Part:BBa_K2371000
N-t7-dCas9
Figure 1. Cartoon expression of the part N-T7-dCas9.
dCas9 is a catalytically dead Cas9 protein that can bind with target sequence with the help of guide RNA(sgRNA). T7 RNA polymerase is a widely used polymerase from the T7 bacteriophage. BGIC-Union split the T7 polymerase into two separate parts(NT7:BBa_2371002 and CT7:BBa_2371003) and connect each of them to dCas9 protein via a linker.{N-T7-dCas9(BBa_K2371000) and C-T7-dCas9(BBa_K2371001)}
Figure 2. The schematic illustration of paired dCas9 system(1). dCas9 is connected to one of the piece of split T7 RNA polymerase(NT7 and CT7). They will combine with sgRNA beforehand and constitute the split T7-dCas9-sgRNA complex.
Each complex by itself is inactive, but when the two complex attached to particular sites we choose for identification of special sequences, they will reassemble to form a completed active T7 polymerase and start transcription in the presence of T7 promotor in cell free environment. Thus, this paired report system can convert the signal of specific cancerous gene into various report signals in cell free system. (i.e. GFP, LacZ, RFP)
Figure 3. The schematic illustration of paired dCas9 system(2). With the presence of target DNA, each complex will bind with pre-designed sgRNA-binding site on the sequence. When the split T7 polymerase approach each other close enough, they will become active and start transcription by adding report gene with T7 promotor in cell free system. After transcription, the mRNA will be translated into report protein like GFP and RFP which generate signal output.
We constructed expression system by inserting the sequence into pet28a plasmid. They were then transformed into Eco.li BL21(DE3), whose genome is genetically modified to contain the coding sequence for T7 RNA polymerase.
Figure 4. Plasmid of NT7-dCas9 on pet28a backbone. Our expression plasmid on pet28a backbone of NT7-dCas9 includes a T7 promotor, a lac repressor, the NT7-Linker-dCas9 coding sequence, a 6 X His-Tag and a T7 Terminator.
Figure 5. Plasmid of CT7-dCas9 on pet28a backbone. Our expression plasmid of CT7-dCas9 on pet28a backbone includes a T7 promotor, a lac repressor, the CT7-Linker-dCas9 coding sequence, a 6 X His-Tag and a T7 Terminator.
Then, we conducted protein induction and purification.
First, a gradient of IPTG was tested to obtain the best induction concentration.
Figure 6. PAGE of NT7-dCas9. The molecular weight of this protein is around 200KD. Alternative of Supernate and sediment with a gradient of IPTG concentrations(100uM, 200uM, 300uM, 400uM) were tested to find the best induction concentration of IPTG.
Figure 7. PAGE of CT7-dCas9. The molecular weight of this protein is around 200KD. Alternative of Supernate and sediment with a gradient of IPTG concentrations(100uM, 200uM, 300uM, 400uM) were tested to find the best induction concentration of IPTG.
With all the four concentration(100uM, 200uM, 300uM, 400uM), we concluded that 200uM is the best induction concentration for four of them.
After determining that we have successfully induced all of the four proteins, we adopted a commercial kit to purify the samples. (Capturem™ His-Tagged Purification Miniprep Kit, Clontech)
Figure 8. The PAGE of NT7-dCas9, CT7-dCas9, Nfluc-dCas9,Cfluc-dCas9 purified solution and Flowthrough. The red circle indicated the expected protein.
As the figure shows, we have successfully purified the protein.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1973
Illegal BamHI site found at 1708 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 2800
Illegal NgoMIV site found at 3904
Illegal NgoMIV site found at 3977
Illegal NgoMIV site found at 4462
Illegal NgoMIV site found at 5371 - 1000COMPATIBLE WITH RFC[1000]
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