Team:SDU-Denmark/Parts
Parts
New Parts
To develop our system, we designed different parts. These parts include both basic and composite parts, where the composite parts consist of our new basic parts and already exciting parts from the iGEM part registry. Our basic parts collection consists of three Cas proteins; Cas13a optimized for E. coli K12, Cas12a optimized for human cells, and Cas12a optimized for E. coli K12. It also includes different sgRNAs and their matching targets. The Cas13a specific sgRNAs are targeting the ETS transcription factor ERG, and both the risk and non-risk allele of the rs6983267 SNP. Cas12a specific sgRNAs target the risk and non-risk allele of the rs6983267 SNP. Matching target sequences can be used as positive controls, to determine the activity of the Cas proteins.
Our composite part collection includes the three different Cas sequences in addition with the positively regulated lac promoter (BBa_R0011), the highly effective ribosomal binding site (RBS) (BBa_B0034), and the (BBa_B1006) terminator which forms an 8 bp stem 6 nt loop.
All new parts:
| Biobrick ID | Type | Description | Designer | Length |
|---|---|---|---|---|
| BBa_K3602001 | Coding | Cas13a w. tags, E. coli K12 optimized | Flora Fuglsang | 3909 bp |
| BBa_K3602002 | RNA | ERG sgRNA Cas13a | Flora Fuglsang | 86 bp |
| BBa_K3602003 | RNA | rs6983267 T-SNP sgRNA Cas13a | Flora Fuglsang | 86 bp |
| BBa_K3602004 | RNA | rs6983267 G-SNP sgRNA Cas13a | Flora Fuglsang | 86 bp |
| BBa_K3602005 | RNA | ERG target Cas13a | Flora Fuglsang | 90 bp |
| BBa_K3602006 | RNA | rs6983267-T Target Cas13a | Flora Fuglsang | 90 bp |
| BBa_K3602007 | RNA | rs6983267-G Target Cas13a | Flora Fuglsang | 90 bp |
| BBa_K3602012 | RNA | rs16901979-A target Cas12a | Flora Fuglsang | 35 bp |
| BBa_K3602013 | RNA | rs16901979-C Target Cas12a | Flora Fuglsang | 35 bp |
| BBa_K3602014 | Coding | Cas12a w. tags, optimized for E. coli K12 | Flora Fuglsang | 4074 bp |
| BBa_K3602015 | Coding | Cas12a w. tags, optimized for human cells | Flora Fuglsang | 4074 bp |
| BBa_K3602020 | Plasmid | Cas13a - composite part | Flora Fuglsang | 4078 bp |
| BBa_K3602021 | Plasmid | Cas12a composite part, optimized for E. coli K12 | Flora Fuglsang | 4243 bp |
| BBa_K3602022 | Plasmid | Cas12a composite part, optimized for human cells | Flora Fuglsang | 4243 bp |
Parts Collection
All parts utilized by PROSTATUS are composed in three-part collections: sgRNA, Target and Cas collection. The Cas collection consists of our three Cas-proteins, both as basic parts and as composite parts. The target collection contains different target sequences complementary to the sgRNAs and is being used as positive controls for our test.
The sgRNA collection contains different sgRNAs for Cas12a and Cas13a, respectively. These are designed for each of our different biomarkers and SNPs. Some of these sgRNAs have been used to demonstrate proof of concept for both Cas13a and Cas12a, as the proteins should only become activated upon complementary binding of sgRNA and target. Some of them have not been used for experiments yet but has been designed in the same way as the other sgRNAs and are thereby expected to work. The sgRNAs all target biomarkers of prostate cancer, but the spacer can easily be replaced to target other biomarkers while keeping our scaffold sequence for either Cas12a or Cas13a can be used. An array of potential biomarkers can be found in the biomarker database made by the 2020 iGEM team of Rochester. Our biomarkers are also present in this biobank, as we have had an ongoing partnership with them. For further information on how to design your sgRNAs, please look at our Contribution site for Cas13a design , and at our theory site for Cas12a design .
Cas protein collection:
| Biobrick ID | Type | Description | Designer | Length |
|---|---|---|---|---|
| BBa_K3602001 | Coding | Cas13a w. tags, E. coli K12 optimized | Flora Fuglsang | 3909 bp |
| BBa_K3602014 | Coding | Cas12a w. tags, optimized for E. coli K12 | Flora Fuglsang | 4074 bp |
| BBa_K3602015 | Coding | Cas12a w. tags, optimized for human cells | Flora Fuglsang | 4074 bp |
| BBa_K3602020 | Plasmid | Cas13a - composite part | Flora Fuglsang | 4078 bp |
| BBa_K3602021 | Plasmid | Cas12a composite part, optimized for E. coli K12 | Flora Fuglsang | 4243 bp |
| BBa_K3602022 | Plasmid | Cas12a composite part, optimized for human cells | Flora Fuglsang | 4243 bp |
sgRNA collection:
| Biobrick ID | Type | Description | Designer | Length |
|---|---|---|---|---|
| BBa_K3602002 | RNA | ERG sgRNA Cas13a | Flora Fuglsang | 86 bp |
| BBa_K3602003 | RNA | rs6983267 T-SNP sgRNA Cas13a | Flora Fuglsang | 86 bp |
| BBa_K3602004 | RNA | rs6983267 G-SNP sgRNA Cas13a | Flora Fuglsang | 86 bp |
Target collection:
| Biobrick ID | Type | Description | Designer | Length |
|---|---|---|---|---|
| BBa_K3602005 | RNA | ERG target Cas13a | Flora Fuglsang | 90 bp |
| BBa_K3602006 | RNA | rs6983267-T Target Cas13a | Flora Fuglsang | 90 bp |
| BBa_K3602007 | RNA | rs6983267-G Target Cas13a | Flora Fuglsang | 90 bp |
| BBa_K3602012 | RNA | rs16901979-A target Cas12a | Flora Fuglsang | 35 bp |
| BBa_K3602013 | RNA | rs16901979-C Target Cas12a | Flora Fuglsang | 35 bp |
Existing parts used:
| Biobrick ID | Type | Description | Designer | Length |
|---|---|---|---|---|
| BBa_J45014 | Coding | alcohol acetyltransferase I; converts isoamyl alcohol to isoamyl acetate (banana odor) | André Green II | 1581 bp |
| BBa_R0011 | Regulatory lacI+pL | Promoter (lacI regulated, lambda pL hybrid) | Neelaksh Varshney, Grace Kenney, Daniel Shen, Samantha Sutton | 55 bp |
| BBa_B0034 | RBS | RBS (Elowitz 1999) -- defines RBS efficiency | Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003. | 12 bp |
| BBa_B1006 | Terminator | Terminator (artificial, large, %T~>90) | Haiyao Huang | 39 bp |
