Team:NAU-CHINA/Integrated HP

NAU-iGEM

Suvery

Purpose

Promote the public's understanding of our project.
Accumulate the public’s feedback of our MARS.
Analyze the factors that influence the public's acceptance of our project.
Improve our design based on the results of our survey.

To achieve the purposes we mentioned above, a survey, with its objectivity, unity, and extensiveness, was taken into our consideration. And below is what we have done:

Preparation for our survey

In order to make our questionnaire scientific and reasonable, we consulted a professor of sociology, who owns expertise in questionnaire design. Then, we improved our survey design from three aspects:

1^st

Make it precise and objective. For clear understanding, we avoided using complicated or ambiguous words in question composition. Concise and objective expressions were carefully chosen in order not to influence the minds of the respondents.

2^nd

Make it scientific. The options in the questionnaire were set to be scientific, avoiding duplication and omissions.
Two identical questions with different orders of options were put in different places of the questionnaire to make sure of the validity of the data. Questionnaires with random or arbitrary answers to these two questions were abandoned in the subsequent statistical analysis.

3^rd

Make it anonymous. To protect the privacy of the respondents and to guarantee the reliability and validity of the feedback, the questionnaire was made anonymous.
Besides, to ensure our survey was ethical enough, all the 360 participants were required to give an informed consent before filling out the questionnaire.

Survey Design

We distributed our survey by Wenjuanwang and iGEMCloud, which is designed by NAU-CHINA 2018.

Our survey design is as follows:

Results

The response for the acceptance of MARS was variable, some thought it's positive and can help to stop malaria while others concern the genetic engineered bacteria may have a negative effect on the ecosystem.

The approval rates of different educational backgrounds are quite interesting. On the one hand, the rates of people who only received secondary degree education are less likely to agree with MARS (Only 62% of them approval MARS). We believe that an inadequate education may explain their reluctancy in understanding MARS. On the other hand, people who received higher education (Bachelors' degree or above), have much higher approval rates around 85%. This backs up our theory above very well.

From the results above we can see，people who or whose relatives ever had a severe disease, no matter malaria or not, show more acceptance of novel technology to treat diseases.

Researchers in biology and doctors find MARS much more approvable than people from other professions. This indicates that the more related the professions are, the more supportive the person is. Since many biological researchers' and doctors’ work has some correlations with deadly diseases, they knew how severe a fatal disease can be and the need of novel ways to control it. That would be the reason why more people of these two professions approve our project.

The total amount of MARS approval rate is about 83% and the disapproval rates is 17%. This is a quite positive result for us, for the supporters is 5 times more than the people who oppose.

Most people are not that satisfied with the current method, and they hope to find a better way to block this disease. Some people can accept the current method, though, they hope a new way can come up.

In order to assess the factors that influence a person’s likelihood of accepting MARS, we designed this question. As the survey indicates, if we want MARS to become more acceptable, the best option is to make sure MARS has a good effect on malaria control. What’s more, we can promote MARS to the public to make people understand it, especially its mechanism. To gain the government’s approval will also be a good choice. So far, no one says he will never accept our project.

As the picture demonstrated above, most people prefer male mosquitoes than female ones, since they don’t bite people and are unable to spread malaria. Female mosquitoes, on the other hand, are only approved by very few people. Besides, many people also agree to choose the one with a better effect.

It can be obviously observed from the chat above that most people agree with the idea to kill mosquitoes. We think the lack of knowledge that mosquitoes can multiply at a high speed after their numbers has been reduced is the main cause of this phenomenon. Thus, we decide to promote this in the following public promotion activities.

When the participants are asked what they like about MARS, most people applied that they think it is creative to use symbiotic bacteria to secrete anti-Plasmodium effector proteins. Some thinks this is much better than using pesticide for it is environmentally friendly. This is a quite promising result for MARS’ design.

Integration into Project Design

To gauge more acceptance, we made a video about our project, using the most accessible words to describe it and posted the video to the most popular social media in China（QQ）.

Besides, our mathematical model predicted that releasing male mosquitoes can get a better effect than releasing female mosquitoes, which is a good news for most of the public since nobody enjoys the biting of females mosquitoes (male mosquitoes do not bite people).

