Team:St Andrews/Notebook
January
Preliminary meetings were to think of potential projects and then narrow them down by considering feasibility and topics of research being done in the university. By the end of January we had narrowed the choices down to three projects: monoclonal antibodies, nerve agents, and the killer peptide. At the same time, we were also applying for funding from the BBSRC.
February
This was quite a major month as it was when we chose the project we would go forward with, monoclonal antibodies. After choosing this we met Uli, a structural biologist at the university. He suggested we look into improving antibodies by trying to introduce a bond called an isopeptide bond into them that would hopefully make them more stable. To this end we started looking at the rational design aspect of our project to decide which proteins were most promising to be able to add an isopeptide bond to. We also started looking for lab space and supervisors to watch over us in the summer.
March
In March we continued planning the project while looking for further funding and lab supervisors. We also planned an iGEM team retreat near Strathpeffer to get to know each other better and create a funding video. March was when we had our half term, so we had a two week break from iGEM.
April
April began with our iGEM retreat which everyone really enjoyed. The rest of the month was spent looking into more specific tasks for the project. We divided the Wet Lab team into subgroups to allow different parts of the project to be worked on at the same time. We also focussed more on human practices by looking into making an impact analysis and researching companies and experts we could reach out to.
May
As everyone had exams in May, iGEM was put on hold until the official start of our project on 3rd June. However, the rational design was completed so that the Wet Lab students could begin experiments immediately.
June
June marked the official beginning of our project and so progress from the different aspects of the project will be split into three parts: Wet Lab, Human Practices, and Modelling
Wet Lab
After designing and ordering the first set of primers, the wet lab team started preliminary lab work by using cell lines provided by Marta to create competent cell aliquots to be used for the rest of the summer. The plasmids that would be the vector for the majority of our constructs (pHisTEV and Bal3 were used for cloning and protein expression respectively) was grown in E. coli and purified for use in future transformations. Once primers arrived, we attempted to clone the desired sequences into the pHisTEV plasmids.
Modelling
The modelling team very quickly split into two sub-teams to focus on different modelling goals for the project. The first team (AI) focussed on learning how to fold proteins using various AI programs and machine learning software available. If they could fold their own proteins, then this would greatly help to see if a slightly edited protein had the potential to form an isopeptide bond. The first protein folding software they looked at was Rosetta, which led to PyRosetta as it allowed more selective folding. They learned how to use the Robetta server to create protein fragments for Pyrosetta and got access to the Edinburgh cluster which allowed them to run thousands of cycles of the protein folding script to see the best outputs much quicker than it would have taken. However, PyRosetta was not able to correctly fold proteins with isopeptide bonds, so the AI team found another AI by Dr M. AlQuraishi which involved training an AI on a list of a large amount of proteins. The team hoped that this would allow the AI to fold proteins correctly that had isopeptide bonds. The second team (Isopeptide Hunters) explored ways to detect isopeptide bonds already present in proteins, the idea being that if they improved it enough, they could use it to find isopeptide bonds in proteins that may have been missed in the pdb database. They started writing a python script that would give them a list of potential proteins with isopeptide bonds, but the initial results were of thousands of potential proteins, so they started looking into what actually made an isopeptide bond.
Human Practices
Human practices made the impact analysis for our projects. This involved reaching out to antibody companies and experts, including Antibody Analytics from whom they were able to have a very insightful call with.
July
Wet Lab
Once sequences were successfully cloned into the cloning plasmid, constructs were transformed into the Bal3 cells to attempt expression. To screen for expression, we used a Nickel pull-down to bind and separate the desired protein. After several tests it was determined that the Bal3 may not be expressing as desired and so a new cell line was acquired. Simultaneously started small-scale expression tests of mutant CH3 domains in SoluBL-21 and Origami cells, and achieved successful expression of AB2, AB7, and the commercial antibody. Cloning of mOrange from the distribution kit for the part improvement medal criteria (yeast-optimised mOrange with SpyTag) was performed in parallel with the main project.
Modelling
The isopeptide hunters spent the first half of July refining their python script to find isopeptide bonds from PDB files. They were continually looking for more parameters they could use to refine their list of potential proteins, such as distances between specific atoms, and the atom locations within the protein. One of the parameters they wanted to use was the fofc electron density maps. They found an expert online called Prof. Robert Hanson who was luckily nearby at the time. The isopeptide hunters were able to meet up with Prof. Hanson and learned how to use Jmol to quantify the electron density data for specific regions of a protein. They then started to incorporate this into their refining process in order to further narrow down the list. Meanwhile the AI team got access to a computer in one of the Computer Science labs, which had the needed software and after some corresponding with Dr AlQuraishi, as well as making a script to converter .tertiary files to .pdb, so the AI team went back to the original AI and managed to get folded proteins.
Human Practices
Human Practices organized a discussion forum, inviting both community members and academics. They decided that this was a great way to discover first, the worries and interests of the general public regarding the project, and second, a way to educate members of the public on what the research entailed. The forum began with presentations by some of the academics, teaching those in attendance both about the specific iGEM project and about biology more generally. The public was engaged and asked many questions. Through the experience, the whole iGEM team took away multiple lessons on how best to communicate their project in the future, and how they could emphasize certain elements to further invigorate the curiosity of interested individuals.
August
Wet Lab
In August expression trials of various antibody mutations continued, and expression of fragments were confirmed using mass spectrometry, however none of the mutants had the desired isopeptide bond as was suggested by prior SDS analysis of cell lysate. We also ran mOrange+SpyTag expression trials in BL21 cells, however it was shown that while the construct was being expressed, it was also being cleaved, resulting in the protein being about half the expected mass upon SDS analysis. To correct this the construct was incubated with OIPD (a SpyCatcher analogue), however results may have been hidden by the presence of an OIPD dimer.
Modelling
The Isopeptide Hunters ran their newly finished code over all of the proteins in the databank. The utilization of JMol to extract information about the fo-fc maps allowed for complete automation of the process. The team found multiple candidates, both for possible pre-existing isopeptide bonds, and for possible proteins to easily insert isopeptide bonds into. They plan to continue this research after iGEM ends.
Human Practices
The Human Practices team ran an outreach session at Edinburgh Zoo in collaboration with Edinburgh UG and OG teams. During the session, members of the public participated in the Microbiome of Scotland project, collecting soil samples, learning about synthetic biology and conducting some experiments.
September
In September, our classes started back up for the year. The Human Practices team attended a Fife teachers training day to showcase the Microbiome of Scotland outreach project in the hopes of encouraging teachers to take part in the project by delivering the sessions themselves. The Modelling team spent the majority of their time creating the wiki in preparation for the wiki freeze. The Wet Lab team compiled the information on the work they had done over the summer and began to edit it into a wiki-ready form.
October
The entire team worked together tirelessly to ensure the wiki was complete and all information accounted for. We then finished the poster and the other last-minute preparations, and headed to Boston for the Jamboree!