Difference between revisions of "Part:BBa K3286008"

Revision as of 21:10, 21 October 2019

IPTG inducible dCas9 expression module

The expression dCas9 is under control of the constitutive TET promoter. Expression regulation is achieved by the lac operator located between dCas9 and pTET. In the un-induced state, the lacI protein is bound to the lac operator inhibiting expression of the dCas9. Upon addition of the inducer IPTG (or Lactose) the repressor lacI binds to the inducer and dCas9 is being expressed. Design of CRISPR interference spacer is and experiment execution are described extensively by Larson et al., 2013.

pTET: Part:BBa_K3286005

lac operator: Part:BBa_K3286004

dCas9: Part:BBa_K3286012

bi-directional terminator: Part:BBa_K3286006

lacI promoter: Part:BBa_K2572009

lacI: Part:BBa_K143033

Prarthana terminator: Part:BBa_K3286007

Sequence and Features

Fluorescence Loss Assays

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.

- sgRNA expression cassette (single target): Part:BBa_K3286003

- dCas9 can further be used in a gene circuit under the control of an Anti-CRISPR. For more information see: Part:BBa_K3286010

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.

- sgRNA expression cassette (double target): Part:BBa_K3286011