In order to better integrate the input of stakeholders into our project, we are combining the Logic Flow and Reaction Timeline. The Logic Flow illustrates the roles of each section and The timeline highlights various interactions with stakeholders, including field research, experiments, advice, and decisions made during the project. It serves as a valuable record of how your team engaged with experts and integrated their feedback into the project.
Each section plays a crucial role in different
phases of the project, from understanding the
problem to defining solutions, ideating solutions, and
ultimately implementing them. Together, these sections form
a comprehensive framework for project development.
• Emphasizes the significance of the team's efforts to deeply comprehend the issues related to the project, ensuring solutions are based on real-world problems.
• Illustrates how the team sought the best solution to address the problem, considering cost-effectiveness and feasibility.
• Describes the team's collaboration with experts to refine experimental methods and explore new solutions to ensure project success.
• Focuses on how solutions were put into practice, including hardware selection and the actual implementation plan.
Each section plays a crucial role in different phases of the project, from understanding the problem to defining solutions, ideating solutions, and ultimately implementing them. Together, these sections form a comprehensive framework for project development.
The perspectives of our stakeholders are intrinsically connected to our project, and the goals that we need to accomplish in the end are directly determined by their suggestions. The guidance that they provide is also very important, and the project that we are working on will take their thoughts into consideration when making decisions. As a result, we developed a reaction timeline to demonstrate how we incorporated the ideas and suggestions of stakeholders into our project and how we put those ideas and proposals into action as the project proceeded.
Our team engaged in an online conversation with Mr. Pan, a government official, to understand their views on using synthetic biology for wastewater treatment, gaining insights into local industrial pollution challenges.
We had an online conversation with Mr. Pan, the director of
the Supervisory Bureau, in order to find out what
the governmental departments think about the application
of synthetic biology to the process of treating
wastewater. During this conversation, we shared
our project with him, and at the same time, we learned from
him about the current focus of our country's
wastewater treatment, as well as the current situation
of local wastewater treatment. In order to find
out what the governmental departments think, we had this
online conversation. He reflected to us that because the
local area is a developed area with an industrial
smelting sector, the local area has been
dealing with a significant amount of difficulty as a result
of the industrial pollution created by the
metallurgy sector. The metallurgical factories
that fall under the authority of the government are all
located in the same industrial park for the convenience of
both the government and the businesses who operate them.
Because of this, the sewage treatment plant that is located
in the industrial park is going to be put through a rigorous
examination. Not only that, but he also noted that cadmium
and arsenic are the most significant types of metal
contamination in the area. After he saw our project, he
considered that the project concept was very nice, and from
the government's point of view, the existing usage
of sewage treatment technologies, including the use of
chemicals, may stabilize the problem of ionic pollution,
but they cannot make the environment entirely
pollution-free. He had high hopes
thatour project would be able to boost the
adsorption rate while simultaneously enhancing the
project's stability. Concerning the issue of
the biosafety of synthetic biology, he thinks that the first
thing that needs to be solved is the leakage problem
that may be caused by the engineered bacteria,
and he hopes that the project will be able to make relevant
limitations on it, so that it can contribute to the goal of
solving the environment with zero pollution. In other words,
he believes that the first thing that needs to be solved is
the problem that may be caused by the engineered bacteria.
Initial field research at the Nanjing City South Wastewater Treatment Plant revealed the challenges of dealing with heavy metal ions in industrial wastewater and the role of microorganisms in conventional sewage treatment.
Our first field research is for the Nanjing City
South Wastewater Treatment Plant. Before we
went in, we did not expect in the domestic sewage in
the heavy metal ions in the low content, and
this wastewater treatment plant as a specialized treatment
of domestic sewage treatment plant, the overall purification
of sewage process is very perfect. At the same time, it is
important to have a basic understanding of the role
that microorganisms play in the conventional
treatment process for sewage, which is to operate as a
removal mechanism for nitrogen and phosphorus present. They
also indicated to us that industrial wastewater, during the
process of sewage treatment of microorganisms, the impact is
more severe due to the concentration of heavy
metals, pH, and temperature, and that even the smallest
mistake can result in death. If we want to use
microorganisms to tackle the problem of heavy metal
ions, then we need to pay attention to the problems that
have been discussed above.
