Parts
We designed several parts, 16 in total, during our iGEM journey, and characterised some of them. These parts were designed to produce full-length antibodies and antibody fragments in the cytoplasm of the SHuffle E.coli system. Details regarding the same are in our Parts page.
SHuffle Troubleshooting Document
This year, our team worked with an engineered strain of E.coli called
SHuffle. SHuffle is specifically engineered to form stable disulfide
bonded proteins in the cytoplasm, by mutating reductive pathways (trx-,
gor-, ahPc*) and over-expressing the disulfide bond isomerase cDsbC.
This creates an oxidative cytoplasm capable of properly folding
disulfide bonded proteins. The inventors of SHuffle have successfully
managed to produce correctly folded, full-length antibodies in the
cytoplasm of SHuffle E.coli cells.
Team Virginia worked on the same chassis as we did this year. We had to
figure out quite a lot along the way, with the help of our mentors and
people who had worked with bacterial over-expression and SHuffle before.
To help other teams who might work with SHuffle in the future, we
decided to compile a document that details some of the things that
worked for us over the course of the project.
The document includes analysis and instruction for the following protocols:
- Considerations for constructing devices in SHuffle
- Transformation methods for SHuffle
- Competency for SHuffle strains
- Electro Competency
- Protein Expression
- Protein Purification
- Harvesting
- Western Blot
A Guide to Avoiding Mistakes in Wet Lab
Stepping into the lab for the first time can be overwhelming. We started out as a group of undergraduates with no knowledge of how to work in a lab. We had great mentors who guided us through every step of our process, but we still made many, many mistakes, from our lack of basic knowledge and carelessness. We have compiled these errors in a guidebook of sorts to help future teams to kickstart their wet lab experimentation with the least amount of failures. We hope that this contribution will help many teams accomplish more with the short time they have, and waste less time and resources in the lab.
Protein Expression - Chassis, Vector and Pull-down
Our team used the prokaryotic E.coli chassis for expressing the
proteins, ie. our antibodies. However, over the course of the project,
we got the opportunity to explore many other systems which can be used
to maximise and optimise protein production. We believe that any future
iGEM team that aims to do protein expression and purification would
benefit from checking out all the available options for the production
and purification of their protein.
To this end, we have compiled a document in collaboration with MIT_MAHE
that broadly covers the possible choices for the same.
Molecular Simulations Handbook
There are many tools available in bioinformatics to assist with carrying
out particular tasks. They vary in their algorithms and depend on
application setting. Given the circumstances of most students, using
these tools and choosing a specific server is difficult for students.
When our team wanted to dock proteins and run Molecular Dynamics
Simulations, there were a plethora of resources at our disposal, but no
clear guidance as to which tool to use when or why.
To address this, we decided to create a handbook on MDS that covers the
capabilities and use-cases of the many available Bioinformatics tools
for the same, in order to allow those who wish to dock their proteins
and perform MDSs to narrow down and simplify the choice of these
servers. Future iGEM teams that seek to speed up the dry lab components
of their project and have a more unified strategy to tackle MDS would
greatly benefit from these manuals. It was compiled with the help of
Team MIT Mahe.
Molecular dynamics simulations guide
Our team, in collaboration with MIT_MAHE again, formulated an elaborate
document on how to go about Molecular Dynamics Simulations with and
without the help of Supercomputing resources. Desmond is a free-to-use
software available in the Schrodinger suite and is used to run Molecular
Dynamics simulations. GROningen MAchine for Chemical Simulations known
as GROMACS is one of the most extensively used and well-liked
open-source physics based bioinformatics programmes. It is typically
employed to simulate macromolecules' molecular dynamics.
These were the two softwares we used - we have documented guidelines for
the same. Like the AlphaFold Guide, the documentation for the MD’s has
also been split into two sections: One for users with Supercomputing
resources and one for users without, along with instructions for running
the same.