Future Plan:
1.Do experiment about the effects of releasing only female mosquitoes or male mosquitoes in the laboratory and verify the prediction of mathematical model.
2.Expand our promotion reach, with various kinds of media such as Wechat, blibli and televation.
3.Communicate with the people who do not approve our project. We will explain our project carefully to them and listen to their opinions.

Yunnan Institute of Parasitic Diseases

Why we visited Yunnan Institute of Parasitic Diseases?

We went to Yunnan Institute of Parasitic Diseases for it is the authoritative health department that eliminated the cases of malaria in Yunnan and had a deep understanding of malaria parasites. It implements the province's parasitic disease prevention and provides healthy and technical services to the society. The earliest system for the prevention and control of malaria in Yunnan is created in this institution. Yunnan, located on the southwestern border of China, was once a high-incidence area of malaria. The experience earned by the Parasitic Disease Control Institute offered references in the prevention and control of parasitic diseases.

What did we do there?

We consulted Director Henglin Yang and Director Hongning Zhou about the design and application of our project. Both of them were expert in malaria control. They had participated in the elimination of malaria in the Mekong River Basin in China, and achieved great success.

Figure 2. the Yunnan Institute of Parasitic Diseases

Feedback of project design

We initially wanted to choose E. coli as the sole biological chassis, considering its clear genetic background and easy to be operated. However, experts suggested that engineering the symbiotic bacteria in the midguts of mosquitoes could ensure a better colonization effect. Besides, they suggested that we could use several kinds of anti-Plasmodium peptides to kill the Plasmodium parasites since many of them have developed drug-resistance. Multiple effectors work together, making it a much more powerful weapon against Plasmodium.

Feedback of project application

They considered our project to be meaningful by providing a new way to solve the global problem. Besides, our idea could also be adopted to develop means to cut off the transmission of mosquito-borne infectious organisms such as Zika Virus.

Interview

As our project proceeds, we encountered some confusing problems for us to solve on our own. Thus, we consulted several professors about our project to make this system it work more efficiently.

Mosquitos: to be or not to be?

//Ai Xia
An associate professor from the College of Plant Protection in Nanjing Agricultural University
Primarily researches on insect pathogen in molecule level, especially the Anopheles sinensis. //

NAU-CHINA:
"To cut off the transmission of Plasmodium parasites, should we kill all the mosquitoes or only the Plasmodium inside them? Which choice can be more responsible for the world?"
Ai Xia:
"The latter would be better. Firstly, if we try to reduce the mosquitoes’ populations, we might cause a series of reactions that lead to a rapid growth of mosquitoes, which may not have the significant result in killing mosquitoes. Also, mosquitoes are part of mother nature, if we try to remove it, we might cause a series of reactions that might affect other species such as dragonflies, flogs, spiders and even birds.
I am not saying it will surely happen. But we should take every necessary measures to prevent these things from happening. It's also in line with the academic ethics we should have."
With this feedback, we set our project's main goal to kill only the Plasmodium inside of the mosquitoes, and therefore, MARS is born.

Figure 3. the College of Plant Protection of Nanjing Agricultural University (the Science and Experiment Building)

We want an efficient scissor

As the nine anti-Plasmodium peptides are co-expressed as a fusion protein, an effective and specific protease is expected.
//Yiping Wang
A professor from College of Life Science in Peking University Primarily working on nitrogen fixing bacteria, the coupling of carbon metabolism and nitrogen metabolism, and the prokaryotic secretion system.//

Figure 4. The College of Life Science of Peking University

Professor Wang looked into his files, and asked some details about our gene design. After a long and careful consideration, the Tobacco Etch Virus protease (TEVp) was chosen to cleave the fusion proteins for its specificity and efficiency in recognition and cleaving. This greatly perfect MARS.

CCIC

CCiC is the abbreviation of iGEM Exchange Conference in China. Every year, dozens of teams from different parts of China participate in it to get the feedback from experts and communicate with other teams. This year, CCiC was held in Shenzhen from August 19th to 23rd, with the guidance of the Professional Committee of Synthetic Biology, Shenzhen Institute of Technology and Chinese Academy of Sciences.

