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.


Parts

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

SHuffle Troubleshooting

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.


Common Wet Lab Mistakes

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.


Protein Production

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.


Guide to Molecular Dynamics