Bioassay for Determining the Concentrations of Caffeine and Individual Methylxanthines in

Complex Samples
Alejandro E. Gutierrez, Prachi Shah, Abigail E. Rex, Tien C. Nguyen, Saamiha P. Kenkare,
Jeffrey E. Barrick, Dennis M. Mishler

Introduction:
• Caffeine (1,3,7-trimethylxanthine) and other methylxanthines are the primary bioactive
chemical components of many beverages
• Caffeine is well known as a neurostimulant frequently used by people to stay awake, but it and
the dimethylxanthines theophylline and theobromine also have other effects on respiratory and
cardiac functions
• Theobromine (3,7-dimethylxanthine) is the molecule responsible for the toxicity of chocolate to
dogs
• Caffeine has been identified as a pollutant in surface waters around urban centers, and it can
be toxic to wildlife
• Theophylline (1,3-dimethylxanthine) and theobromine are used as bronchodilators in human
and veterinary medicine
• Measuring the amounts of these drugs in blood and urine can be important for monitoring
correct dosage
• The pharmacological effects of methylxanthines can also lead to illicit uses. While not currently
banned, caffeine is on the World Anti-Doping Agency’s 2018 list of monitored substances
• Use of caffeine and other methylxanthines as performance-enhancing drugs has also been
reported in horse racing

Objective:
• To accurately determine the methylxanthine content – quality assurance and safety –
compounds are lethal at high concentrations

Previous Methodology:
- While this bioassay is useful for measuring the amount of caffeine in a formulated beverage or
unknown sample that contains only caffeine, it does not discriminate between different
methylxanthines, and any xanthine or guanine present in the sample will also support growth
• Biosensor based on coupling auxotrophy to a caffeine demethylation pathway
• First component - it uses an Escherichia coli strain with its de novo guanine synthesis pathway
knocked out via removal of the guaB gene, which encodes the enzyme that converts inosine-5ʹ-
phosphate to xanthosine-5ʹ-phosphate (XMP). These ΔguaB E. coli are guanine auxotrophs.
They also retain the ability to synthesize guanine from xanthine through its conversion into
XMP. Therefore, growth of this ΔguaB strain is directly proportional to the concentration of
guanine or xanthine present in the culture medium, as long as it is the limiting nutrient
• Second component - pDCAF3 plasmid, which encodes a synthetic operon for demethylating
caffeine and other methylxanthines to xanthine. This operon contains genes from Pseudomonas
putida CBB5, an organism that is capable of living off caffeine as its sole carbon and nitrogen
source
• The pDCAF3 plasmid and ΔguaB strain of E. coli were combined to create a cell that can
demethylate caffeine and other methylxanthines to xanthine, which can then be used to
synthesize the guanine needed for its growth. This bacterial strain was used to implement a
quantitative bioassay for caffeine and related molecules in which the number of E. coli cells after
growth to saturation can be used to calculate the number of methylxanthine molecules that were
originally present in a culture