Feedback from Mr. Xiao confirmed the relevance of the team's project in the market, focusing on the importance of eliminating heavy metals in electroplating wastewater within government-mandated limits.
As a result of our research on the industrial wastewater
treatment process utilized by this company, we have realized
that the membrane that we aim to manufacture will be
very comparable to the membrane that is presently being
utilized within the plant. That is, RO
membrane, and RO membrane is more
expensive in comparison to the items that we
create, both in terms of its cost and its
efficiency. The cost is always the first
issue to consider when determining the
standard, and the plating plant that they recommended to us
is the current factory that uses both chemical and physical
methods to precipitate heavy metals. Despite the fact that
there are situations when total absorption does not occur.
However, the cost of sludge that is created by the
treatment of sewage will fluctuate depending on the
content of heavy metals. When the concentration
of metals rises, the cost of sludge treatment will be
reduced instead; therefore, he believes that our
algorithm for cost can be evaluated from a different
perspective. During this time, we were able to
communicate with Mr. Xiao, the principal designer, and he
stated that the method of ion membrane adsorption
that is now in use should serve as the counterpart to
our product. Because of this, we were able to
validate our position in the relevant
market. Additionally, the electroplating plant
informed us that this was for the treatment of wastewater
from electroplating operations. The elimination
efficiency is particularly crucial, and it must
be less than 1 microgram per liter; otherwise, in accordance
with the government's rule, each instance of exceeding the
requirement will result in shutdown.
The Sanyou Wastewater Treatment facility, responsible for industrial and domestic wastewater in an industrial park, is impacted by weather, with heavy metal contamination from the environment, and integrating a project in the final treatment stage is proposed for achieving near-zero discharge.
The Sanyou Wastewater Treatment facility is the primary
wastewater treatment facility for the nearby industrial
park. It is responsible for the treatment of all wastewater
generated inside the industrial park, including both
industrial and domestic wastewater. Through extensive
conversation with Mr. Tang, who is the plant's
director, as well as studies conducted in the
field. We discovered that the weather
is not only responsible for the delivery of sewage but
also for the creation of sewage itself. When
there is a significant amount of precipitation, the
heavy metals that are in the air and on the ground will
fall into the water, then travel via the sewer
pipes to the sewage treatment facility, where they will
undergo centralized treatment. Heavy metals can also
be found in the soil. He is of the opinion that our
project is suited to be incorporated into the final
stage before the outlet. This would allow the
concentration of ions that cannot be entirely
treated by current treatment methods to be
further adsorbed with our goods, so reaching essentially
zero discharge. He believes that this would be the best way
to accomplish this goal.
To accurately measure metal ion concentrations, the team consulted Professor Zheng Chen, considering the use of an inductively coupled plasma-mass spectrometer (ICP-MS).
Because our biosorbent is capable of lowering the
concentration of metals in the water to an extremely low
level, we were interested in determining the
precise concentration of metals in the wastewater that is
processed by our facility. We asked Professor Zheng
Chen of the College of Environmental Sciences,
a senior researcher who has studied heavy metals in soil,
about the problem we want to solve this year, and he thought
that there is a research instrument in the College of
Environmental Sciences that can help us accurately measure
the content of metals in water. This is because
ordinary instruments for determining the content of
metals in water are ambiguous and may not be
able to measure them effectively. This instrument, known as
an inductively coupled plasma-mass spectrometer (or ICP-MS
for short), is capable of making precise
measurements of the concentration of metal
ions. This allows us to establish whether or not the ions
that we are able to absorb meet the standards for emission.
Challenges arose in growing different strains for experiments, and consultation with Professor Zhu Yongtao helped adjust shaking times and plate scratching frequencies to ensure successful experiments.
