Proof of Concept

Overview

Based on the background of the SARS-CoV-2, it is urgent to solve the problem of how to restore the happiness of human life as soon as possible. With the availability of vaccines and various preventive devices, it is particularly important to develop an innovative device that can provide more accurate and efficient warnings. Because in the future there will be not only the threat of COVID-19, but also other threats that humans cannot imagine. Based on the concept of WBE and synthetic biology, we hope to create a library of short peptides that can be screened for binding to a future viral protein, thus providing early warning of the next pandemic outbreak.

Experiment

Random Peptide Library

Of course, this is not a simple matter, we first constructed the random peptide library fragment and constructed the initial random peptide library fragment on pDONR207 vector by gateway method, then on pGADT7-GW vector by LR reaction, and finally transferred into the yeast strain. Then we co-transformed the Y187 strain containing the random peptide library with the AH109 strain containing the bd-bait protein, and finally screened the strain using a triple deletion plate, and the screened strain was subjected to PCR and sent for sequencing, and finally we analyzed the random peptide library fragment that interacted with the spike RBD structural domain based on bioinformatics.

Figure1: AH109 strain and Y187 strain binding subs

Figure2: Growth of the transformed strain

Detection system ( two-component system )

After receiving the strain from Tsinghua, we changed the sensing sequence in PmrB to our random peptide library. and started the next step of the experiment.

In order to establish the relationship between extracellular antigen concentration and final fluorescence intensity. We established a series of models by mathematical modeling. Finally, the theoretical relationship between the antigen concentration and the final fluorescence was established as a function of the antigen concentration. And the theoretical detection limit of this cell sensor was determined to be 1000ng/ml. the linear range was 1000~1700ng. in accordance with our expectation.

Figure2:Relationship between spike protein concentration and fluorescence intensity(left：spike induction results under fluorescence microscope. The engineering bacteria was induced with 1.0 μg/mL~3μg/ml spike(after codon optimization). Photos were taken under fluorescence microscope.right：the theoretical relationship between spike protein concentration and fluorescence intensity was made by mathematical modeling method based on the principle of two-component system)

In the next step we constructed strains and tested the action of the part with different gradients. As a result, although showing similar trends to the mathematical modeling, required overall higher concentrations of antigen to excite the same intensity of fluorescence. This may be due to the wrong settings of our peptide library and antigen binding parameters. However, we successfully excited fluorescence of different intensities with different antigen concentrations.

Escape Model

We need E. coli to roll over in place when not irradiated with blue light, which requires us to knock out the original CheZ sequence in E. coli. We wanted to knock out this gene with CRISPER/Cas9 technology, but got nothing. So we let the blue light switch regulate the expression of both CheZ and suicide genes. Let E. coli lyse and die while being irradiated by blue light for a long time.

After constructing the escape circuit, we incubated E. coli with light avoidance/blue light. Meanwhile, E. coli will keep releasing chromoprotein, and we use this as a basis to observe the group movement phenomenon. of E. coli.

We can observe that the blue pigment is more expressed and darker in the areas not irradiated with blue light. But there is also chromoprotein production in the area irradiated with blue light, which may be due to the fact that the death of E. coli requires a certain relaxation time.

And we found that the boundary between blue light and non-blue light was blurred, which is possibly because the E. coli at the boundary escaped back to the blue light region.

So we can conclude that our escape model can make most of the bacteria escape to the blue light-free region. However, knocking out CheZ is necessary for the overall experimental accuracy.

Hardware

Preface: What our project does is to sensitively detect the presence of pathogens in sewage for regionalized epidemic warning. We sincerely expect that our project will bring a healthy and safe life to human beings.Based on this project, we designed a hardware device and expected this device to be installed near sewage treatment pipes in residential areas to detect virus.

Name：Jiang Ziya2.0

Top cover: The top cover is the location of our bacteria enrichment and is fixed with 6 self-tapping screws, water is fed from the lateral side and it integrates 3 normally closed solenoid valves. A central solenoid valve for blocking wastewater and bacteria-rich liquid, and two small solenoid valves for wastewater discharge with controlled reaction intervals. According to the tendency of bacteria to light, the top is made of transparent glass laminated with led light strips to attract the movement of bacteria for the purpose of enrichment. An opening is made on the side for the connection of the solenoid valve lines.

The middle body: the reaction position is located in the middle of the 6 cylindrical holes. Detecting independently and constantly for multiple times is what we are pursuing. To realize this assumption, we designed a rotating scheme that the motor at the bottom drives the whole reaction device to rotate, which cooperating with the solenoid valve in the top cover to deliver the liquid to the reaction vessel hole, with the reaction stock solution placed inside the hole in advance. In order to avoid the device too heavy, opening is made on reaction vessel to reduce the weight in order to reduce the load of the motor and ensure the accuracy. The Below the reaction device is the observation room, the liquid after the reaction is finished flows from the reaction tank to the observation room, the top of the observation room adopts inclined openings and covered with glass, so that the reaction results can be seen clearly and intuitively.

