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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Genetics

Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection

Published: September 27th, 2016

DOI:

10.3791/54426

1Electrical Engineering, National Tsing Hua University

We describe a low cost, configurable morbidostat that enables the characterization of antibiotic drug resistance by dynamically adjusting the drug concentration. The device can be integrated with a multiplexed microfluidic platform. The approach can be scaled up for laboratory antibiotic drug resistance studies.

We describe a low cost, configurable morbidostat for characterizing the evolutionary pathway of antibiotic resistance. The morbidostat is a bacterial culture device that continuously monitors bacterial growth and dynamically adjusts the drug concentration to constantly challenge the bacteria as they evolve to acquire drug resistance. The device features a working volume of ~10 ml and is fully automated and equipped with optical density measurement and micro-pumps for medium and drug delivery. To validate the platform, we measured the stepwise acquisition of trimethoprim resistance in Escherichia coli MG 1655, and integrated the device with a multiplexed microfluidic platform to investigate cell morphology and antibiotic susceptibility. The approach can be up-scaled to laboratory studies of antibiotic drug resistance, and is extendible to adaptive evolution for strain improvements in metabolic engineering and other bacterial culture experiments.

Since the introduction of the first antibiotic drug penicillin, microbial antibiotic resistance has developed into a global health problem1. Although the acquisition of antibiotic resistance can be retrospectively studied in vivo, the conditions of these experiments are often not controlled throughout the entire evolution2. Alternatively, adaptive laboratory evolution can reveal the molecular evolution of a microbial species under environmental stresses or selection pressure from an antibiotic drug3. Recently, many well-controlled evolutionary experiments of antibiotic drug resistance have elucidated the emergence of antibioti....

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1. Assembly and Pretesting of the Morbidostat Device

  1. Assembly of the Morbidostat
    1. Punch 3 holes on the cap of the culture vial with an 18 G syringe needle. Cut three pieces of polyethylene tubing ~7 cm in length. Insert these three pieces of polyethylene tubing on the cap.
    2. Use tape to wrap the edge of the cap to serve as the cast for the polydimethylsiloxane (PDMS) mixture. Mix 5 g of A component and 0.5 g of B component of the PDMS in a 150 ml plastic container by stirring manually with.......

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The above-described morbidostat is schematized in Figure 1. The common morbidostat operations, including experimental evolution, antibiotic susceptibility test and cell morphology checking, were validated in an E. coli MG1655 culture exposed to trimethoprim (TMP), a commonly used antibiotic drug5,6. TMP induces very distinctive stepwise increases in drug resistance, and the mutations are clustered around the dihydrofolate reductase (DHFR) gene. Therefo.......

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A low-footprint morbidostat device from low-cost components is demonstrated. The increases in drug resistance level registered by the device are consistent with those of previous reports5. Designed for evolutionary studies of drug resistance, the device is potentially applicable to many other experiments. First, a comprehensive database of drug-induced mutations can be established for a large set of clinically relevant antibiotics. For example, the evolutionary pathway of multiple drug resistance can be studie.......

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The authors would like to thank Prof. Sze-Bi Hsu and Ms. Zhenzhen for useful discussions and help in the theoretical analysis and numerical simulation. Y. T. Y. would like to acknowledge funding support from the Ministry of Science and Technology under grant numbers MOST 103-2220-E-007-026 and MOST 104-2220-E-007-011, and from the National Tsing Hua University under grant numbers 103N2042E1, 104N2042E1, and 105N518CE1.

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Name Company Catalog Number Comments
Environmental Shaker Incubator BioSan ES-20
Arduino Leonardo board Arduino Leonardo
680 Ohm Carbon Resistor Digikey Bias resistor for LED
100k Ohm Carbon resistor Digikey Bias resistor for phototransistor
940 nm light emitting diode Bright LED Electronic BIR-BM13E4G-2 Optical density measurement
940 nm phototransistor Kodenshi  ST-2L2B Optical density measurement
Darlington pair IC Toshiba Mouser ULN2803APG  this IC drives micropumps and magnetic stirring unit
5V DC brushless fan  ADDA AD0405LX-G70 spec: 5V supply voltage and 80mA available www.jameco.com
Piezoelectric micropump CurieJet PS15I-FT-5L Pressure >3kPa  Flow rate >5 ml/min
Tygon 3350 Tuning Saint Gobain ABW00001 ID: 1/32" OD: 3/32" L:50' 
Magnetic Stir bar COWIE tapered shape dim: 10 mm x 4mm
Glass scintillation 20ml vial DGS Pyrex glass 28mm(dia.)x 61 mm(h)
Culture vial holder Custom made from Polyformaldehyde 
Silicone  Dow Corning Sylgald 184 used to seal the glass vial
Medium bottle VWR 66022-065
Difco M9 minimal salt 5x BD Medium
Cadamino Acid BD Medium
glucose Sigma
Agar Bateriological Oxoid for agar plate
Luria Bertani medium
Inverted microscope Leica Microsystems Leica DMI-LED used for microfluidic measurement Use X40 objective NA=0.55
Microscope Incubator Live Cell Instrument CU-109 used for microfluidic measurement
Solenoidal valves Pneumadyne S10MM-31-12-3 Normally open 1.3 Watt 12 Vdc
USB interface card Hobby Engineering USBIO24-R Digital I/O Module  for microfluidics measurement
Air compressor Rocker Scientific ROCKER 440 Pressure source for microfluidcs Max. Pressure 80 Psi
Male luer-lock fittings to 1/8" barb ValuePlastics.com MTLL230-1 used for microfluidic control
1/8" barb to 10-32 threaded port ValuePlastics.com B-1 used for microfluidic control
Female luer-lock fittings to 10-32 threaded port ValuePlastics.com KFTL-1 used for microfluidic control
NPN darlington transistor 500mA, 40V (2N6427) DigiKey.com 2N6427GOS-ND used for microfluidic control
10kOhm, carbon film resistor, 0.25W DigiKey.com P10KBACT-ND used for microfluidic control
Tantalum capacitor, 10uF, 25V, 10% DigiKey.com 478-1841-ND used for microfluidic control
Andor CCD camera Andor Zyla 4.2 Plus SCMOS used for microfluidic on chip imaging
ELISA plate reader
two component Silicone  Momentive RTV 615 used for microfluidic chip fabrication
SU-8 photoresist Micrchem SU8 2015 used for microfluidic chip fabrication
AZ4620 photoresist Clariant AZ 4620 used for microfluidic chip fabrication
Plasma cleaner Harrick Plasma PDC 32G used for microfluidic chip fabrication
20 Gauge Syringe Needle BD used for microfluidic chip fabrication
Labcycler Sensoquest Labcycler PCR 
DNA polymerase Toyobo KDO Plus PCR amplification
Trimethoprim Sigma
Plate reader Biotek Synergy H1 hybrid  antibiotic resistane measurement

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