Team:XJTU-China/Contribution
Contribution
Part
Overview
This year, XJTU-China does the literature review about the existing part (BBa_K3196028) held in the iGEM part repository. BBa_K3196028 is a low-pH induced promoter, Pgas, originally registered by iGEM19_HUST-China, and XJTU-China appends its functional parameters characterized by Yin et al. (2017), to the footer of the original webpage. Below exhibits the information we add on.
Functional Parameters of Pgas
As is shown in figure below (Yin et al., 2017), the strength of Pgas is mainly dependent on the pH, while limited influenced by the types of acids.
Fig. 1 Factors affecting
relative Pgas-inducing capacity. (A) Influence of acid
type on
Pgas-inducing capacity. (B) Influence of citrate concentration on
Pgas-inducing capacity. (C) Fold changes in CAD expression at
different pHs. Error bars represent the standard errors of the means
of results for three biological replicates.
The author gives an equation with linear regression analysis to describe the relationship between the relative expression level and pH:
log(E)=[(6.042/pH)-0.9059]
in which, E represents the relative expression level compared with expression at pH 7.0. The correlation coefficient factor R2 is 0.9962.
Hardware
1. Overview
In order to realize the coordination of hardware circuit and gene circuit, we have made an automatic culture device. At the same time of detecting the growth and production status of bacteria, the device can feedback and adjust the conditions of culture, thus controlling the toggle-switch circuit to allow cells to enter different states between “proliferation” and “production”. Through the fitting of experimental results and modeling prediction, we can calculate the best time to change the cultivation conditions, and write it into the control program to realize the automatic control of the production process.
2. Design
Fig. 2.1 The design of
hardware
2.1 Detecting Module
We use a simple spectroscopic device to monitor the cell density and the concentration of tryptophan in the medium. Our detecting module includes two sets of tungsten light sources, filters of the corresponding wavelength and CCDs. By measuring the absorbance of light filtered to 600nm wavelength, the cell density can be represented. While with the presence of tryptophan detection circuit, the light passing through the 485nm optical filter can excite GFP and its emission light will be detected by the CCD after a 510nm filter. After receiving by CCDs, all optical signals of this module are converted into corresponding circuit signals and transmitted to the control module for processing.
2.2 Controlling Module
This module contains a single chip microcomputer (SCM) with its controlling program, controlling the parts in cultivation module by receiving and analyzing the signals from detecting module. After receiving the signal, according to the program written into the SCM, it can calculate the state of the cell density and product concentration in the culture medium. When certain conditions are met, corresponding instructions are issued to control the temperature of culture medium and the pumping of inducer, with the information fed back to users in real time.
2.3 Cultivation Module
All fermentation and culture conditions are provided by this module. The electric heater and ventilator maintain the temperature of the incubator and are controlled by the SCM to switch the temperature between 37 and 42 degrees Celsius. A ration pump can be used to add IPTG to the medium upon receiving the SCM signal. The main part is a sterile tank made of plexiglass, including a transparent and equal thickness area for spectrophotometry and fluorescence detection. Plus, magnetic stirrer, ventilation device and other devices for cultivation are also contained in this module.
3. Program
The core function of this module is realized by STM32 single chip microcomputer. The logic control program is designed as follows: the digital temperature controller continuously monitors the temperature changes in the container, and keeps communicating with the micro controller. Micro controller controls the relay to turn on the light source and photocell briefly every minute to detect the growth of biological community in the container. If the electromagnetic wave of 600 nanometer wavelength reaches a certain threshold, the micro controller opens the relay of the electric heating rod through the digital temperature controller; If the wavelength of 485 nanometer to 510 nanometer, the micro controller through the digital temperature controller to disconnect the relay of the electric heating rod, and prompt to replace the solution.
4. Showcase
Worked by our skillful teammates in hardware group, the prototype of our hardware is built and tested. We record and document a clip of video of this machine, upload it to iGEM repository, and append it below.