Part:BBa_K2558003:Experience
Fluorescence Loss Assays
characterised by iGEM19_Wageningen
dCas9 - monoplex
The strength of dCas9 inhibition was measured in an Fluorescence Loss Assay. GFP, under the control of the lacUV5 promoter, is genomically inserted in an E. coli DH10ß strain. Three spacer were designed (Sp1 - 3) targeting either the -10 element of the lacUV5 promoter (Sp1) or the first 100 nucleotides of the GFP cds. A non-targeting Spacer (SpNT) was included as a control. All spacers were expressed via a single guide (sg)RNA expression cassette. Expression of dCas9 is IPTG inducible via the lacI/lac operator system.
Sp1 - 3 show clear reduction of GFP levels. Leakiness of lacI/lac operator system results in GFP repression even under un-induced conditions. The choice of spacers shows an effect on the efficiency of repression. Targeting the promoter region is more promising for repression of gene expression (Sp1) compared to targeting the beginning of the cds (Sp2&3). For recommendations regarding spacer design see Larson et al., 2013.
- Complete dCas9 expression cassette: https://parts.igem.org/Part:BBa_K3286008
- sgRNA expression cassette (single target): https://parts.igem.org/Part:BBa_K3286003
- dCas9 can further be used in a gene circuit under the control of an Anti-CRISPR. For more information see: https://parts.igem.org/Part:BBa_K3286010
The expression of dCas9 is regulated by an IPTG inducible expression system. Sp1 - 3 represent three different spacers targeting GFP or the associated promoter. SpNT represents a non-targeting spacer serving as a control. 
dCas9 - multiplex
In a similar manner the strength of dCas9 inhibition on two targets, GFP & RFP, was measured simultaneously. GFP & RFP were under the control of the constitutive phage lambda PR & PL promoter, respectively . Sp1 - 3 refer to three different combinations of two spacers targeting each either GFP or RFP. A non-targeting Spacer (SpNT) was included as a control. Sp1 - 3 were expressed via a double sgRNA expression cassette with each sgRNA being linked to either a GFP or RFP targeting spacer. Expression of dCas9 is IPTG inducible via the lacI/lac operator system.
- Complete dCas9 expression cassette: https://parts.igem.org/Part:BBa_K3286008
- sgRNA expression cassette (double target): https://parts.igem.org/Part:BBa_K3286011
The expression of dCas9 is regulated by an IPTG inducible expression system. Sp1 - 3 refer to three different combinations of two spacers targeting each either GFP or RFP. SpNT represents a non-targeting spacer serving as a control. 
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