New Methodology
1. 7 new plasmids containing all subsets of 3 demethylase genes (ndmA, ndmB, ndmC)
2. Growing pDCAF strains in media and creating standard curves - The pDCAF strains plus
ΔguaB and BW25113 E. coli were grown overnight in 2 ml of M9CG medium supplemented with
a 125 μM concentration of the specific methylxanthine that each strain can degrade (BW25113
received an equal volume of water) and 20 μg/ml CAM (except for the ΔguaB and BW25113
strains). The plates were divided into nine sections, and the saturated cultures from each of the
strains were streaked out in the same spot on each plate. The plates were incubated at 30°C
overnight and photographed the next day
3. pDCAF-BC demethylation analysis - Growing pDCAF strains in media and creating standard
curves.pDCAF strains were individually streaked out from glycerol stocks on M9CG agar plates
supplemented with 20 μg/ml CAM and 100 μM concentrations of the methylxanthine specific to
each strain (xanthine was used for the ΔguaB and pDCAF-A strains). Note that 50 μg/ml
kanamycin (KAN) must be used instead of CAM for the ΔguaB strain. Plates were incubated
overnight at 30°C. Colonies were picked from each plate and put into separate 2-ml M9CG
cultures supplemented with 50 μg/ml KAN, 20 μg/ml CAM, and 100 μM concentrations of the
methylxanthine specific to each strain. Again, CAM was not used for the ΔguaB strain. Each
culture contained only one pDCAF strain, and cultures were grown overnight at 30°C with
shaking at 200 rpm. These cultures were then diluted 1:1,000 into 2-ml triplicate M9CG cultures
supplemented with KAN and CAM (as described above) and a range of concentrations from 0 to
250 μM for the different methylxanthines based on the linear range of the standard curve
reported previously for the ΔguaB E. coli with the pDCAF3 construct (22). These cultures were
then grown overnight shaking at 30°C and 200 rpm for 22 h. The next day, 150 μl of each
culture was pipetted into a 96-well plate, and the OD600 was measured using an Infinite 200
PRO Tecan microplate reader. These endpoint OD600 measurements were then plotted against
the final concentration of methylxanthine present in each culture, and a linear trendline was fit to
each plot
4. HPLC analysis of methylxanthines in media - To test the pDCAF-BC strain in media
containing multiple methylxanthines, we first isolated three fresh colonies of the strain. These
colonies were grown to saturation overnight as described above. The saturated cultures were
then added 1:100 to liquid medium containing 20 μM 1,7-MX, 3,7-MX, and/or 1,3,7-MX. Growth
conditions and the recording of OD600 values were performed as described above. To
determine whether pDCAF-BC could target the caffeine molecule for demethylation, we grew
the strain in the presence of both 20 μM 3,7-MX and 20 μM 1,3,7-MX as described above. The
cultures were then spun down, and the supernatant was removed and put through a 0.22-μm
filter. The sterilized medium was transferred to fresh culture tubes, and a second pDCAF strain
was added to this sterile media and grown as described above
5. Bioassay usage with a coffee sample. To confirm our bioassay findings, we ran a sample of
the sterile media containing the methylxanthines through an HPLC before and after incubation
with pDCAF-BC (as described above). A Shimadzu SIL-20AC HPLC system equipped with an
SPD-M20A photodiode array detector and a Zorbax StapleBond C18 column (4.6 mm by 250 
mm, 5 μm) was used. All samples were filtered through a 0.22-μm filter prior to analysis. For all
samples, methanol-water-acetic acid (30:70:0.5, vol/vol/vol) was used as the mobile phase, and
each run was performed at 1 ml/min for 15 min (22). The results were read at a wavelength of
280 nm, and methylxanthine retention times and maximum wavelengths were compared to
standards of media supplemented with different compounds and previous studies to confirm
their identities (8, 23, 29, 45)
6. To prove the bioassay functions in a complex solution, we used coffee from a caramel-flavored
Starbucks Keurig K-cup supplemented with 2 mM either 1,3-MX or 1,3,7-MX. The ΔguaB, pDCAF-AB,
and pDCAF-ABC strains were then grown in media containing these samples, as described above with
minor changes. Importantly, instead of using casein, we used Casamino Acids (Fisher, BP1424-500) to
minimize background growth. Since growth is noticeably slower when using Casamino Acids, we
increased the growth times to ∼48 h to allow for growth to saturation. Otherwise, we performed the
bioassay in triplicate, as described above. HPLC analysis of diluted coffee samples and methylxanthine
standards used an Agilent Technologies 1260 Infinity II HPLC system equipped with a Poroshell 120 EC-
C18 column (4.6 mm by 100 mm, 2.7 μm). For all samples, methanol-water-acetic acid (15:85:0.5,
vol/vol/vol) was used as the mobile phase, and each run was performed at 1 ml/min for 15 min. We used
methylxanthine standards of 10, 100, 500, and 1,000 μM to determine the amount of each methylxanthine
in the coffee samples, analyzing all coffee samples three times at a wavelength of 280 nm
7. Calculations of methylxanthine concentrations.To determine the concentration of a specific
methylxanthine in a mixture, the growth of each strain must be compared to its specific standard
curve (described above). Once the total methylxanthine concentration has been determined for
the strain in question, the concentrations of all lower-order molecules must be subtracted out.
This includes guanine and xanthine. In Table 3, we show how to do this by calculating the
concentration of each individual molecule, as detected by each strain. For example, to
determine the amount of 1,3,7-MX present, we first calculate the concentration of each molecule
present in the mixture using every strain, including the ΔguaB strain. We start by calculating
how much guanine plus xanthine (guanine+xanthine) are present using the ΔguaB strain. We
then calculated how much 3-MX and 7-MX are present using the pDACF-B and pDCAF-C
strains, respectively, and subtracting from those values the amount of guanine+xanthine
measured. To determine the concentrations of the higher-order methylxanthines, we subtracted
the concentrations of each of these lower-order molecules, where appropriate. For example, to
determine how much 3,7-MX is present, we need to subtract the concentration of 3-MX, 7-MX,
and guanine+xanthine in the liquid. However, to determine how much 1,3-MX is present, we
only subtract the 3-MX and guanine+xanthine concentrations. This logic is then applied for
determining the concentration of 1,3,7-MX (Table 3). Note that due to some terms cancelling
out, it is sometimes possible to calculate the concentration of a target methylxanthine without
individually measuring the concentrations of every one of the lower-order methylxanthines