Overview

We strongly believe that the overall idea of our project — to develop a pathogen-specific drug and a pathogen-specific drug delivery system, worth of further investigation by future iGEM teams. Approaches related to this idea may reduce many infections caused by drug-resistant bacteria and antibiotic misuse. In addition, the successful production of AMPC in E. coli indicates that this approach may be useful for the production of peptides generally toxic to E. coli using common recombinant DNA technologies. The microneedle-based drug delivery system constructed in this project can be a useful platform for targeting diseases not only S. aureus but also many other diseases. Lastly, our troubleshooting notes and python code for future iGEM teams can help them quickly understand the model-building skills and directions.

AMPC - Microneedle System

We constructed the AMPC-microneedle system by adding AMPs as drugs and HAMA coated on the microneedle surface, providing a platform for targeting diseases caused by S. aureus. This also proposes the system for future iGEM teams to manufacture drug delivery strategies for curing other diseases. In addition, the strategies with the specific cleavage sites were designed to convert inactive and active states for the AMP, providing a means for the iGEM team to prepare for future AMP sterilization.

The Strategies for Different Condition

NTHU_Taiwan constructed a “trigger release system”. In Fig.1 there are two stages in one system. For future iGEM teams, they can design the linker for different pathogens or using specific materials to build a pathogen-specific drug or pathogen-specific drug delivery system for different condition.

Fig.1 We proposed the system for future iGEM teams to manufacture drug delivery strategies for curing other diseases.

Fig.2 Solve different condition by replacing different film and linker

Antibiotics Overuse Issue

Critically, we tried to solve the issue of antibiotics overuse, with the development of precision medicine which is controlled by microneedles. It has been identified as a forward-looking approach to achieving optimal personal health decisions, antimicrobial peptide complex (AMPC) should be the better therapy.

Environmentally Friendly Device

The AMPC-Microneedle are eco-friendly device. All parts of device are constructed by biocompatible material, such as gelatin, hyaluronic acid, CMC, AMPs and etc.

Replace sharp injection devices with microneedle patches can reduce the possibility of injury for recycling.

Troubleshooting Notes

Wiki

The GitLab platform is new to iGEMers this year and considering that there may be many iGEMers don’t have experience in coding. We hope some of our solutions for errors can help future teams. If you want to see how to make those amazing animation or blocks, please refer to our GitLab repository. The structure of our code and some comments may help you code smoothly.

Highly suggest using Ubuntu or a terminal to run the code locally. The steps for setting up the environment and running codes are based on the steps of iGEM official but with more details. Note that this tutorial is using Linux instructions in Ubuntu.
1. Copy the path of the repository you want to clone.
2. Right click on where you want to clone the files into and open the git bash.
3. (in git bash) git clone https://gitlab.igem.org/2022/nthu-taiwan
4. (in Ubuntu open the location you clone the path into) cd /mnt/c/iGEM2022/wiki/nthu-taiwan
5. (do as root) sudo -s
6. python3 -m venv venv
7. . venv/bin/activate
8. pip install -r dependencies.txt
9. python3 app.py
If you change the code when executing, press Ctrl+C to end the process and do step 8 again to run again to see the result of your change.
If encounter error code like this when doing python3 -m venv venv:
E: Could not open lock file /var/lib/dpkg/lock - open (13: Permission denied)
E: Unable to lock the administration directory (/var/lib/dpkg/), are you root?
1. Check if the python version is higher than 3
2. Try sudo -s to be root to do the step
3. Next, try apt-get update
If the slide in slideshow disappears after 1 second in view, but shows when clicking next or prev:
1. Try comment those codes about .fade or .fade.in. in your modified .css file because the bootstrap.css file had a couple rules for them.
Make good use of “vw” or “vh” rather than “px” when defining width or length
1. It is convenient that “vw” and “vh” can change the size of the element when changing the browser size easily.
2. Remember to check if the element messed up when change to smartphone view.

Protein Docking

For a beginner in the field of protein analysis, we strongly suggest that they find a full and step-by-step tutorial online. On ResearchGate, there may be lots of people who have the same question as you and can find useful answers there. We put the corresponding YouTube channel in reference. These are the errors and questions we met during our work.

Autodock error: autodock4: too few values read in. Check grid map 'a'.
1. Check if you remove the ions and do the right steps in the protein preparation step.
2. Check if the grid box is set in a reasonable size and position.
3. Try more positions and sizes or search for papers to set the grid box of the protein.
With a very high positive energy of binding in Autodock?
1. Check if the grid box is set in a reasonable size and position.
2. Sometimes it's not an error but has to change the ligand or grid box.
Autodock error: V8protease:A:HOH1000:O and V8protease:A:HOH1000:O have the same coordinates
1. Maybe you did something wrong in the step of cleaning the protein or protein preparation.
2. Check if you remove the ions and water in the protein preparation step.

Building Mathematical Models

Fig 2. iGEM Engineering Cycle can be applied anywhere.

Before starting building models, the first and the most important thing is to think about the purpose of the model. Doing more case studies as we can is helpful for getting inspired. Ask ourselves that

Is this work can be done easier by biology experiments?
If yes, then it might need to be modified. If we don't have enough time to do it all in the wet lab, try to predict the result in the model.
What is the direct answer you want to find?
Sometimes we might find another related solution or result for the problem. However, we should set a goal for ourselves and go in the right direction.
What related conditions might affect the result?
Take our project as an example, the degradation rate and the V8 protease activity are two variables that we are highly concerned with. Therefore, we modified the equations and let the lines or the graph we draw would change because of the two variables. Those equations should be explained reasonably or cited from past research to convince others.
What data is needed?
The imagination is great, but we should think more about how to get the parameters and data. We can design an experiment and do measurements to get it. If it is difficult to obtain from the wet lab, try to find them in past research or past iGEM teams' projects. “Standing on the Shoulders of Giants” is always useful in doing research.
What will we obtain by building a model?
After setting the right direction for ourselves and designing the skeleton of the model well, we should run the process in our brain and think if this system is possible to give us want we want in the end.

During building models, as beginners, we might feel overwhelmed about what are the steps we should follow. Actually, is not that difficult at all. Try the following steps, they may help.