Team:NAU-CHINA/Parts
parts
anti-GFP-mnotch-TEV protease--NLS
Long description：The Notch receptor is a natural cell surface receptor whose extramembrane domain can be replaced by a variety of receptors, and the intramembrane domain can be replaced with a variety of transcription factors to construct a customized cell signaling pathway. We retained the conserved mmotch transmembrane domain, replacing the extracellular domain with anti-GFP and the intracellular domain with the TEV protease, thereby converting the extracellular signal into an intracellular signal.
Source：We borrowed the Anti-GFP-mnotch-tetR-VP64-V2 plasmid from Fudan University. After the TEV protease sequence used by the iGEM17_Oxford group was found on the iGEM website, it was sent to Kingsray for synthesis.
Design considerations：Since we need TEV protease to enter the nucleus, we added SV40 NLS to the back of the TEV protease.Since we used animal cells as the chassis organism, the coding sequence was humanized.
Figure 1. Effects of cell surface-expressed GFP on synNotch-TEV. A. 1.Fluorescence microscope observation of HEK 293 T transfected with plasmid containing synNotch. The cells were cross-linked with GFP. The results showed that synNotch can be located to the membrane. 2.Blank control (without transfection). B. Assay of the synNotch-TEV and FLAG-tagged TEV concentration affected by cell surface-expressed GFP. C,D. Relative level of the synNotch-TEV and FLAG-tagged TEV affected by cell surface-expressed GFP. synNotch-TEV affected by surface-expressed GFP can be resolved into FLAG-TEV and V5-mNotch. Data are mean ±S.E. (n=3). **, p < 0.01; N.S., no significance.
Bxb1 recombinase
Long description：Bxb1 is a widely studied recombinase that we use to mediate intracellular signals.
Source：We found the coding sequence of Bxb1 on NCBI and, after humanization, sent it to Kingsray for synthesis.
Design considerations：Since we used animal cells as the chassis organism, the coding sequence was humanized.
Bxb1 attB-RFP-Bxb1 attP
Long description：We added Bxb1 recombination sites, attB and attP, at both ends of the reporter gene RFP (Inverted). When Bxb1 is expressed, attB and attP are recognized, and the sequence of RFP is inverted to enable normal expression.
Source：RFP? After the recombination site of Bxb1 was found in the literature, it was sent to Kingsray for synthesis.
Design considerations：The sequence of the RFP is inverted. To facilitate cloning, we randomly added some bases on both sides of attB and attP.
Bxb1 attB-Bxb1 attP
Long description：Bxb1-attB and Bxb1-attP are recognition and recombination sites for Bxb1 recombinase.
Source：After the recombination site of Bxb1 was found in the literature, it was sent to Kingsray for synthesis.
Design considerations：To facilitate cloning, we randomly added some bases on both sides of attB and attP.
PhiC31 recombinase
Long description：PhiC31 is a widely studied recombinase that we use to mediate intracellular signals.
Source：We found the coding sequence of PhiC31 on NCBI and, after humanization, sent it to Kingsray for synthesis.
Design considerations：Since we used animal cells as the chassis organism, the coding sequence was humanized.
PhiC31 attB-mCherry-Bxb1 attP
Long description：We added PhiC31 recombination sites, attB and attP, at both ends of the reporter gene mCherry (Inverted). When PhiC31 is expressed, attB and attP are recognized, and the sequence of RFP is inverted to enable normal expression.
Source： After the recombination site of PhiC31 was found in the literature, it was sent to Kingsray for synthesis.
Design considerations：The sequence of the mCherry is inverted. To facilitate cloning, we randomly added some bases on both sides of attB and attP.
PhiC31 attB-Bxb1 attP
Long description：PhiC31-attB and PhiC31-attP are recognition and recombination sites for PhiC31 recombinase.
Source：After the recombination site of PhiC31 was found in the literature, it was sent to Kingsray for synthesis.
Design considerations：To facilitate cloning, we randomly added some bases on both sides of attB and attP.