It frequently occurred that the strains were unable to grow
vigorously during the implementation of our
experimental design, and while we were
conducting the experiments, the OD600 exceeded 0.8
on several occasions. This was due to the fact
that different strains are located in different growth
environments, which is why we tested different strains in
terms of the adsorbed ions. In addition, we hoped to
determine a highest protein expression by using de-expressed
proteins of different strains. However, because different
strains are located in different environments, Because of
this, we consulted with Professor Zhu Yongtao, and with his
assistance, we reduced the amount of time that the
bed was shaken in order to maintain an OD600 of
approximately 0.6, and we decreased the frequency with
which the plate was scratched to once every two
days, both of which contributed to the
successful completion of the experiment.
Precision issues in determining protein expression led to consulting Professor Peter, who suggested using Congo red staining for clarity in identifying the strain with the highest protein expression.
When we attempted to determine the level of protein expression produced by the strain, we ran into the issue of lack of precision. Additionally, the liquid medium had a tendency to interfere with the value of the measurement of protein expression. Therefore, we sought assistance from Professor Peter of Harvard University, who provided us with a number of different approaches to decision. Both the Congo red staining method and the Thioflavin T (fluorescent dye) approach can be utilized to get the desired results. Thioflavin T is able to do quantitative analysis more accurately than the others. The Congo red staining method is also simpler and more expedient. In addition to that, he suggested that we make use of PBS buffer. PBS buffer contributes to a reduction in the amount of non-specific CR binding. He proposed that we utilize cells scraped immediately off the induction agar plate and resuspend them directly into PBS+CR in order to limit the influence of liquid media's interference on the results of the measurement. This would allow us to keep the results of the test as accurate as possible. The interference caused by the liquid medium might be reduced as a result of this. Following the advice of Prof. Peter, we decided to adopt the Congo red staining approach in the end since it was the method that provided us with the clearest picture of which strain had the highest level of protein expression while also being the most time and labor efficient. The answer to this question will tell us which strain to utilize as our strain to produce protein.
Questions about protein structure led to consulting Dr. Han Lanlan, who believed that adding cysteines at specific positions wouldn't form disulfide bonds due to their distance.
We planned to introduce mutations into the Smta
protein, mostly by adding cysteines to the
positions Arg26, Lys8, and Lys22, and Lys445
respectively. Therefore, our modeling group
used alphafold2 to make predictions about it; however, after
observing the structure of the protein both before and after
the mutation, we came up with a new question: we
couldn't be certain whether or not these sites of the
protein would form new disulfide bonds after they were
changed to cysteines; this was a problem for
us. Because of this, the structure of our protein
would be altered; thus, we questioned Dr. Han
Lanlan, who was of the opinion that it is highly improbable
that the mutation of these sites will result in the
formation of disulfide bonds and influence the binding
energy. because of the great distance that separates
each of these locations from the others. There
is a very slim possibility that disulfide links will
be formed, which would have an effect on the
structure of the protein. In addition, he advised
that we replace the amino acids at positions 16-20 and
then 46-50 with cysteines to see whether or not
the structure of the protein can be flipped so as to
increase the number of binding sites.
Discussions with Mr. Xiao influenced the selection of polycarbonate and cellulose acetate membranes, taking into account impact resistance and cost-effectiveness.
Regarding the selection of membranes, we envisioned using a design that was analogous to that of RO membranes; however, we were still uncertain as to how exactly to determine which membrane would serve as a carrier for our proteins. Therefore, we had a conversation with Mr. Xiao, the chief engineer of Taicang Huihu Electroplating Plant. He informed us that while selecting a membrane, one must take into account both the cost-effectiveness of the membrane and the realistic application situations that may occur in the wastewater. For instance, the water flow for the impact of the membrane during the process of sewage treatment. Because of this, the membrane needs to have a high impact toughness in order to be effective. At the same time, the sewage is full of chemicals that cause corrosion; hence, the membrane also needs to have a particular level of resistance to chemical corrosion. Therefore, as a result of a combination of the factors discussed above, our Noble ious metal membrane carrier chose polycarbonate membrane. He possesses good impact stability and chemical corrosion resistance, and his aging resistance is strong; it can be reused to cut costs, and it can be extracted after the Nble metals have been absorbed and then reused. Heavy metals can be eluted and utilized out of the cellulose acetate membrane, which not only has a high tensile strength and flexibility but also has a low cost and the ability to naturally decompose in the environment. Cellulose acetate membranes are the vehicle for transporting heavy metals.
