Difference between revisions of "Part:BBa K3332000"
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<partinfo>BBa_K3332000 short</partinfo> | <partinfo>BBa_K3332000 short</partinfo> | ||
| − | It is a fusion protein of TEX and cpYFP that can be purified with the function of His-Tag on the N-terminal. We use it to construct a new part that can be purified with the function of His-Tag on the N-terminal and detect NADPH. | + | It is a fusion protein of TEX and cpYFP that can be purified with the function of His-Tag on the N-terminal. We use it to construct a new part that can be purified with the function of His-Tag on the N-terminal and detect NADPH. |
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iNap is a chimera of circularly permuted YFP (cpYFP) and the NADP(H) binding domain of Rex from ''Thermus aquaticus'' (T-Rex). iNap has an excitation peak around 420 nm and one emission peak near 528 nm. Upon NADPH binding, iNap show apparent fluorescence changes. <ref>Zou Y, Wang A, Shi M, et al. Analysis of redox landscapes and dynamics in living cells and in vivo using genetically encoded fluorescent sensors[J]. Nat Protoc, 2018, 13(10): 2362-2386.</ref><ref>Zhao Y, Hu Q, Cheng F, et al. SoNar, a Highly Responsive NAD+/NADH Sensor, Allows High-Throughput Metabolic Screening of Anti-tumor Agents[J]. Cell Metabolism, 2015, 21(5): 777-789.</ref><ref>Tao R, Zhao Y, Chu H, et al. Genetically encoded fluorescent sensors reveal dynamic regulation of NADPH metabolism[J]. Nat Methods, 2017, 14(7): 720-728.</ref> | iNap is a chimera of circularly permuted YFP (cpYFP) and the NADP(H) binding domain of Rex from ''Thermus aquaticus'' (T-Rex). iNap has an excitation peak around 420 nm and one emission peak near 528 nm. Upon NADPH binding, iNap show apparent fluorescence changes. <ref>Zou Y, Wang A, Shi M, et al. Analysis of redox landscapes and dynamics in living cells and in vivo using genetically encoded fluorescent sensors[J]. Nat Protoc, 2018, 13(10): 2362-2386.</ref><ref>Zhao Y, Hu Q, Cheng F, et al. SoNar, a Highly Responsive NAD+/NADH Sensor, Allows High-Throughput Metabolic Screening of Anti-tumor Agents[J]. Cell Metabolism, 2015, 21(5): 777-789.</ref><ref>Tao R, Zhao Y, Chu H, et al. Genetically encoded fluorescent sensors reveal dynamic regulation of NADPH metabolism[J]. Nat Methods, 2017, 14(7): 720-728.</ref> | ||
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| + | <html> | ||
| + | <figure> | ||
| + | <img src="https://2020.igem.org/wiki/images/9/94/T--XMU-China--XMU-China_2020-iNAP_Mechanism.png" width="40%" style="float:center"> | ||
| + | <figcaption> | ||
| + | <p style="font-size:1rem"> | ||
| + | </p> | ||
| + | </figcaption> | ||
| + | </figure> | ||
| + | </html> | ||
| − | :Fig 1. iNap mechanism | + | :'''Fig 1.''' iNap mechanism |
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Here, 6His-Tag is fused on the C-Terminal of iNap to help purify it, which can be used in vitro characterization of iNap sensors. Then, coding sequence of target gene iNap-Histag was inserted into an expression vectors with J23100 and RBS(<partinfo>BBa_K880005</partinfo>) to obtain <partinfo>BBa_K3332045</partinfo>. We transformed the constructed plasmid into ''E. coli'' BL21 (DE3) to verify its expression. | Here, 6His-Tag is fused on the C-Terminal of iNap to help purify it, which can be used in vitro characterization of iNap sensors. Then, coding sequence of target gene iNap-Histag was inserted into an expression vectors with J23100 and RBS(<partinfo>BBa_K880005</partinfo>) to obtain <partinfo>BBa_K3332045</partinfo>. We transformed the constructed plasmid into ''E. coli'' BL21 (DE3) to verify its expression. | ||
| + | |||
| + | <html> | ||
| + | <figure> | ||
| + | <img src="https://2020.igem.org/wiki/images/5/5a/T--XMU-China--XMU-China_2020-J23100_B0034_inap-his-tag_B0015_and_J23100_B0034_his-tag-inap_B0015.png" width="70%" style="float:center"> | ||
| + | <figcaption> | ||
| + | <p style="font-size:1rem"> | ||
| + | </p> | ||
| + | </figcaption> | ||
| + | </figure> | ||
| + | </html> | ||
| − | Fig 2. Gene circuit of iNap with Histag | + | :'''Fig 2.''' