Team:Crete/Contribution
Introduction
Living through the negative effects of the coronavirus in our daily lives, both in the health and in the social and political fields, vaccines seemed like the ultimate salvation!Although many times opinions are divided, as the feeling of fear and insecurity overwhelms people, leading them to resort to denial, the best treatment is information! Therefore, through our project, we present the positive effect of vaccines from our point of view, with the primary aim of informing and familiarizing our fellow human beings with vaccination. At the same time, we provide a step-by-step description of the whole process of producing an edible vaccine against SARS-CoV-2, thus leaving a library of tools for later groups or researchers who want to deal with edible vaccines.As igem crete we are the first team from Crete to participate in the IGEM competition.Despite the generous help of IGEM HQ, we had no model team before us to guide us. Therefore, we started to take things step by step from the beginning, reading at the same time different Dos & Don'ts and studying tips which helped us both during the project and in the part of communication with different forums and in the interaction with specific social groups such as young children, COVID-19 sufferers, doctors etc. in order to inform them about the competition and our project. Wishing to contribute to the effort of a later igem group, we have created this Booklet which provides some tips and guidelines to facilitate the work of each group. Along with the booklet, we have decided to give the next igem crete team a part of the money raised by the Fundraising Team, in order to give a boost to the next generation to make the procedural part easier, focusing more on the Labs and Human Practices part.
Introduction for experimental animal handling guide
Our initial reasoning was to divide the project into two experimental procedures.
The first would be the preparation of the edible vaccine, while the second would be the feeding of the edible vaccine to mice in which the antigen protein would be expressed transmembrane by immunization against SARS-CoV-2 via the intestinal mucosa followed by quantification of the antibodies in experimental animals.Despite the difficult conditions that prevailed due to the quarantine, we hoped to be able to carry out both experimental procedures. For this reason, at a very early stage, we began to study in detail the handling of experimental animals both in terms of safety and in terms of laboratory, in order to have covered any eventuality and to be prepared.
However, given the current situation with coronavirus, most laboratories could accept a small number of people, with the result that the evolution of the experimental process was delayed. So, as time was running out and we did not want to exceed the time limits, we decided not to perform the second series of experiments involving the immunization of mice as it was a process that required a lot of time, about a month at best.Βut we are looking forward to continue this project after the end of the competition if the conditions are more ideal for us.
Nevertheless, we considered that the knowledge and information we received both from our study and from discussions with competent professors and veterinarians, such as Ms. Tsoukatou and Mr. Spilianakis would be quite useful for future groups that may be dealing with vertebrates and specific mice.So we decided to gather all this information in an "experimental animal handling guide" wanting to facilitate the work of future groups and give them the trigger to move forward and develop our idea.
Troubleshooting problems that may help future teams inside and outside the lab
1)This year, the world has been hit by a global pandemic due to the coronavirus. Our team was created in December, while Greece was in total lockdown, with the result that in the first months we have never met the team members up close, even for a first acquaintance. The situation worsened when we were called to meet as a group in order to carry out the brainstorming session and to choose our project. Unfortunately, the minimal interpersonal contact with the members of the team made the communication between us even more difficult. At the same time access to the lab in the summer was very uncertain, which made us all even more anxious and stressed. Time passed very quickly and as the requirements for choosing the right project grew we decided to set a weekly schedule to complete each task on time. So every Monday all the members of the team completed a to-do list of all the responsibilities they had to do. Every Sunday we set weekly two-hour meetings in which each member presented his work in the first hour and in the second hour we discussed issues that we reflect on the project and set new weekly responsibilities.
In addition, we considered that as members of a team it would be good to know each other better in order to achieve the best possible cooperation.
But because the coronavirus conditions did not allow many people to gather, we chose to have an online meeting once a month in which we discussed various non-project issues, told our news, and played games in order to get closer to each other.
2)Unfortunately the pandemic had a negative impact even on the Fundraising team, as we all had to adapt to the new measures and reduce interpersonal contacts to a minimum. Thus, members of this sub-team were unable to visit companies and businesses at close quarters in order to inform those responsible about the competition, the project and the costs that would be involved in running it.
These circumstances played a decisive role in raising the necessary funds as the role of this team had become very difficult.The good news is that the required amount was raised, but this was achieved after persistent and countless rejections as virtual contact reduces trust and reliability.The key to dealing with this difficult situation was our patience and consistency in making phone calls and sending the necessary emails.At the same time, in order to make it easier to inform our community about the project, we have created a short and comprehensive presentation of the project.
This presentation consisted of two strands:
The first was a simple description of our idea, and the second was a detailed presentation of the ultimate purpose of the revenue we were seeking.
Consequently what we would suggest to future groups would be not to lose patience and hope as with perseverance , consistency and organization the desired results will come !
3)Of course, the wet lab part of the project was not unaffected by SARS-CoV-2, as our entry into the lab was delayed due to the extraordinary measures taken by the state.
Unfortunately, the number of people a laboratory could accept was very limited, and we decided that the ideal case was to have two members of the wet lab team in the laboratory to monitor the preparation of the edible vaccine, while observing the necessary measures against the spread of coronavirus.As time went by and our anxiety to carry out all the necessary procedures for the perfection of the project increased, we considered this case ideal in order to start the experimental process in time.
A)In most of the edible vaccines made to date, it was a prerequisite to dissolve the cell walls in order to release the antigen protein for immunisation through the intestinal mucosa, as mammals and especially humans find it difficult to discard plant tissues because they were usually taken from the intestine without breaking the cell walls.
To solve this important problem we thought that the protein-antigen should be transmembrane, i.e. protruding from the cell, therefore the antigen is outside the plasma membrane, so that the dissolution of plant tissue does not need.
This way all the cells will have the antigen on their surface so that they do not have to break down to be released.
B)One of the most widely used fluorescent proteins used as spectroscopic tags that reveal the localization, mobility and interactions of proteins in cells is GFP.
Although we initially considered using GFP, we objected that as a dimeric protein within the cell it can create stereochemical inhibition on how the trimeric spike protein will form if two receptors are stuck together.
So we decided to use a monomeric protein like mcherry which does not have this problem.
C)As we have mentioned above, one of the key features of our idea that made it innovative was the immunity against three strains of SARS-CoV-2.
The protein antigen is synthetic and consists of a signal peptide (SP), three copies of the S1 subunit of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) subunit linked to an amino acid linker, a transmembrane (TM) domain, an HA ( Human Haemoglobin Influenza Label) and a fluorescent protein (mCherry). The three S1 subunits are derived from the spike protein (S) and resemble the wild-type version of this subunit, the alpha variant (lineage B1.1.1.7) identified in the UK and the beta variant (lineage B1.351) identified in South Africa.
For cloning we were looking for a way to amplify the segments easily and quickly, using as few enzymes as possible.
In addition, it was necessary to intensify the segments in a specific order.So after research we came up with Golden Gate Cloning which is a method of assembling multiple DNA fragments simultaneously with the help of two main enzymes. Type IIS restriction enzyme and T4 DNA ligase.
The main advantage of this method is that this type of restriction enzyme cuts out the recognition sequence and as a result the restriction site does not appear in the generated sequence.
In addition, by this mode the subunits could be easily and directly switched upon the appearance of a newly threatening mutation.
It was therefore obvious that this method greatly aided the development of the project both in terms of time and flexibility.
The iGEM Human Practices Handbook
Even though we’re a new team, we were extremely proud of our human practices work. It is our firm belief that we have succeeded in that sector by following, respecting and expanding upon the guidelines of the iGEM competition. This process was, nonetheless, a time consuming one since we had to process a fairly big amount of information and interpret them in the right-according to our opinion- way so we could end up with the desired result. Mid-way through the competition we realised that by documenting our takes on several aspects of the Human Practices, we could save precious time from future teams. Moreover as a newcomer to the iGEM scene, we believe that our fresh take on such matters may help future teams that also participate in the competition for the first time. From the rule of 3 Rs to our take on Ethics, we present to you our short guide for our Human Practices!
Parts registered and described
|
Type |
Part Name |
Kind |
Description |
Length |
|
Basic |
Plasmid Backbone |
pBluescript II SK(-)_BsaI domesticated |
2962 |
|
|
Basic |
DNA |
Linker for 3x Spike S1 proteins |
18 |
What parts did we register?
pBluescriptII/SK(-)_BsaI domesticated:
This vector has a BsaI recognition site which is a restriction enzyme compatible with the method of Golden Gate cloning. Golden Gate is a very efficient and relatively quick method of cloning. So any future team which will use this method can consider this vactor for their project.
Linker for 3x Spike S1 proteins:
This type of linker in the context of our project is used to join and trimerise the 3 spike S1 proteins from the 3 different SARS-CoV-2 variants. Nonetheless the linker can be used to connect different type of proteins.