Conversations with Mr. Tang clarified hardware design principles, such as double pipes, and the importance of water flow direction in wastewater treatment, informing the team's hardware implementation.
What sort of hardware operation principle is a tough matter for us, so we spoke with Tang, the director of Sanyou Environmental Protection, who explained to us that they use a kind of hardware equipment in the factory that adopts the design of double pipes. As a result of this consultation, we now know that they use a kind of hardware equipment that operates on the principle of double pipes. The water that is treated and flowing through the outer pipe is under low pressure, while the sewage that is flowing through the inner pipe is under high pressure. A microfiltration membrane, which is used to clean the wastewater, is positioned on the surface of the inner pipe of the system. After going through this procedure, the sewage will be able to be treated in a manner that is more effective. However, Mr. Tang also remarked to us that we have to pay attention to the direction in which the water is flowing, as it is likely that a cycle will not entirely clean the water, and the effect of the water flow makes it simple to wash away the protein. He cautioned us to pay attention to the flow of water. As a result, the hardware component of our project has benefited from the knowledge gained from the device that treats sewage and has been motivated by its design. We use two waterproof motors, one rotating counterclockwise and the other rotating clockwise, in order to achieve the continuous circulation of water in the hardware. At the same time, in order to prevent the protein from being washed away by the water, we use a porous material to sandwich the protein membrane, in order to prevent it from being washed away by the water flow. This allows us to achieve both of these goals simultaneously.
The feedback that we receive from our stakeholders and the help that is generated for our project will be utilized to direct the implementation of stage 1 of our project, and this feedback will also be used to improve our experiments, modeling, and other aspects of the project.
XJTLU-CHINA 2023 shows all the stakeholders that may affect the project as well as the output and input they may cause to our project through HPcycle(figure1), and also through the simultaneous labelling of each line with the specific aspects that they affect, in order to solve this problem which is closely related to human beings. This is done in order to solve this problem which is closely related to human beings. This offers a more all-encompassing perspective of each stakeholder. We developed a Reaction timeline in order to include their suggestions, and we utilized the Kano model(figure2) to prioritize their requirements and establish the order in which our answers were to be prioritized in order to guarantee that we had a complete understanding of their requirements.
This cycle reflects the crucial roles played by these
stakeholders in the project, providing
input, guidance, and inspiration, while also benefiting
from the project's development and addressing
environmental issues. Additionally, the cycle
provides a clearer depiction of the relationship between our
project and the stakeholders.
Figure1(The use of arrows and labels shows the relationship
between the stakeholder and the project)
Expert is the advisor who provides academic guidance to our projects and provides timely help and insight when we want to take a practical step forward or when we have new ideas to share and get advice on. They serve as the main input to our team.
Whenever we run into difficulties with our experiments and modeling and hardware, we turn to expert for help. When they make a suggestion to us, we evaluate the suggestion in terms of its impact on the values of our project and explore how it can be incorporated into the project to enhance and improve our project.
Government laws give benchmarks for the purpose of our entire project, and our project is required to meet the national standards based on the relevant laws as the goal to provide the direction of the project's efforts. At the same time, to achieve true industrialization, understanding government regulations and communicating with government professionals can also enhance the progress of project engineering. Our project will also enable the government to provide updated insights into the direction of synthetic biology applications. They play not only the role of input but also the role of output to our team.