Bottom: The bottom layer is fixed with 4 screws and the middle body. Waterproof treatment is done in the middle of the two layers to prevent water leakage which will burn the circuit board and battery at the bottom.

Product Parameters：
Overall size radius 85mm, height 235mm
Top Cover: height 80mm, radius 85mm; volume of enrichment zone: 80mm3
Rotating reaction area: height 55mm radius 70mm; reaction cell height:45mm radius 15.5mm volume 46mm3
Middle body: Height 140 Radius 85 Thickness 4mm
Bottom: Height 15 Radius 85

(the physical image printed out by 3D modeling)

Development Boards

The Arduino Uno is a development board based on the microcontroller ATmega328P. It has 14 digital input/output pins (6 of these pins can be used as PWM output pins), 6 analogue input pins, a 16 MHz quartz crystal, a USB interface, a power supply interface, support for in-line serial programming and a reset button. All we need is to connect the development board to the PC via the USB interface and it is ready to use. Here are its main parameters.

Microcontroller ATmega328P
Working voltage 5 volts
Input voltage (recommended) 7 to 12 volts
Input voltage (limit) 6 to 20 kv
Digital input output pin 14 (of which 6 pins can be used as PWM pins)
PWM pin 6
Analog input pin 6
Input/output pin DC current 20 ma
3.3V pin current 50 ma
A Flash Memory. 32 KB (ATmega328P) of which 0.5 KB is used for system boot
SRAM(static memory) 2 KB (ATmega328P)
EEPROM 1 KB (ATmega328P)
Built-in LED pins 13
Long 68.6 mm
Wide 53.4 mm
Heavy 25 g
The clock frequency 16 MHz

Stepper motor

Our sampling module is a six-hole, equally divided disc. After each sampling is complete, a stepper motor drives the disc to rotate by 60°. This allows the next sampling hole to be aligned with the valve

Drive Boards

The DRV8825 is an integrated motor driver chip designed by Texas Instruments. The chip has two H-bridges and a 1/32 microstepping indexer to drive one bipolar motor or two DC brushed motors. The input voltage range is from 8.2 to 45V, providing a drive current of 1.75A and a peak current of 2.5A at 24V 25°C. The on-state resistance of 0.2 ohms ensures good thermal stability. The chip also has integrated short circuit, overheat, undervoltage and cross-conduction protection circuits to detect fault conditions and quickly cut off the H-bridge, thus protecting the motor and driver chip.

DRV8825driver module pinout diagram

The pin connections of the DRV8824/8825 and even the common A4988 motor driver modules are not very different. In the diagram above, M0, M1 and M2 are not connected then they are high and the operating mode of the driver board is 32 subdivision. M0, M1 and M2 can be connected to ground separately or all together to achieve different working modes. The following is the subdivision setting table for our DRV8825 driver module. 

M0 M1 M2 TYPE
Low Low Low Full Step
High Low Low Half Step
Low High Low 1/4 Step
High High Low 1/8 Step
Low Low High 1/16 Step
High Low High 1/32 Step
Low High High 1/32 Step
High High High 1/32 Step

Arduino CNC

Arduino CNC motor expansion boards are commonly used to drive NEMA17 motors (42 stepper motors) in 3D printers, robotic arms or robotic systems. We use the AFMotor motor expansion board to drive the 28BYJ-48 stepper motor. The CNC expansion board can support stepper motor driver boards such as the A4988 and DRV8825. We have paired it with a 12V external power supply.

Software

Angle

#include "AccelStepper.h" 
#include "MsTimer2.h"
#include "elapsedMillis.h"
// Define constants for motor control
const int enablePin = 8;  
 
const int xdirPin = 5;    
const int xstepPin = 2;   
const int ydirPin = 6;     
const int ystepPin = 3;   
const int zdirPin = 7;    
const int zstepPin = 4;   
 
const int moveSteps = 200;    //The number of running steps used to test motor operation
 
