Team:EPF Lausanne/Part Collection
E. coli
dCas9-ω
dCa9s-ω is our main tool to build our project in E. coli. This catalytically dead Cas9 fused to the RNA polymerase ω subunit can act as an activator or as an inhibitor when provided the right guide RNA.
PAM rich URS J23117 promoter
This promoter is composed of the weak J23117 promoter, BBa_J23117, fused to a PAM rich upstream regulatory region (URS) that is needed for dCas9 to bind as dCas9 cannot bind without a PAM sequence.
sgRNA Z0 expressing cassette
the sgRNA (single guide-RNA) Z0 inhibit PAM rich URS J23117 promoter when in complex with dCas9-ω. This part is an expressing cassette under the constitutive promoter pBAD. The sequence Z0 makes this part unique, and the scaffold is a common part to all the sgRNAs and is recognized by dCas9-ω.
sgRNA Z4 expressing cassette
the sgRNA Z4 activates PAM rich URS J23117 promoter when in complex with dCas9-ω. This part is an expressing cassette under the constitutive promoter pBAD. The sequence Z4 makes this part unique, and the scaffold is a common part to all the sgRNAs and is recognized by dCas9-ω.
This part is not RFC10. Removing bad restriction site would imply a lose of function.
sgRNA Z35 expressing cassette
the sgRNA Z35 inhibit PAM rich URS J23117 promoter when in complex with dCas9-ω. This part is an expressing cassette under the constitutive promoter pBAD. The sequence Z35 makes this part unique, and the scaffold is a common part to all the sgRNAs and is recognized by dCas9-ω.
PAM rich URS J23117Alt promoter
This promoter was made on the same template as PAM rich URS J23117 promoter, but was mutated to obtain different sequences for sgRNA targeting. It is also fused to a PAM rich upstream regulatory region (URS) that is needed for dCas9 to bind, as dCas9 cannot bind without a PAM sequence.
sgRNA X0 expressing cassette
the sgRNA (single guide-RNA) X0 inhibit PAM rich URS J23117Alt promoter when in complex with dCas9-ω. This part is an expressing cassette under the constitutive promoter pBAD. The sequence X0 makes this part unique, and the scaffold is a common part to all the sgRNAs and is recognized by dCas9-ω.
sgRNA X4 expressing cassette
the sgRNA X4 activates PAM rich URS J23117Alt promoter when in complex with dCas9-ω. This part is an expressing cassette under the constitutive promoter pBAD. The sequence X4 makes this part unique, and the scaffold is a common part to all the sgRNAs and is recognized by dCas9-ω.
sgRNA X35 expressing cassette
the sgRNA X35 activates PAM rich URS J23117Alt promoter when in complex with dCas9-ω. This part is an expressing cassette under the constitutive promoter pBAD. The sequence X35 makes this part unique, and the scaffold is a common part to all the sgRNAs and is recognized by dCas9-ω.
S. cerevisiae
dCas9_VP64
dCa9s-VP64 is the main tool to build our project in S. cerevisiae. This catalytically dead Cas9 fused to a nuclear localization sequence (NLS) and the eukaryotic transcription factor VP64 can act as an activating (or as an inhibitin) transcription factor when forming a complex with the appropriate guide RNA.
This part is not RFC10.
c3_0 gRNA expressing sequence
This sequence produces guide RNA c3_0 when put under the control of a RNA polymerase II promoter. gRNA c3_0 can form a complex together with dCas9_VP64 which activates promoter CYC_0.
c3_1 gRNA expressing sequence
This sequence produces guide RNA c3_1 when put under the control of a RNA polymerase II promoter. gRNA c3_0 can form a complex together with dCas9_VP64 which activates promoter CYC_1.
c3_2 gRNA expressing sequence
This sequence produces guide RNA c3_2 when put under the control of a RNA polymerase II promoter. gRNA c3_0 can form a complex together with dCas9_VP64 which activates promoter CYC_2.
c3_3 gRNA expressing sequencee
This sequence produces guide RNA c3_3 when put under the control of a RNA polymerase II promoter. gRNA c3_0 can form a complex together with dCas9_VP64 which activates promoter CYC_3.
c6_0 gRNA expressing sequence
This sequence produces guide RNA c6_0 when put under the control of a RNA polymerase II promoter. gRNA c6_0 can form a complex together with dCas9_VP64 which inhibits promoter CYC_0.
c6_1 gRNA expressing sequence
This sequence produces guide RNA c6_1 when put under the control of a RNA polymerase II promoter. gRNA c6_1 can form a complex together with dCas9_VP64 which inhibits promoter CYC_1.
c6_2 gRNA expressing sequence
This sequence produces guide RNA c6_2 when put under the control of a RNA polymerase II promoter. gRNA c6_2 can form a complex together with dCas9_VP64 which inhibits promoter CYC_2.
c6_3 gRNA expressing sequence
This sequence produces guide RNA c6_3 when put under the control of a RNA polymerase II promoter. gRNA c6_3 can form a complex together with dCas9_VP64 which inhibits promoter CYC_3.
c7_0 gRNA expressing sequence
This sequence produces guide RNA c7_0 when put under the control of a RNA polymerase II promoter. gRNA c7_0 can form a complex together with dCas9_VP64 which inhibits promoter CYC_0.
c7_1 gRNA expressing sequence
This sequence produces guide RNA c7_1 when put under the control of a RNA polymerase II promoter. gRNA c7_1 can form a complex together with dCas9_VP64 which inhibits promoter CYC_1.
c7_2 gRNA expressing sequence
This sequence produces guide RNA c7_2 when put under the control of a RNA polymerase II promoter. gRNA c7_2 can form a complex together with dCas9_VP64 which inhibits promoter CYC_2.
c7_3 gRNA expressing sequence
This sequence produces guide RNA c7_3 when put under the control of a RNA polymerase II promoter. gRNA c7_3 can form a complex together with dCas9_VP64 which inhibits promoter CYC_3.
CYC_0 promoter
CYC_0 promoter acts as a transistor in yeast. It is activated by the complex c3_0-dCas9_VP64 but repressed by complexes c6_0-dCas9_VP64 and c7_0-dCas9_VP64. When any combination of those three complexes is expressed, inhibition prevails.
CYC_1 promoter
CYC_1 promoter acts as a transistor in yeast. It is activated by the complex c3_1-dCas9_VP64 but repressed by complexes c6_1-dCas9_VP64 and c7_1-dCas9_VP64. When any combination of those three complexes is expressed, inhibition prevails.
CYC_2 promoter
CYC_2 promoter acts as a transistor in yeast. It is activated by the complex c3_2-dCas9_VP64 but repressed by complexes c6_2-dCas9_VP64 and c7_2-dCas9_VP64. When any combination of those three complexes is expressed, inhibition prevails.
CYC_3 promoter
CYC_3 promoter acts as a transistor in yeast. It is activated by the complex c3_3-dCas9_VP64 but repressed by complexes c6_3-dCas9_VP64 and c7_3-dCas9_VP64. When any combination of those three complexes is expressed, inhibition prevails.
