Published October 27, 2018

The Oscillostat

Our device, the Oscillostat, continuously evolves bacteria while oscillating between selective pressures to mimic fluctuating environments.

IntermediateWork in progress3,039

Things used in this project

Hardware components

Futaba S3110 servo motor

Farnell MC02068 perisaltic pump

Koge RP9879 air pump

Digikey 1528-1402-ND red laser

Mouser 856-TSL235R-LF light sensor

Digikey 259-1461-ND CPU fan

Arduino UNO

Versilon SPX-60 FB silicone tubing (4mm)

Story

Summary

Using continuous microbial cultures it is possible to study the course of evolution in the laboratory. In vitro evolution experiments have revealed principles of selection under constant conditions and provided a tool for developing new biomolecules. However, natural evolution happens under fluctuating environments. We propose to build a low-cost, continuous culture apparatus with temporally modulated environments for experimental evolution. We will do this by expanding on existing designs for turbidostats, devices that maintain a constant density of microbial cultures. Our device, the Oscillostat, will switch between inputs to generate user-specified selective pressures of defined frequencies and amplitudes. We will demonstrate the potential of this system by exposing bacteria to periodic pulses of oxidative stress in order to evolve a recently-described stress response that is under stochastic regulation. Our general-purpose platform will benefit a range of biologists wishing to harness evolution in the laboratory.

Prototype Oscillostat during a laboratory test

Growth chamber assembly

We designed a chamber for growing 10-40 ml cultures of cells based on a design reported by Hoffmann et al. 2017 (10.1371/journal.pone.0181923). As seen in the 3D print files below, a central cylinder houses a sterile disposable culture tube, with windows included to monitor growth and fit optics. A pair of windows connect to a 650nm laser and light sensor for measuring the optical density of the cell culture. Below the central housing is a base plate that screws into a CPU fan, which acts as a magnetic stirrer to keep the culture well-mixed.

The culture tube is closed by an air-tight seal. As media is pumped in, the added pressure pushes liquid out a small outlet line into a waste or sample-collection container. This allows us to maintain a constant volume in the growth chamber without requiring additional powered components.

Currently, we have tested we can control the laser and stirring fan, and collected preliminary measurements with the light sensor. The next task will be to calibrate how different light sensor readings correlate to cell densities, to ensure our device can keep the cell density at a constant rate.

Fluid pumping

To maintain a sterile setup, we decided to use a peristaltic pump to control all flows into the culture vessel. The motor is connected on one end to a T-junction that combines the inputs from the valve, and on the other end to the tube where cell growth is occurring. We power the 12V DC motor through an adapter connected to a wall socket.

Using an L293D chip, we can control both the speed and direction of the pump. We have performed early tests to match the speed of the pump with the flow rate of our passive waste outlet in order to maintain a constant volume.

Valve switching

We implemented a valve that can select between all 4 possible states of two input lines: on-on, on-off, off-on, and off-off. The design, modified from a valve suggested by Mikey77 (Instructables.com), achieves this by simply rotating a shaft that can pinch closed silicone tubing. We 3D printed our modified design and connected the shaft to a small servo with sufficient torque to switch between states.

After several iterations of testing and redesigning parts of our 3D prints, we found the optimal dimensions to robustly close selected tubing lines in the "off" states while remaining fully open in the "on" states. Using our valve and peristaltic pump, we have been able to cleanly switch between two inputs over several cycles, as would occur in a directed evolution experiment with oscillating conditions.

Future work

Currently, we have assembled a prototype that demonstrates the core functionalities of the full Oscillostat, including sustaining robust osculations between two media inputs. Next, we will need to do further work integrating our light sensor to prepare it for monitoring live bacteria. Using the flow speed controls we already set up, we will then need to program the feedback circuits that maintain constant growth. Finally, we aim to add a user-friendly interface to allow biologists to easily program and set up their own experiments with our device.

Code

PinchValv_PerisPump.ino

#include <Servo.h>

//Pins for peristaltic pump
const int OUT6 = 6; //1EN
const int OUT10 = 10; //2A
const int OUT11 = 11; //1A
int IN0 = 0; //Analog IN, potentiometer
int IN4 = 4; //Digital IN 4, pushpin

//Pins for pinch valve
int potPin = 1;
int servoPin = 9;
Servo servo;


void setup() {
  //Peristaltic pump setup  
  pinMode(OUT6,OUTPUT);    //sets dpin as output
  pinMode(OUT10,OUTPUT);    //sets dpin as output
  pinMode(OUT11,OUTPUT);    //sets dpin as output
  pinMode(IN4,INPUT_PULLUP);    //sets dpin as output

  //Pinch valve setup:
  servo.attach(servoPin);

}

void loop() {
  //Peristaltic pump with potentiometer for speed and reverse button
  int speed = analogRead(IN0) / 4;
  boolean reverse = digitalRead(IN4);
 
  analogWrite(OUT6,speed); //Motor turn
  digitalWrite(OUT10,reverse); //Motor turn
  digitalWrite(OUT11,!reverse); //Motor turn

  //Pinch valve operation with potentiometer:
  int reading = analogRead(potPin);     // 0 to 1023
  int angle = reading / 6;              // 0 to 180-ish
  servo.write(angle);
}

Credits

Jarrod Shilts

2 projects • 3 followers

Om Patange

1 project • 1 follower

The Oscillostat