TP901 recombinase
Long description：TP901 is a widely studied recombinase that we use to mediate intracellular signals.
Source：We found the coding sequence of TP901 on NCBI and, after humanization, sent it to Kingsray for synthesis.
Design considerations：Since we used animal cells as the chassis organism, the coding sequence was humanized.
TP901 attB-mCherry-TP901 attP
Long description：We added TP901 recombination sites, attB and attP, at both ends of the reporter gene mCherry (Inverted). When PhiC31 is expressed, attB and attP are recognized, and the sequence of mCherry is inverted to enable normal expression.
Source： After the recombination site of TP901 was found in the literature, it was sent to Kingsray for synthesis.
Design considerations：The sequence of the mCherry is inverted. To facilitate cloning, we randomly added some bases on both sides of attB and attP.
TP901 attB-TP901 attP
Long description：TP901-attB and TP901-attP are recognition and recombination sites for TP901 recombinase.
Source：After the recombination site of TP901 was found in the literature, it was sent to Kingsray for synthesis.
Design considerations：To facilitate cloning, we randomly added some bases on both sides of attB and attP.
TetO-miniCMV promoter-Bxb1
Long description：The TetO sequence, SV40 NLS, flag tag, miniCMV promoter, Bxb1 recombinase are sequentially linked and involved in intracellular signal processing. According to the feedback of the digital model, the different strength promoters upstream of the recombinase and the recombination efficiency of the recombinase will affect the stability of the system. Therefore, in order to optimize our system, we used three promoters and three recombinases, hoping to find the optimal solution.
Source:The TetO sequence was cloned from a Commercialized plasmid. The miniCMV promoter was cloned from ?We found the coding sequence of Bxb1 on NCBI and, after humanization, sent it to Kingsray for synthesis.
Design considerations：SV40 NLS and flag was added by PCR.
TetO-EF1αpromoter-Bxb1
Long description：The TetO sequence, SV40 NLS, flag tag, EF1α promoter, and Bxb1 recombinase are ligated in sequence, and participate in intracellular signal processing.According to the feedback of the digital model, the different strength promoters upstream of the recombinase and the recombination efficiency of the recombinase will affect the stability of the system. Therefore, in order to optimize our system, we used three promoters and three recombinases, hoping to find the optimal solution.
Source:The TetO sequence was cloned from a Commercialized plasmid. The EF1αpromoter was cloned from ?We found the coding sequence of Bxb1 on NCBI and, after humanization, sent it to Kingsray for synthesis.
Design considerations：SV40 NLS and flag was added by PCR.
TetO-Ubc promoter-Bxb1
Long description：The TetO sequence, SV40 NLS, flag tag, Ubc promoter, and Bxb1 recombinase are ligated in sequence, and participate in intracellular signal processing.According to the feedback of the digital model, the different strength promoters upstream of the recombinase and the recombination efficiency of the recombinase will affect the stability of the system. Therefore, in order to optimize our system, we used three promoters and three recombinases, hoping to find the optimal solution.
Source:The TetO sequence was cloned from a Commercialized plasmid. The Ubc promoter was cloned from ?We found the coding sequence of Bxb1 on NCBI and, after humanization, sent it to Kingsray for synthesis.
Design considerations：SV40 NLS and flag was added by PCR.
TetO-miniCMV-PhiC31
Long description：The TetO sequence, SV40 NLS, flag tag, minCMV promoter, and PhiC31 recombinase are ligated in sequence, and participate in intracellular signal processing.According to the feedback of the digital model, the different strength promoters upstream of the recombinase and the recombination efficiency of the recombinase will affect the stability of the system. Therefore, in order to optimize our system, we used three promoters and three recombinases, hoping to find the optimal solution.
Source:The TetO sequence was cloned from a Commercialized plasmid. The miniCMV promoter was cloned from ?We found the coding sequence of PhiC31 on NCBI and, after humanization, sent it to Kingsray for synthesis.
