Best Environment Nomination!!
Partnership
Nanjing-China
WHY
Nanjing-China utilized Shewanella to bioconjugate silver nanoparticles (heavy
metal enrichment) to improve electrical conductivity. We knew each other since we
both intended to reduce silver ions to nano-silver for wastewater
treatment. Thus, our project’s theme is highly relevant.
However, the mechanism of reducing silver ion is totally different in
terms of peptide and metabolism accordingly.
ABOUT
The principle of is Nanjing-China’s project to use short peptides
to adsorb silver ions and then generate electrons through the metabolism of
Shewanella to reduce them so that the silver atoms are separated from the
short peptides and aggregated and mineralized into nano-silver
particles. NJU enriches the bacterial liquid biofilm on the electrode
to decompose organic wastewater and uses its decomposition to generate energy,
which is used as a microbial fuel cell.
EXPERIMENT
Share plasmid
When it comes to collaboration, XJTLU shared the sequence of silver ion
detection plasmid silver sensor-GFP in pGEX-4T-1 with NJU.
Please refer to part page from Nanjing-China.
E. coli growth curve measurement
We discussed with NJU the method for determining the growth curve of E. coli in the presence of silver ions. NJU recommends that we use a microplate reader to measure, thereby reducing the experimental workload of spectrophotometer testing. In addition, in order to prevent the absorbance from reaching 1.5 (due to the inaccurate reading of the microplate reader when the absorbance exceeds 1.5), NJU recommends that we dilute the bacterial solution to an absorbance of 0.05 before starting the culture and measuring the growth curve. We adopted the method of NJU, measured the absorbance of E. coli every two hours, and finally drew the growth curve of E. coli under silver ions, see the experimental result for details.
SEM nanosilver verification
We all use SEM to observe the morphology of nano-silver on the biofilm, and use EDS to analyze the elements, but we do not see silver lines. We asked NJU for possible reasons, and their SEM results. From their results, we know the distribution and morphology of nanosilver on the cell surface.
Competent bacteria preparation
In the experiment, we encountered an emergency when the competent kit (zymobroth reagent) was used up. Since the kit took two weeks to arrive, we borrowed the kit for preparing competent bacteria from NJU. NTU enthusiastically shared the method of directly preparing competent bacteria with calcium chloride. As a result, we successfully prepared competent bacteria according to the protocol provided by NTU.
Method for measuring silver ion binding to short peptides
We asked NJU about the method of measuring the amount of silver adsorption, and NJU shared the experimental method of silver binding protein affinity. NJU wanted to measure the absorption peaks of silver using ultraviolet spectroscopy, and determine the concentration of silver ions after reaction immobilization based on the absorption peaks. We exchanged the failed experience of measuring nano-silver by ultraviolet spectroscopy (the reactant contains short peptides, bacteria, culture medium, and it is not pure, so the absorption peak is not obvious). Finally, we recommend NJU to use ICP-MS to measure the content of silver ions in bacteria and supernatants, as well as the binding rate of reactive silver and short peptides.
MODELING
NJU also shared the SAD model of bacterial growth curve under
metal ion stress with corresponding codes in terms of bacterial growth curve
modeling.
HP and COMMUNICATION
We shared the effluent standard documents with NJU for
reference. We also held an online zoom meeting and
established a WeChat group for long-term communication.
In addition to working together, our hp team members have developed a deep friendships with students in the Nanjing University varsity team. We communicate with each other in all aspects of our studies, life, etc., and travel together when we have the opportunity. One of the fascinating features of iGEM is that it provides a platform for communication and expression, where people with similar souls can meet and become friends, which is part of the point of the competition.
Nanjing-NFLS
ABOUT
Nanjing_NFLS is committed to tackling algal toxin pollution. We came together because of our projects to deal with water pollution. Fortunately, the students in our team who are mainly in charge of HP are from Nanjing, China. This mutual understanding is another opportunity for us to achieve full cooperation.
MENTORSHIP
We conducted mentorship with Nanjing_NFLS on modeling. We first recommended some books about modeling to Nanjing_NFLS, including “Introduction to Synthetic Biology: About Modeling, Computing and Circuit Design” and “Fundamentals of Systems Biology: From Synthetic Circuits to Whole-cell Models”. We then sent Nanjing_NFLS videos of our modeling workshops that covered introductory material like Law of Mass Action, Michaelis-Menten Equation, and Hill Equation, which supplied NFLS with basic knowledge and helped them construct the foundation of our model.
