The parts we created for our project.
| New BASIC PARTS (n=17) | ||||
|---|---|---|---|---|
| Part Name & Number | Type | Description | Designer (iGEM Group) | Length (bp) |
| f3-55mn-MCS, BBa_K4415000 | Plasmid backbone | Chimeric phage with a multiple cloning site, ability to express protein on the pIII coat protein - tet resistance | McMaster SynBio | 8639 |
| Anti-CD22 antibody scFv, BBa_K4415001 | Coding | scFv with the ability to bind to the B-cell protein CD22, which is internalized when bound | GenBank - KM196172.1 | 741 |
| Granzyme B, BBa_K4415002 | Coding | Apoptosis-inducing granzyme most commonly released by NK cells and cytotoxic T cells | CCDS - 9633.1 | 744 |
| TNF-alpha, BBa_K4415007 | Coding | Apoptosis-inducing cytokine | GenPept - NP_000585 | |
| 2A Linker, BBa_K1537017 | Coding | Cleavage linker with improved ability to separate two protein products expressed under the same promoter | Jie Li (iGEM14_UESTC-GreenLife) | 63 |
| GSG+E2A, BBa_K4415003 | Coding | Self-cleaving peptide similar to T2A, with slightly reduced efficacy | https://www.biorxiv.org/content/10.1101/2022.03.08.482734v1.full.pdf | 69 |
| Kozak sequence, BBa_K4415004 | RBS | Eukaryotic protein translation initiation sequence. Base pairs before, including, and after the start codon are part of this sequence | McMaster SynBio, based on https://www.nature.com/articles/308241a0 and https://onlinelibrary.wiley.com/doi/full/10.1046/j.1365-2141.2003.04754.x | 13 |
| Human codon optimized eGFP, BBa_K4415005 | Coding | Fluorescence protein sequence derived from pAAV-GFP for identification of cells that translated the transfected cassette. Codon optimized for expression in human cells. | https://www.addgene.org/32395/ | 720 |
| NEW COMPOSITE PARTS (n=8) | ||||
| Part Name & Number | Type | Description | Components | Length (bp) |
| Granzyme B TNFa Expression Cassette, BBa_K4415006 | Composite | Transfected expression cassette for delivery of apoptosis inducing genes | TNFa-GSG+T2A-GrzB-GSG+E2A-eGFP | 2292 |
Main page: f3-55nm-MCS is a chimeric phage made using 2 fd-tet derived phage species; f3-55nm, and fMCS. Together, these phages allow us to create a cloning vector with the ability to display a protein on the pIII coat protein. SfiI sites flank a stuffer sequence that once removed, can be replaced with a suitable protein that will be expressed on the N-Terminus of the pIII proteins. The MCS contains multiple unique sites, allowing for insertion of any sequences of interest.
Design: To include both the MCS and the protein display insertion sequence, f3-55nm and fMCS were combined. Both were digested with BamHI-HF and SacII; with the larger fragment of f3-55nm used, and the shorter fMCS fragment used. They were then mixed and ligated with T4 ligase.
Main page: scFv with the ability to bind to the B-cell protein CD22. Once bound, the CD22, the scFv, and anything bound to it are internalized into the B-cell.
Design: Designed for insertion into the f3-55nm-MCS phage vector for display on the pIII coat protein. The protein coding sequence is bound by 2 SfiI restriction enzyme sites, with the stop codon removed to allow for continuous synthesis of the pIII protein. 2 addition base pairs were added after the last codon, which puts the insert into frame.
Main page: Granzyme B is a protein typically released by NK cells and cytotoxic T cells, mediating apoptosis. Used here to induce apoptosis in cells that express the transfected gene.
Design: Stop codon removed to allow for usage with 2A linker sequences
Main page: TNFa is a cytokine with a variety of different uses in the body. Used here to induce apoptosis in cells that express the transfected gene.
Design: Stop codon removed for usage with 2A linker sequences
Main page: E2A is a 2A self-cleaving peptide, similar to the commonly used T2A peptide but coming from equine rhinitis A virus. These peptides cause ribosomal skipping, allowing for multiple proteins to be generated from 1 mRNA sequence. Only 1 of each of these sequences can be used in a sequence at a time however, so different 2A peptides such as E2A must be used if generating more than 2 proteins. GSG amino acids before the 2A sequence increase cleaving efficacy.
One significant benefit of E2A over other 2A peptide sequences is its legality for export out of the USA, while P2A is not.
Design: Sequences coming before the 2A sequence must have their stop codons removed to allow for continuing translation after the final amino acid.
Main page: The Kozak consensus sequence is the protein translation initiation site in eukaryotic cells. It includes 9 base pairs before the ATG start codon, and 1 base pair. This particular sequence has been optimized for proteins whose first amino acid after Methionine has a codon that starts with A by matching the -3 base pair.
Main page: eGFP is a fluorescent protein that releases 509nm wavelength light when excited. This eGFP sequence has been codon optimized for expression in human cells. The stop codon has not been removed, making it suitable for insertion without its addition, though it can also be used at the end of multiprotein sequences linked with 2A self cleaving peptides.
Design: Amino acid sequence was codon optimized for expression in human cells using IDT's tool.
Main page: This composite part was designed for use as an expression cassette for transfection of apoptosis-inducing genes. These include both TNFa and Granzyme B to cause the apoptosis, T2A and E2A self cleaving peptides, as well as eGFP for transfection tracking. Delivery of these sequences is done using the f3-55nm-MCS chimeric phage vector, which will include an Adeno-associated virus phage which the expression cassette has been ligated into.
Design: The 2A cleaving peptides between the coding sequences for TNFa, Granzyme B, and eGFP allow for all 3 proteins to be expressed from one mRNA sequence. The proteins that come before the 2A sequences require their stop codons to be removed, as the cleavage happens in the 2A peptide sequence.