Design considerations：SV40 NLS and flag was added by PCR.
TetO-EF1α-PhiC31
Long description：The TetO sequence, SV40 NLS, flag tag, EF1α promoter, and PhiC31 recombinase are ligated in sequence, and participate in intracellular signal processing. According to the feedback of the digital model, the different strength promoters upstream of the recombinase and the recombination efficiency of the recombinase will affect the stability of the system. Therefore, in order to optimize our system, we used three promoters and three recombinases, hoping to find the optimal solution.
Source:The TetO sequence was cloned from a Commercialized plasmid. The EF1α promoter was cloned from ?We found the coding sequence of PhiC31 on NCBI and, after humanization, sent it to Kingsray for synthesis.
Design considerations：SV40 NLS and flag was added by PCR.
插细胞荧光图
TetO-Ubc promoter-PhiC31
Long description：The TetO sequence, SV40 NLS, flag tag, Ubc promoter, and PhiC31 recombinase are ligated in sequence, and participate in intracellular signal processing. According to the feedback of the digital model, the different strength promoters upstream of the recombinase and the recombination efficiency of the recombinase will affect the stability of the system. Therefore, in order to optimize our system, we used three promoters and three recombinases, hoping to find the optimal solution.
Source:The TetO sequence was cloned from a Commercialized plasmid. The Ubc promoter was cloned from ?We found the coding sequence of PhiC31 on NCBI and, after humanization, sent it to Kingsray for synthesis.
Design considerations：SV40 NLS and flag was added by PCR.
TetO-miniCMV promoter-TP901
Long description：The TetO sequence, SV40 NLS, flag tag, miniCMV promoter, and TP901 recombinase are ligated in sequence to participate in intracellular signal processing. According to the feedback of the digital model, the different strength promoters upstream of the recombinase and the recombination efficiency of the recombinase will affect the stability of the system. Therefore, in order to optimize our system, we used three promoters and three recombinases, hoping to find the optimal solution.插细胞荧光图
Source:The TetO sequence was cloned from a Commercialized plasmid. The miniCMV promoter was cloned from ?We found the coding sequence of TP901 on NCBI and, after humanization, sent it to Kingsray for synthesis.
Design considerations：SV40 NLS and flag was added by PCR.
Figure XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
TetO-EF1α-TP901
Long description：The TetO sequence, SV40 NLS, flag tag, EF1α promoter, and Tp901 recombinase are ligated in sequence, and participate in intracellular signal processing. According to the feedback of the digital model, the different strength promoters upstream of the recombinase and the recombination efficiency of the recombinase will affect the stability of the system. Therefore, in order to optimize our system, we used three promoters and three recombinases, hoping to find the optimal solution.
Source:The TetO sequence was cloned from a Commercialized plasmid. The EF1α promoter was cloned from ?We found the coding sequence of TP901 on NCBI and, after humanization, sent it to Kingsray for synthesis.
Design considerations：SV40 NLS and flag was added by PCR.
Figure XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
TetO-Ubc promoter-TP901
Long description：The TetO sequence, SV40 NLS, flag tag, Ubc promoter, and TP901 recombinase are ligated in sequence, and participate in intracellular signal processing. According to the feedback of the digital model, the different strength promoters upstream of the recombinase and the recombination efficiency of the recombinase will affect the stability of the system. Therefore, in order to optimize our system, we used three promoters and three recombinases, hoping to find the optimal solution.
Source:The TetO sequence was cloned from a Commercialized plasmid. The Ubc promoter was cloned from ?We found the coding sequence of TP901on NCBI and, after humanization, sent it to Kingsray for synthesis.
Design considerations：SV40 NLS and flag was added by PCR.
miniCMV promoter-Bxb1-RDF
Long description：Bxb1-RDF recognizes the Bxb1 attL and Bxb1 attR sequences, so the intermediate sequence can be flipped back and the reporter gene expression is turned off. According to the feedback of the digital model, the different strength promoters upstream of the RDF and the recombination efficiency of the RDF will affect the stability of the system. Therefore, in order to optimize our system, we used three promoters and three RDFs, hoping to find the optimal solution.