Meanwhile, NFLS provided us feedback on the lectures on how they could be improved to adapt to beginners. They suggested starting each section with the basis behind each equation, such as the background in high school biology and chemistry, or providing a list of “prerequisite” topics that may be useful in understanding this section. They also noted that analogies or connections with prior knowledge would be very helpful in directing the listener to a comfortable and familiar mindset. Their feedback help us a lot in terms of further use of the materials for education in future seasons!
We also helped NFLS troubleshoot in an online Q & A session, and point out directions for further work. NFLS explained to us that when building their model, one of the parameters was different from the values presented in previous literature, and they could not find the reason to account for it. On this situation, we assisted NFLS at identifying a difference in the experiment settings and the environment surrounding the enzyme. We offered suggestions on how to fully address the assumptions behind each model, and how to reasonably interpret the model’s results. XJTLU-CHINA’s input further validified our model and its compatibility with our experimental conditions. We hope our feedback would help NFLS validiy their model and its compatibility with experimental conditions.
FBA
Water environment and biological solutions have been a big part of iGEM projects yearly. Nanjing_NFLS and XJTLU-CHINA believed that by assembling more teams in this area, we would be able to generate rigorous impact both in public education and in contributing to the iGEM environment community. Therefore, we co-initiated with the Freshwater Bioremediation Alliance.
Nanjing_NFLS wrote the first promotion article and went on point-to-point invitations with other teams. We successfully organized 5 other teams, including CHINA-FAFU, UM_Macau, GXU-China, Nanjing-China, and NPU-CHINA. We then hosted the 1st meetup for each team to introduce their projects and explore further opportunities.
CO-INTERVIEW
We also co-interviewed professor Shen from the Nanjing Institute of Water Resources. She told us people tend to use ionic complexation, and sedimentation to remove the heavy metal ions. Through exchanges with experts, we have a more comprehensive understanding of the application of biological methods to treat water pollution in different regions, climates, and environments in real life.
BIOSAFETY MEETING
We host biosafety conferences in the freshwater bioremediation alliance to discuss options for dealing with biosafety issues with various teams. After the meeting, we made a handbook to record the plans of each team and the main content of the meeting. This handbook can be referred to by the following teams as a contribution to iGEM.
QUESTIONNAIRE
In addition, we discussed questionnaire design specifications and helped each other forward questionnaires to expand our target audience. High school teams have larger secondary and elementary audiences. Therefore, the age group of the questionnaire audience will be more balanced so as to improve its reliability.
DKU
WHY
We discussed with DKU mostly on projects. Due to the proximity, the members of DKU visited XJTLU and we developed friendships with each other.
ABOUT & EXPERIMENT
Experimental questions & Advice From XJTLU:
We discussed the specific design of our plasmids. Specifically, it's about how and why they choose each part of the plasmid for surface display.
DKU applied three microorganisms and the whole team was divided into three groups, each responsible for E. coli, Yeast cells, and Lactococcus lactis. All groups applied a strategy that conjugates the targeted nanobody with a surface protein that is to be expressed, transported, and located in the extracellular space. The surface protein and the targeted nanobody were connected with a linker sequence, and each nanobody is marked with at least one protein tag for their identification. We also provided links to igem parts so they can check it directly.
Experimental questions & Advice From DKU:
DKU is curious about whether the biofilter can be blocked due to E. coli proliferation.
We replied that the microorganisms are immobilized in filters, and the filtration condition may not allow E. coli to grow at such a speed. Moreover, the CsgA protein's ability to adsorb heavy metal ions under different conditions (like pH) can be tested through experiments.
MODELING
We exchange ideas on models for bacteria growth. We developed a model for predicting bacteria growth, and it may also be applied to DKU’s situation by adjusting some of the parameters.
The main topic focuses on the quantified expression of csg through controlling IPTG concentration.
We intended to control the expression of csg protein by adjusting the amount of IPTG inducer, which can also be a helpful approach for DKU to check the relationship between the cell-cell adhesion affinity and the number of expressed nanobodies. However, as DKU also applied the TlpA promotor, it may influence the actual expression to a great extent and cause a big difference from the theoretical calculation. DKU Fully considered our modeling ideas with their project.
SAFETY
DKU advised that the heavy metal ions will remain captured, as protein itself has a great affinity to the heavy metal ions. However, it remains to be seen whether the released protein will leak out from the filter.
EDUCATION
We talked about how to interact with and educate the public. They told us they would make questionnaires to evaluate their knowledge related to antibiotics, probiotics, and microorganisms. They were also planning to make some handouts and deliver a lecture about it in high school.
