Team:Toronto/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

Figure 1.1: 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

Figure 1.1: Other parts