3 Title: Engineered probiotics that sequester arsensite in a mouse gastrointestinal system

Overview

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Key numbers

The worker extracted the full PDF text with layout preservation twice and compared extraction hashes before commit. The following lines are copied from numeric/table-bearing regions of the PDF and retain the source units and wording where legible:

• 35 yielding a non-toxic chelator suitable for intracellular sequestration. The resulting engineered
• 160 circuit were exposed to a range of sodium arsenite concentrations (0 to 5 µM) overnight, and
• 162 mCherry fluorescence averaged 975 ± 41 a.u. (mean ± S.D.), whereas exposure to 5 µM
• 178 (RBS) strength across a range of
• 199 cultures were collected and mean ± S.D. of three biological replicates.
• 219 range of sodium arsenite concentrations, and cellular mCherry fluorescence was quantified. The
• 272 exhibited the greatest dynamic range, with mCherry fluorescence increasing from 54,481 ±
• 278 expression from host transcriptional system, thereby expanding its dynamic range and
• 305 of arsR expression by 0.2% arabinose. Notably, most substitutions alleviated cytotoxicity, except
• 375 step, we sought to identify an arsenite dose that would generate a detectable but unsaturated
• 383 levels rose rapidly to 181 ± 66 ppb after 1 hour and continued increasing to 306 ± 25 ppb at 8
• 384 hours, remaining elevated (198 ± 44 ppb) even after 24 hours, indicating systemic saturation
• 386 dynamic and responsive profile: blood arsenite reached 15 ± 11 ppb at 1 hour, peaked at 34 ±
• 387 12 ppb by 4 hours (remaining 31 ± 4 ppb at 8 hours), and returned near baseline (4.6 ± 1.2 ppb)
• 414 of arsenite within 2 hours, whereas 5 × 109 cells would capture ~82% (Figure 4D). Increasing
• 415 the bacterial dose further to 1 ×1010 cells produced only modest additional benefit (~91%).
• 430 were 4.5 ± 1.6 ppb, significantly lower than those in mice given wild-type EcN (37.4 ± 9.1 ppb)
• 431 or no bacteria (34.5 ± 10.7 ppb). This protective effect persisted at the 8-hour time point, with
• 432 engineered EcN-treated mice maintaining low arsenite levels (4.3 ± 1.0 ppb) compared to wild-
• 433 type EcN (33.5 ± 4.0 ppb) and no-bacteria controls (33.1 ± 3.1 ppb). By 24 hours, arsenite was
• 434 undetectable in the blood of mice treated with engineered EcN, whereas low but measurable
• 435 levels remained in the wild-type EcN (4.2 ± 1.0 ppb) and no-bacteria (4.7 ± 1.0 ppb) groups.
• 510 which consisted of 1× M9 salts, 2 mM MgSO4, 0.1 mM CaCl2, 0.2% (w/v) glucose, and 0.2%
• 570 containing a range of sodium arsenite concentrations (0, 0.16, 0.31, 0.63, 1.25, 2.5, or 5 µM).
• 623 was added to a final concentration of 0.2% (w/v) to induce expression. Following 3 hours of
• Table 1. Graphite furnace parameters for arsenite quantification.
• 731 prevent clotting, each sample was immediately mixed with 10 µL of 2% (w/v) EDTA. Samples
• 750 1.08 L/min. These parameters ensured stable plasma conditions, efficient ionization of analytes,
• 752 mixed with 5 mL of a diluent containing 0.5% HNO3 and 2% ethanol (v/v). The diluted mixture
• 755 element stock standard (Inorganic Ventures, Christiansburg, VA) in 0.5% HNO3 and 2% ethanol
• 757 analysis with calibration levels set at 0.01, 0.1, 0.5, 1, 2.5, 5 and 15 µg/L. An internal standard
• 760 quantification. The detection limit for arsenic was 0.018 µg/L (2.34 ×10-4 µM).
• 917 stable CRISPR-based kill switches for engineered microbes. Nat Commun 13, 672

Methods (brief)

• 26 strategies to prevent dietary arsenite from entering the human body during digestion. Here, we
• 62 spectrometry (11, 12) and atomic absorption spectroscopy (13-15) have value but are costly,
• 114 effective clearance of residual toxicants and extending the protective effect during digestion.
• 199 cultures were collected and mean ± S.D. of three biological replicates.
• 248 in a non-growing state, saturated cultures were collected daily and resuspended in an equal
• overnight induction with 5 µM arsenite, cells were collected to measure mCherry levels with flow cytometry
• 311 supernatants were collected to quantify residual arsenite levels (Figure 3C). Among the three
• above the corresponding plots. (B) In vitro arsenite sequestration by engineered EcN was quantified using ICP-
• gray shaded region indicate samples with undetectable arsenite. Two-way ANOVA revealed significant effects of
• 355 sodium arsenite and collected samples to measure arsenite concentration in supernatant at
• 424 samples were collected at 1, 4, 8, and 24 hours. At every time point, blood arsenite levels in the
• 564 samples were collected for flow cytometry analysis to quantify cellular mCherry fluorescence.
• 581 log phase cultures (OD600 ≈ 0.6) were exposed to 5 µM sodium arsenite. Samples were
• 582 collected at 0, 0.5, 1, 2, 3, and 4 hours post-induction, and cellular mCherry fluorescence was
• 600 medium to a final volume of 3 mL. Samples were collected before each medium exchange (0,
• 604 sodium arsenite, and cell samples were collected after 1 and 24 hours of induction for
• 606 Cellular fluorescence measurement by flow cytometry. To prepare samples for flow
• 608 The samples were analyzed using an ACEA NovoCyte 3000VYB flow cytometer (ACEA

Implications

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Wiki pages this source may touch

• Fish — marine, predatory (tuna, swordfish, shark, king mackerel)
• Fish — marine, non-predatory (sardines, anchovies, salmon, cod)
• Mercury
• Cadmium
• Arsenic
• Nickel

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Update history

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