1.Collaboration with Harvard
The grand iGEM Jamboree is coming, and a lot of teams have put out applications for various collaborations on the iGEM website. Fortunately, we got an opportunity to be part of the team Harvard (Fighting Stigma Against Synthetic Biology), and we also wanted to promote communication between us through the cooperation.
This year, Harvard wants to work with other teams to end the misunderstanding of synthetic biology. We found that human exposure to bacteria tends to have negative effects, which is as something dangerous. As an iGEM team, we understand that genetically engineered bacteria are largely untapped resources, helping us show the world the true potential of using such a bacteria for treatment, diagnosis, environment and so on.Harvard plans to release a series of articles to show all the cool things and germs the team is working on. Besides, they want introductions of other teams’projects, so we send ours to them on request.
Here is the introduction of our project. In the wake of the increasing greenhouse exerting more serious impacts on the global environment, the question of how to efficiently deal with CO2 is particularly critical. However, the traditional gas collection technology featured by high energy consumption and low efficiency is not yet mature enough. Meanwhile, it is these characteristics that have limited the development of itself.Our project is to obtain carbonic anhydrase (CA-2) with high thermal stability through computer simulation of the protein molecules expressed by carbonic anhydrase gene locus mutation, and import it into escherichia coli cells to achieve efficient collection of CO2.
We are honored to participate in this cooperation, and hope that through this channel we can reduce the misunderstanding of genetic engineering.
We noticed the cooperation request issued by Linking University's iGEM team (Linkoping_Sweden). Linkoping_Sweden has a new idea of cooperation.They want all the teams to use letters or emails to show interesting things happening inside and outside the team. So we participated in this cooperation. This is an interesting collaboration, as it may be boring during the experiment, but the stories that happen in the experiment make the experiment fun.And by organizing these stories, we learned more about our experiments.
3 . Callobration with NEU-China
On August, 2018, our team established a connection with iGEM group of NEU-China-A and thus a couple of video conferences have been made so far. The two teams introduced their own projects in detail; the project of NEU-China was to design a biological system against IBD and prevent CRC by using engineered probiotics. Since the concentration of NO in the intestines of IBD patients was about 100 times higher than that of normal people; NO molecules were chosen as inflammatory signals, and probiotics (Nissle 1917) were selected as chassis organisms. The project consisted of two sets of devices, one of which was an anti-inflammatory equipment, including a sensor for detecting inflammatory signals, an effective enhancer and an effector secreting interleukin 10 (il-10). Another one was an anti-cancer device,which is able to express a kind of myrosinase from cruciferous vegetables by virtue of engineering probiotics. Meanwhile, it can transfer glucosinolate, a natural ingredient in cruciferous vegetables into sulforaphane. Such a kind of product was a type of organic molecule with cancer-fighting ability and at the same time it could inhibit oxidative stress. Similarly, it could work against intestinal inflammation. In addition, we used cold shock kill switches (cold shock expression Lysis and maz-ef) to prevent our engineer bacteria from escaping into the environment and causing DNA contamination.
During exchange of project experience with each other, we learned that NEU-China-A had a little bit of a problem in one of the plasmid insertion experiments, so they asked us for help and wanted us to conduct the construction experiment of secreting the yebf-homoil10 plasmid. Our counterpart and we made it clear about what we were going to cooperate followed by three video meetings. NEU-China-A helped us carry out experiments on optimizing the conditions of prokaryotic expression of CSCA protein and purifying expression. In a similar way, we helped NEU-China-A perform fragment-linking experiment. NEU-China-A provided us with original plasmid of PCDF-Duet1 (dry powder tube), taught us to overlap the secretion label Yebf (from BL21(DE3)DNA, GenBank: EF648003.1) with human-derived interleukin 10 (from BBa_K554004, Spring 2018 Distribution 21K 2018 Kit Plate 1), and required us to add the FLAG label at the N end of homoIL10 for subsequent protein detection. According to the requirements of our friend, we conducted the experiment on construction of secreting the plasmid Yebf-homoIL10. Having sequenced and verified the plasmid construction successfully, we mailed no-less-than-100ng plasmid dry powder to them.
NEU-China-A helped us implement experiments on optimizing the conditions of prokaryotic expression of CSCA protein and purifying expression. Our friendship has thus been strengthened and the projects have also been pushed forward as the result of this collaboration, which is as a successful cooperation by both of us.
4 . IIT-Madras
As the project continues to proceed, HP's exchanges and cooperations between teams are more frequent and in-depth. After seeing the cooperation application issued by the iGEM team of the Indian Institute of Technology (IIT-Madras) at the iGEM official website, we contacted IIT-Madras to discuss cooperation actively.
We are pleased to collaborate with the iGEM team of the Indian Institute of Technology, Madras (IIT-Madras) to make a video on the science of synthetic biology in the mother tongue. The Indian Institute of Technology is also working with other teams in English, Tamil, Telugu, Kannada, Malayalam, Marathi, Bengali, Gujarati and Hindi.
IIT-Madras sends an English script to our team. We need to match the provided slideshow video with the provided text and add the translated text to the subtitles, and finally make it into Chinese video.
We thought it was a simple matter, but there were some problems at the beginning.The problems is summarized as follows:
1. The English script does not exactly match the sound in the video, and some paragraphs are missing.
2. English and Chinese belong to different language families. They are slightly different in grammar, and the word order is reversed in translation.
3. Some video pictures and sounds are out of sync and need to be adjusted.
4. Our team members are not familiar enough with the Indian accent in English, and some words cannot be completely accurate.
