Team:Stanford/Contribution


Contribution

In order to help assist future iGEM teams in their projects, Stanford’s 2021 iGEM team has left very useful contributions. While there are many aspects of our project that future teams can emulate and shadow, our main contribution is our yeast plasmid that we designed. Not only have we shared the yeast plasmid we created, but we also included the protocol in case other teams want to try attempting something similar. Additionally, we have included a version of a reductase enzyme in house flies that is codon-optimized for expression in yeast.

Contribution Part:BBa_K4009004: Yeast Plasmid

Functionality: This plasmid contains the CYP6B1 DNA sequence that allows the Saccharomyces cerevisiae to produce the CYP6B1 protein in the Papilio polyxenes, the black swallowtail butterfly.

Design Notes: 1. The following cleave sites are avoided: BamHI, SpeI, and XhoI.

2. We optimized the CAI and GC content through IDT, NCBI Blast Protein, and Genscript.

3. The parts URA3, AmpR, and DGH/TDH3p are chosen to minimize the plasmid burden on the Saccharomyces cerevisiae

Source: the current CYP6B1 DNA sequence is based on the CYP6B1 amino acid sequence from the uniprot

Contribution Part:BBa_K4009003: Reductase from House Fly

We created the yeast plasmid as part of our engineering design cycle. We wanted to include reductase as this would be helpful in the co-expression of the CYP6B1 black swallowtail butterfly enzyme. Students can use this if they are interested in exploring expression in yeast, as well as other types of bacteria. This would be most saliently used in applications for the gut microbiome, however, we would hope that other applications would arise that could be within and outside the domain of studying the gut microbiome and human health.

The source of the part is from the housefly cytochrome P450 reductase, as this has been shown to be appropriate for co-expression. We sourced it from UniProt. For our design considerations, we had to consider how we would perform codon optimization, and additionally, we experimented with the addition and removal of restriction enzymes. Based on our optimization, we were able to achieve a part that met our needs.



OTHER CONTRIBUTION: PLASMID DESIGN AND CODON OPTIMIZATION PROTOCOL

Additionally, we’ve created a protocol for plasmid design that we have adapted based on advice that we have received from our mentor. We used this process multiple times for our experimental design, and we hope that this will be useful for teams in the future as well.


*** This protocol is based on Dr. Prashanth Srinivasan’s tutorial on codon optimization that we have adapted and tested for our project and can be used as a reference for future iGEM teams. The protocol was used for CYP6B1 plasmid design and NADPH--cytochrome P450 reductase from Musca domestica (House fly). Rationale for designing these two plasmids can be found here on our project description page. ***

Protocol

1. Find target DNA/amino acid sequence on database
2. Import to Benchling
3. Annotate Important Sequence

Codon Optimization (tool: VectorBuilder; jcat)
1. Avoid cleavage sites of restriction enzymes:
  • BamHI: 5'-GGATCC-3'
    EcoRI: GAATTC ; CTTAAG
    EcoRV: 5'-GATATC-3'; 3'-CTATAG-5'
    SpeI: 5′-ACTAGT-3′
    PstI: 5′-CTGCAG-3′
    XbaI： 5′-TCTAGA-3′; 3’-AGATC|T-5’
    XhoI: CTCGAG
    • 2. Check Codon Adaptation Index (CAI) and GC content
    3. Sanity check using NCBI Blast Protein for checking if translation of optimized DNA sequence matches the original amino acid sequence from the database.
    4. Check for rare codons (tool for rare codon analysis)
    5. Add in the 5’ and 3’ handles for the optimized DNA sequence.


    Going through this protocol was a significant part of our Engineering Process. We designed, built, and tested many iterations of our plasmids by going through this. We are very grateful to Dr. Srinivasan, who helped us develop this protocol and engineering strategy, and ultimately allowed us to build the plasmid we did, which is linked in the "Part Contribution: Yeast Plasmid" link at the top of the page and can be found in the Parts Registry.

    To see more about the Engineering Success we had when using this protocol, and in actually getting to the Part that we were able to contribute, check out our Engineering Success page where we describe our journey through the Design-Build-Test cycle.