AccelStepper stepper1(1,xstepPin,xdirPin);//Create stepper motor object 1
AccelStepper stepper2(1,ystepPin,ydirPin);//Create stepper motor object ２
AccelStepper stepper3(1,zstepPin,zdirPin);//Create stepper motor object ３

elapsedMillis sinceTest1;
elapsedMillis sinceTest2;

int state = 0;
int ok = -1;
void setup() {

  // Serial.begin(9600);

  pinMode(xstepPin,OUTPUT);     // Arduino control DRV8255x stepper pins for output mode
  pinMode(xdirPin,OUTPUT);      // Arduino control DRV8255x direction pins for output mode
  pinMode(ystepPin,OUTPUT);     // Arduino control DRV8255y stepper pins for output mode
  pinMode(ydirPin,OUTPUT);      //  Arduino control DRV8255y direction pins for output mode
  pinMode(zstepPin,OUTPUT);     //  Arduino control DRV8255z stepper pins for output mode
  pinMode(zdirPin,OUTPUT);      // Arduino control DRV8255z direction pins for output mode
  
  pinMode(enablePin,OUTPUT);   // Arduino control DRV8255 enable pin for output mode
  digitalWrite(enablePin,LOW); // Set the enable control pin to low to put the motor driver board into operation
                                
                             
  stepper1.setMaxSpeed(3000.0);     //  Set maximum motor speed 300 
  stepper1.setAcceleration(200.0);  // Set motor acceleration 200.0
  stepper1.setSpeed(300.0); 
 
  stepper2.setMaxSpeed(3000.0);     // Set maximum motor speed 300 
  stepper2.setSpeed(300.0); 

  stepper3.setMaxSpeed(3000.0);     //  Set maximum motor speed 300  
  stepper3.setAcceleration(20.0);  //Set motor acceleration 20.0

  

}

void loop() {
  
  if (ok == -1){
    stepper1.moveTo(33);
    ok = 0;
  }else if (ok == 0){
    if (stepper1.currentPosition() == 33){
      stepper1.setCurrentPosition(0);
      ok = 1;
      sinceTest2 = 0;
    }
  }else if(ok == 1){
    if(sinceTest2>=3000){
      stepper1.moveTo(33);
      ok = 0;
    }
  }
  if (sinceTest1 >= 5000) {
    sinceTest1 = sinceTest1 - 5000;
  
  }
  stepper1.run();   //No.1 motor operation
  stepper2.run();   // No. 2 motor operation
  stepper3.run();   // Motor No. 3 running
 
}

Timer

#include "AccelStepper.h"
#include "MsTimer2.h"
#include "elapsedMillis.h"
const int enablePin = 8;  
 
const int xdirPin = 5;    
const int xstepPin = 2;   
const int ydirPin = 6;     
const int ystepPin = 3;   
const int zdirPin = 7;    
const int zstepPin = 4;  

AccelStepper stepper1(1,xstepPin,xdirPin);//Create stepper motor object 1
AccelStepper stepper2(1,ystepPin,ydirPin);//Create stepper motor object ２
AccelStepper stepper3(1,zstepPin,zdirPin);//Create stepper motor object ３

elapsedMillis sinceTest1;
elapsedMillis sinceTest2;
elapsedMillis sinceTest3;

int state1 = 0;
int state2 = 0;
int ok = -1;
void flash() {
  if (state1 == 0){
    state1 = 1;
    stepper1.stop();
  }else{
    state1 = 0;
    stepper1.runSpeed();
  }
  Serial.println("Test1 (1 sec)");
}

void setup() {
  pinMode(xstepPin,OUTPUT);     // Arduino control DRV8825x stepper pins for output mode
  pinMode(xdirPin,OUTPUT);      // Arduino control DRV8825x direction pins for output mode
  pinMode(ystepPin,OUTPUT);     // Arduino control DRV8825y stepper pins for output mode
  pinMode(ydirPin,OUTPUT);      // Arduino control DRV8825y direction pins for output mode
  pinMode(zstepPin,OUTPUT);     //Arduino control DRV8825z stepper pins for output mode
  pinMode(zdirPin,OUTPUT);      // 
  
  pinMode(enablePin,OUTPUT);   //
  digitalWrite(enablePin,LOW); // 
                               // 
                                
                             
  stepper1.setMaxSpeed(3000.0);    
 

  stepper2.setMaxSpeed(3000.0);     
  stepper2.setSpeed(300.0); 

  stepper3.setMaxSpeed(3000.0);     
  stepper2.setSpeed(300.0);

}

void loop() {
  if (sinceTest1 >= 5000) {
    sinceTest1 = sinceTest1 - 5000;
  Serial.println("Test1 (1 sec)");
    if (state1 == 0){
      state1 = 1;
      stepper1.setSpeed(300.0);
    }else{
      state1 = 0;
      stepper1.setSpeed(0.0);
      sinceTest1 =4000;
    }
  }
  Serial.println("Test2 (1 sec)");
    if (state2 == 0){
      state2 = 1;
      stepper2.setSpeed(300.0);
    }else{
      state2 = 0;
      stepper2.setSpeed(0.0);
      sinceTest2 =4000;
    }
  stepper1.runSpeed(); 
  stepper2.runSpeed();
  stepper3.runSpeed();

}