After consultation and repeated revisions by the iGEM team members of the Anhui University of Technology, four days later, the results were satisfactory and the video was sent to the iGEM team of the Indian Institute of Technology. We have established a good relationship with the iGEM team (IIT-Madras) of the Indian Institute of Technology, Madras.
With the successful completion of the HP cooperation, the Anhui University of Technology iGEM team (AHUT-CHINA) looks forward to more cooperation opportunities with the Indian Institute of Technology iGEM team (IIT-Madras).
5 . Cooperation with Nanjing University
With the shortening of the time, our iGEM team encountered difficulties. The plasmid solution used in the experiment procedure in interlab was not effective after many times of cultivation, so the next experiment could not be carried out. We communicated the results with other iGEM teams. Nanjing-China of Nanjing university knew about it and then provided the bacteria liquid we needed. Our team members went to the school of life science of Nanjing university to obtain the bacteria liquid .
With the help of Nanjing-China, we successfully carried out the next experiment of Interlab, and finally achieved success. We would like to express our hearty thanks to Nanjing-China for their help!
We contacted the iGEM team of Northeast Forestry University (NEFU-China) on August 2018, and our two teams shared each other some detailed descriptions of our projects .
NEFU modeling: They store information in a yeast, and the time of information storage is the key to the case. The information storage time (a yeast growth time) needs to consider some parameters ,such as the initial inputs of α yeast and a yeast, the concentration of a-factor, and the expression efficiency of Bax gene. Firstly, partial correlation analysis is used to analyze the local sensitivity of the complex relationship between parameters and results, which provides a basis for accurate and efficient modeling and reliability analysis of simulation results. The results of sensitivity analysis showed that the secretion amount of α-factor and the growth time of a yeast had a higher sensitivity . Therefore, α- factor is refined into dry powder, which excludes the influence on the information storage of other interference factors such as the growth of α yeast . Secondly, since the main body of case is that the two kinds of yeast cultured in the medium ,and the growth of α yeast has some effects on the growth of a yeast should also be taken into account. So the competition model and the gun battle model are established. Finally, they provided a web-based, fully interactive modeling platform that provided information for wet experiments.
According to the case process we guess that : the amount of information stored (a growth time of yeast) is related to the initial inputs of two yeasts, The amount of α-factorsecreteby α yeast, and the promoting efficiency of the promoter. Therefore, the information storage is set as the output value, and the other parameters are set as the input values.Then the partial correlation coefficient is performed to verify the accuracy of the verification and which input is most relevant to the output.
The relationship between the time of information storage and the amount of α factor, time, promoting efficiency, the amount of yeast, and amount of alpha yeast was determined by model 1. They divided the experiment into two parts. The first part is that: a yeast and alpha yeast compete for growth without secretion of alpha factor. The second part is that: in the case of α-factor secretion, the two yeast growth for fighting, and the death rate of α yeast is the expression rate of Bax gene, and the expression rate of Bax gene is related to the start efficiency of the Fig2c promoter. In order to measure the efficiency of the start of the Fig2c promoter, the promoter efficiency of the promoter was measured by the amount of fluorescence by inserting the fluorescent protein gene of EGFP.
Through the above experimental process, we have established two mathematical models:
The change trend of OD value of yeast with time was reviewed, taking the maximum value of the slope r1=r2
2) fighting model:
They putted in different proportions of yeast and calculated the information storage time by modeling. At the same time, the ratio of yeast can be predicted by different storage times. NEFU-China provided a web-based, fully interactive modeling platform that not only provided some information for their wet experiments, but also enabled future iGEM teams to build more efficiently based on our work.
In the laboratory,our team has simulated the exhaust gas ratio which can be found in literatures .The corresponding proportion of the gas was mixed manually and the gas was injected into water to prepare an unsaturated solution. The content of carbon dioxide in the solution was tested continuously according to the time of the gas entering, and this was used as the data source of our mathematical modeling.
First of all.By using MATLAB to conduct the correlation analysis of the experimental data,we found that the color readings (five dimensions: B, G, R, H, S) showed a certain linear correlation with the concentration of carbon dioxide.This conclusion is consistent with the other conclusion ,which obtains its conclusion by using lambert-beer's absorption law. That is, there is a certain relationship between the substance concentration and the color reading. Secondly, using the multiple regression of statistics to carry out regression analysis on the data, the relationship between the material concentration and the color reading (five-dimensional) is obtained, and the appropriate mathematical expression (or mathematical model) between them is determined as the empirical formula or regression equation.
A mathematical model for determining color readings and carbon dioxide concentration - a linear regression equation. Firstly, a linear regression model between carbon dioxide concentration and color reading is established. The residual of the model is large and the fitting effect is not good.
Considering establishing a nonlinear quadratic regression model.Using the rstool function modeling in the MATLAB statistical toolbox, and evaluating the pros and cons of the model by residual standard deviation and residual. In the final nonlinear quadratic regression model, the residual standard deviation is small, the prediction model is very good, and the residual of the model is reduced by an order of magnitude compared with the multiple linear regression model. Therefore, the linear quadratic regression model is better than the linear regression model. The comparison of the errors of the two models shows that the nonlinear regression quadratic equation has higher precision.
In the process of project communication with each other, we learned that they had a little trouble in modeling. It may be that Northeast Forestry University-China did not fully consider the influence of external factors on the experiment, which led to some errors in the model establishment. Through our tests, we found that the lethal efficiency of their calculations was wrong. After we found the problem, we told them in time and helped them improve the model.
The Northeast Forestry University iGEM team (NEFU-China) also helped us to test the safe reliability of our model. Through this cooperation, the friendship between our two teams was strengthened and the progress of the project was promoted. Both sides regard it as a fairly successful collaboration.
Thanks to the iGEM team of Northeast Forestry University (NEFU-China) for their help！