The objectives of the project will be to achieve maximum coincidence and parallelism with the regulatory emission standards. At the same time, as our projects mature, we will consult with government authorities and experts to ensure that our projects do not violate legal and biosafety laws, and that they enhance the substantive problem-solving capabilities of our projects. They will also give us commercial advice and insights. At the same time, we use our own project as an output to make the government pay attention to the problem that our project is trying to solve, so as to raise the attention to the problem itself and to the potential of synthetic biology applications in reality.
They are the beneficiaries of our projects, and we have to learn from them and understand their needs in order to successfully commercialize our projects. They are also the people we learn from and the source of our inspiration. We know that if we want to make a product that really works, we must have a deep knowledge of the various aspects of the wastewater treatment process that we want to embed in the product of our project. The field study of the wastewater treatment plant also helped us to deepen our knowledge of the market position, and the experiments and hardware were able to provide direct input for improvement. The wastewater treatment plant plays both an input and output role in our project.
While communicating with the wastewater treatment plant, we have a meticulous investigation of the treatment aspects of the plant, so that each step of the plant is taken into account. Also with the current treatment experience of the wastewater treatment plant and the inspiration it provides, thus making the industrialization of the hardware more perfect. And through the research of the treatment process of the sewage treatment plant, the real environment of the sewage treatment is fully considered, so that the design of the experiment can be better, so that it can really be put into the sewage to use. And after the initial results of the experiments are produced, we will also return to them again to determine whether our project is mature enough to be put into use, or to seek further suggestions for improvement.
The public living in the surrounding watersheds and watersheds are the most directly affected by our project; they are dependent on the environment of the surrounding watersheds and will be directly affected by the increase and decrease of pollution in the watersheds. Synthetic biology is uniquely suited to address this issue. Through their attitudes towards synthetic biology, we can** get their opinions on how to deal with the problem.** We can also promote synthetic biology to them, which will increase the acceptance of synthetic biology to the general public. They play an input role in our program.
Our projects take into account the local environment and the different priorities of the public for the problems to be solved, so the solutions we need to adopt will also change. We use actual interviews to communicate with the public in different directions. We will use actual interviews with the public from different directions to better understand what they really need and how to deal with it. We will also collect different opinions and approaches to synthetic biology from them.
When it comes to a project, one of the first things that has
to be prioritized is the requirement to get our
product finished and promoted in a targeted manner
within the time limitations as soon as
possible. For this reason, it is imperative that we put the
requirements of our stakeholders first. As a result, we
utilized the KANO model(Figure) to conduct an
in-depth analysis of each requirement and organize
it according to one of four categories. In
order to determine which of these four
characteristics are more important, the
Better-Worse coefficient was applied to the
data
When this kind of capability is offered, a rise in user satisfaction is shown by a high value for the Better category as well as a high absolute value for the Worse category in Quadrant I. Therefore, characteristics that fulfill requirements that fall under this quadrant are desirable.
The second quadrant has a high value of Better and a low absolute value of Worse, which indicates that the level of customer satisfaction will greatly rise when this kind of capability is made available. Therefore, needs that fall under this quadrant are considered to be glamorous characteristics.
A low value for Better and a low absolute value for Worse in the third quadrant imply that there will not be a significant difference in the level of user satisfaction with or without this sort of feature; hence, the criteria that fall within this quadrant are considered to be features that make no difference.
Because the Better value in Quadrant 4 is low and the Worse value is high, which indicates that customer happiness would considerably drop if this sort of feature is not given, requirements falling inside this quadrant are essential qualities to have.
To summarise, for the requirements in Quadrant IV, we have the highest priority because users will not buy our product if they are not completed. Moving on to Quadrant II, completing these requirements will make our product substantially more attractive to users, which will promote our project for the better. After completing the requirements in these two quadrants, then we can consider the first quadrant, and for the third quadrant, we can exclude these requirements because users are dispensable for these requirements. We hope that more iGEMers will use this methodology to better classify different requirements.