Source:Bxb1-RDF is a fusion protein of Bxb1 and the corresponding RDF. We obtained their sequences from the literature and, after humanization, sent them to Kingsray for synthesis.
Design considerations：In order to allow Bxb1-RDF to enter the nucleus, we added SV40 NLS after PCR.
EF1αpromoter-Bxb1-RDF
Long description：Bxb1-RDF recognizes the Bxb1 attL and Bxb1 attR sequences, so the intermediate sequence can be flipped back and the reporter gene expression is turned off. According to the feedback of the digital model, the different strength promoters upstream of the RDF and the recombination efficiency of the RDF will affect the stability of the system. Therefore, in order to optimize our system, we used three promoters and three RDFs, hoping to find the optimal solution.
Source:Bxb1-RDF is a fusion protein of Bxb1 and the corresponding RDF. We obtained their sequences from the literature and, after humanization, sent them to Kingsray for synthesis.
Design considerations：In order to allow Bxb1-RDF to enter the nucleus, we added SV40 NLS after PCR.
Ubc promoter-Bxb1-RDF
Long description：Bxb1-RDF recognizes the Bxb1 attL and Bxb1 attR sequences, so the intermediate sequence can be flipped back and the reporter gene expression is turned off. According to the feedback of the digital model, the different strength promoters upstream of the RDF and the recombination efficiency of the RDF will affect the stability of the system. Therefore, in order to optimize our system, we used three promoters and three RDFs, hoping to find the optimal solution.
Source:Bxb1-RDF is a fusion protein of Bxb1 and the corresponding RDF. We obtained their sequences from the literature and, after humanization, sent them to Kingsray for synthesis.
Design considerations：In order to allow Bxb1-RDF to enter the nucleus, we added SV40 NLS after PCR.
miniCMV promoter-PhiC31
Long description：PhiC31-RDF recognizes the PhiC31 attL and PhiC31 attR sequences, so the intermediate sequence can be flipped back and the reporter gene expression is turned off. According to the feedback of the digital model, the different strength promoters upstream of the RDF and the recombination efficiency of the RDF will affect the stability of the system. Therefore, in order to optimize our system, we used three promoters and three RDFs, hoping to find the optimal solution.
Source:PhiC31-RDF is a fusion protein of PhiC31 and the corresponding RDF. We obtained their sequences from the literature and, after humanization, sent them to Kingsray for synthesis.
Design considerations：In order to allow PhiC31-RDF to enter the nucleus, we added SV40 NLS after PCR.
EF1αpromoter-PhiC31
Long description：PhiC31-RDF recognizes the PhiC31 attL and PhiC31 attR sequences, so the intermediate sequence can be flipped back and the reporter gene expression is turned off. According to the feedback of the digital model, the different strength promoters upstream of the RDF and the recombination efficiency of the RDF will affect the stability of the system. Therefore, in order to optimize our system, we used three promoters and three RDFs, hoping to find the optimal solution.
Source:PhiC31-RDF is a fusion protein of PhiC31 and the corresponding RDF. We obtained their sequences from the literature and, after humanization, sent them to Kingsray for synthesis.
Design considerations：In order to allow PhiC31-RDF to enter the nucleus, we added SV40 NLS after PCR.
Ubc promoter--PhiC31-RDF
Long description：PhiC31-RDF recognizes the PhiC31 attL and Bxb1 attR sequences, so the intermediate sequence can be flipped back and the reporter gene expression is turned off. According to the feedback of the digital model, the different strength promoters upstream of the RDF and the recombination efficiency of the RDF will affect the stability of the system. Therefore, in order to optimize our system, we used three promoters and three RDFs, hoping to find the optimal solution.
Source:PhiC31-RDF is a fusion protein of PhiC31 and the corresponding RDF. We obtained their sequences from the literature and, after humanization, sent them to Kingsray for synthesis.
