Difference between revisions of "Team:Toronto/Parts"
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Revision as of 01:18, 1 November 2017
Parts
Submitted Parts
| pSB1C3 | Registry number | Type | Length (bp) | Function | Assembly Method | UNS |
|---|---|---|---|---|---|---|
| LacILOV-mCherry | BBa_K2469003 | Composite | 1615 | Light regulated reporter | Digestion and ligation | 2, 3 |
| HIS-AcrIIA4 | BBa_K2469002 | Basic | 398 | Inhibits the function of S. pyogenes Cas9 and dCas9. | Gibson assembly of amplified AcrIIA4 from G-block and amplified pSB1C3 backbone from LacILOV-mCherry Construct in pSB1C3 | 2, 3 |
| LacILOV | BBa_K2469001 | Basic | 728 | A fusion protein of the light, oxygen and voltage-sensitive domain of the Avena Sativa phototropin 1 (LOV2) and the LacI domain. This transcriptional regulator represses promoters under control of LacI. In the presence of blue light this protein will act as a repressor, and in its absence the protein will dissociate from the promoter | Gibson assembly of amplified LacILOV part from LacILOV-mCherry construct in pKDL071 and pSB1C3 backbone from LacILOV-mCherrt construct in pSCB1C3 | 2, 3 |
| reporter construct | BBa_K2469004 | Composite | 3253 | Light regulated reporter switch, alternating between mCherry and YFP flourescence | Gibson assembly of amplified reporter construct out of pkDL071 and amplified pSB1C3 backbone from the LacILOV-mCherry construct in pSB1C3 | 2, 3 |
Other Parts
| pKDLO71 | Type | Length | Function | Assembly Method | UNS |
|---|---|---|---|---|---|
| LacILOV-mCherry | Composite | 1615 | Light regulated reporter | Gibson g-Block + Backbone with UNS | 2, 3 |
| reporter mCherry | reporter construct, consisting of the CI repressor (with LVA degradation tag) and mCherry under the control of the Ptrc-2 promoter; used as a negative control in the sgRNA assay | Gibson g-Block + Backbone with UNS | 2, 4 | ||
| reporter mCherry + sgRNA | Half of the full construct, to test the function of the sgRNA (ensure it is targetting the correct sequence) | Gibson assembly of sgRNA g-Block into reporter mCherry, with UNS 5 extension on backbone | 4, 5 | ||
| reporter mCherry + sgRNA+ AcrIIA4 | Incorporation of the Anti-CRISPR protein into the sgRNA construct to have both expressed simultaneously and test the function of the Anti-CRISPR | Gibson assemly of AcrIIA4 g-block into reporter mCherry + sgRNA, with UNS 6 extension on backbone | 4, 6 | ||
| reporter construct | Composite | 3253 | Light regulated reporter switch, alternating between mCherry and YFP flourescence | Gibson assembly of reporter YFP (LacILOV + YFP) into reporter mCherry with UNS 3 extension on backbone | 3, 2 |
| full construct | Light-regulated switch controlling the activity of the CRISPR-Cas9 system. | Gibson assembly of AcrIIA4 subpart (LacILOV + AcrIIA4 + YFP) into reporter mCherry + sgRNA, with UNS 3 extension on bacbone | 3, 6 |
