Design og brug af en Low Cost, Automated Morbidostat for Adaptive Evolution Bacteria Under antibiotikum Drug Selection
Summary
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.
Abstract
Vi beskriver en lav pris, kan konfigureres morbidostat til karakterisering den evolutionære vej for antibiotikaresistens. Den morbidostat er en bakteriekultur enhed, der kontinuerligt overvåger bakterievækst og dynamisk justerer lægemiddelkoncentrationen til konstant at udfordre bakterier, som de udvikler sig til at erhverve resistens. Enheden er udstyret med en arbejdsvolumen på ~ 10 ml og er fuldt automatiseret og udstyret med måling optisk tæthed og mikro-pumper til medium og drug delivery. For at validere platformen, vi målte den trinvise overtagelse af trimethoprim modstand i Escherichia coli MG 1655, og integreret enhed med en multiplex mikrofluid platform til at undersøge celle morfologi og antibiotisk modtagelighed. Tilgangen kan være op-skaleret til laboratorieundersøgelser af antibiotikaresistens narkotika, og er udskydelige for adaptive evolution til strain forbedringer i metabolic engineering og andre bakterielle kultur eksperimenter.
Introduction
Siden indførelsen af den første antibiotikum lægemiddel penicillin, har mikrobiel antibiotikaresistens udviklet sig til en globalt sundhedsproblem 1. Selv om overtagelsen af antibiotikaresistens kan efterfølgende undersøges in vivo, er betingelserne for disse eksperimenter ofte ikke kontrolleres gennem hele evolution 2. Alternativt kan adaptiv laboratorium evolution afslører molekylær evolution af en given mikroorganisme under miljømæssige belastninger eller selektionspres fra et antibiotikum stof 3. For nylig har mange velkontrollerede evolutionære eksperimenter af antibiotikaresistens stof belyst fremkomsten af antibiotikaresistens stof. For eksempel, Austin gruppe demonstrerede hurtige fremkomst i en ordentligt manipuleret mikrofluid compartmented miljø 4. Den nyligt udviklede morbidostat inducerer systematiske mutationer under narkotika selektionspres 5,6. Den morbidostat, et mikrobielt selection enhed, der kontinuerligt justerer antibiotikum koncentration for at opretholde en næsten konstant befolkning, er et stort fremskridt fra udsving test, der anvendes i mikrobiologi 7,8. I udsving test, er et antibiotikum lægemiddel injiceret i høj koncentration, og de overlevende mutanter screenes og talt. I stedet mikrober i en morbidostat konstant udfordret og erhverve flere mutationer.
Den morbidostat fungerer på samme måde som kemostat, en kultur anordning opfundet af Novick og Szliard i 1950, der holder en konstant befolkning ved kontinuerligt at levere næringsstoffer, samtidig fortynde den mikrobielle population 9. Siden introduktionen har kemostat været fremført og forbedret. Aktuelle mikrofluide kemostater har nået nanoliter og encellede kapacitet. Imidlertid er disse indretninger er uegnede til adaptive evolution eksperimenter, som kræver en stor cellepopulation med mange mutationsbegivenheder 10,11. For nylig, mini-kemostater med arbejder mængder ~ 10 ml er også blevet udviklet til at udfylde hullet mellem liter skala bioreaktorer og mikrofluid chemostaten 12,13.
Her præsenterer vi udformning og anvendelse af en billig, automatiseret morbidostat for et antibiotikum resistens undersøgelse. Den foreslåede modul kan anvendes i en rysteinkubator i en mikrobiologiske laboratorium med minimalt behov hardware. Open source firmware er også let skræddersys til specifikke anvendelser af adaptiv evolution, såsom metabolic engineering 3. Endelig er det morbidostat integreret i en multiplex mikrofluid platform for antibiotika resistensbestemmelse 14.
Protocol
Representative Results
Discussion
En lav-fodaftryk morbidostat enhed lavpris komponenter demonstreres. Stigningerne i resistens niveau registreres af enheden er i overensstemmelse med de tidligere rapporter 5. Designet til evolutionære studier af resistens, enheden er potentielt anvendelig til mange andre eksperimenter. Først kan etableres en omfattende database af lægemiddelinducerede mutationer for et stort sæt af klinisk relevante antibiotika. For eksempel kan den evolutionære vej af multipel lægemiddelresistens studeres ved simpelth…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| 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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