the metabolism of purines (like guanine, which our Caffeinated Coli produce from methylxanthines). We are investigating how the purR mutation affects the growth of ΔguaB E. coli. NdmE Project. Caffeinated Coli are able to metabolize caffeine using an operon (group of genes) from the bacteria Pseudomonas putida CBB5. When Version 1.0 was created, we didn’t have the full sequence for one of these genes (ndmE), so we substituted a similar gene from a different species. We now have the ndmE sequence and are investigating the effect of “restoring” ndmE to the Caffeinated Coli operon.