Design considerations：In order to allow PhiC31-RDF to enter the nucleus, we added SV40 NLS after PCR.
miniCMV promoter-TP901-RDF
Long description：TP901-RDF recognizes the TP901 attL and TP901 attR sequences, so the intermediate sequence can be flipped back and the reporter gene expression is turned off. According to the feedback of the digital model, the different strength promoters upstream of the RDF and the recombination efficiency of the RDF will affect the stability of the system. Therefore, in order to optimize our system, we used three promoters and three RDFs, hoping to find the optimal solution.
Source:TP901-RDF is a fusion protein of Bxb1 and the corresponding RDF. We obtained their sequences from the literature and, after humanization, sent them to Kingsray for synthesis.
Design considerations：In order to allow TP901-RDF to enter the nucleus, we added SV40 NLS after PCR.
EF1αpromoter--TP901-RDF
Long description：TP901-RDF recognizes the TP901 attL and TP901attR sequences, so the intermediate sequence can be flipped back and the reporter gene expression is turned off. According to the feedback of the digital model, the different strength promoters upstream of the RDF and the recombination efficiency of the RDF will affect the stability of the system. Therefore, in order to optimize our system, we used three promoters and three RDFs, hoping to find the optimal solution.
Source:TP901-RDF is a fusion protein of TP901 and the corresponding RDF. We obtained their sequences from the literature and, after humanization, sent them to Kingsray for synthesis.
Design considerations：In order to allow TP901-RDF to enter the nucleus, we added SV40 NLS after PCR.
Ubc promoter--TP901-RDF
Long description：TP901-RDF recognizes the TP901 attL and TP901 attR sequences, so the intermediate sequence can be flipped back and the reporter gene expression is turned off. According to the feedback of the digital model, the different strength promoters upstream of the RDF and the recombination efficiency of the RDF will affect the stability of the system. Therefore, in order to optimize our system, we used three promoters and three RDFs, hoping to find the optimal solution.
Source:TP901-RDF is a fusion protein of TP901 and the corresponding RDF. We obtained their sequences from the literature and, after humanization, sent them to Kingsray for synthesis.
Design considerations：In order to allow TP901-RDF to enter the nucleus, we added SV40 NLS after PCR.
TetO-miniCMV promoter- EGFP
Long description：The miniCMV promoter and the EGFP coding sequence were ligated in sequence for characterizing the promoter strength.
Source:The miniCMV promoter was cloned from? The EFGP was cloned from?
Design considerations：In order to facilitate the construction of TetO-miniCMV promoter -recombinase parts, We added the TetO sequence before the miniCMV promoter. However, in the absence of TetR, The TetO sequence does not affect the strength of the promoter, that is, it does not affect the expression of EGFP.
Figure. miniCMV-EGFP
Figure. miniCMV-EGFP + TetR
TetO-EF1α promoter -EGFP
Long description：The EF1α promoter and EGFP coding sequences were ligated in sequence to characterize promoter strength.
Source:The EF1α promoter was cloned from? The EFGP was cloned from?
Design considerations：In order to facilitate the construction of TetO-EF1αpromoter -recombinase parts, We added the TetO sequence before the EF1α promoter. However, in the absence of TetR, The TetO sequence does not affect the strength of the promoter, that is, it does not affect the expression of EGFP.
Figure. EF1α-EGFP
Figure. EF1α-EGFP + TetR
TetO-Ubc promoter- EGFP
Long description：The Ubc promoter and EGFP coding sequences were ligated in sequence for characterizing promoter strength.
Source:The Ubc promoter was cloned from? The EFGP was cloned from?
Design considerations：In order to facilitate the construction of TetO-Ubc promoter -recombinase parts, We added the TetO sequence before the Ubc promoter. However, in the absence of TetR, The TetO sequence does not affect the strength of the promoter, that is, it does not affect the expression of EGFP.
Figure. EF1α-EGFP
Figure. EF1α-EGFP + TetR