During the conference, we gave a Powerpoint presentation of our project, put up our posters and explained it to the other teams. Experts from different fields challenged us with some questions about our design and model. Their feedback reminded us of some points that we did not think of before, and inspired us to further improve our project.

Feedback for wet lab:

1.To figure out whether the engineered Serratia marscence will generate biosafety problems;
2.To clarify whether we need the fold-stabilized protein to keep the fusion protein of anti-Plasmodium peptides unfolded to avoid the condition that Tobacco Etch Virus protease(TEVp) cannot recognize the cleavage site;
3.To make engineered Serratia marscence colonize in the wild mosquitoes' midgut, and decide which would be better: to release infected mosquitoes or to use powdered sugar with engineered Serratia marscence.

Improvement in our project based on the suggestions above:

1. As for biosafety:

The specificity of anti-Plasmodium peptides had been taken into consideration when we designed and discussed our project. Several peptides have been documented to target only Plasmodium, but other peptides may have the possibility to harm the other organisms.
Serratia are abundant in the soil, however, our engineered Serratia marscence have a relatively large plasmid, which make it less competitive in the soil than the wild strains.
To put our project into practice, small-scale experiments are still needed. But we did not get the permission from iGEM HQ, who recommended only verifying that the system can work in Serratia. We adopted their suggestion.

2. Through the projection of bioinformatics, Tevp can recognizes the cleavage site.
Also, the cleavage effect of Tevp was proved to be good by analyzing our experimental result.
So, we do not have the fold-stabilized protein.

3.We chose to release the infected mosquitoes.
Releasing the infected mosquitoes is safer, since powdered sugar with engineered Serratia marscence may be eaten by the other insects, which can cause unnecessary effects on other organisms.
And the transmission efficiency is high by the projection of mathematical model.

Feedback for mathematical model:

Future plan:
Peptide expression model:
Suggestion: To simulate the expression of Anti-Plasmodium peptide better, not only a better simulation of the expressed protein molecules per bacterium is required, but also a relatively accurate estimation of the number of bacteria is needed.
Check: The experimental group provided us with the data of the amount of proteins secreted by a single Serratia marcescens.
By literature retrieval, we get the number of Serratia marcescens per mosquito. Then we investigate the amount of the S. marscence in mosquito’s midgut so that we can estimate the density of S. marscence.
Suggestion: To predict the expression efficiency of peptides, accurate promoter strength data is needed.
Check: By referring to cell biology by the numbers we get the range of transcription rate with different promoters. Three most effective promoters were selected, and our experimental group verified them.

Cellular automaton:
Suggestion:Besides functioning as simulating the epidemic feature of engineered bacteria, there are lots of other potential roles that cellular automaton model can play. For example, searching a better mosquito release strategy, and using parameter sensitivity analysis to give a certain orientation for the experiment group.
Improvement:
We set a series of release strategies, including: quantity of release spots, quantity of released mosquitoes, the mating of released mosquito, times of release. By testing combinations of these strategies, we finally obtained a well performed strategy.

Agar art

What is 'agar art'?

https://www.asm.org/Events/2019-ASM-Agar-Art-Contest/Home
“Have you ever seen art created in a petri dish using living, growing microorganisms? That's agar art! Creators use either naturally colorful microbes, like the red bacteria Serratia marcescens, or genetically modified microbes, like the yeast Saccharomyces cerevisiae transformed with violacein genes, as 'paint' and various types, shapes and sizes of agar as a 'canvas.' ”

This year, we held this meaningful competition in our school.

We gave the students a PowerPoint presentation to introduce Agar Art to them. They were free to team up, and every team was required to hand in one work. When preparing for their work, they were encouraged to retrieve and read articles by themselves. And excellent works were selected to exhibit on Wechat Official account. These excellent participants underwent our next stage of training which is provided by us to complete more outstanding works.

Hundreds of people participated in our competition and really enjoyed it. Many lovely works were submitted. “Study in Agricultural university, I am already accustomed doing experiment”, a student said, “but it is the first time that I find experiment can be an art.”