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. It is speculated that in the presence of blue light, LacILOV dissociates, causing it to unbind from DNA and allow transcription. In the absence of light, the protein dimerizes and binds to the promoter of lac operon, inhibiting the transcription of downstream genes.	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

Table 1: List of parts that was submitted to iGEM registry.

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

Table 2: Other parts used in our project that was not submitted to iGEM registry.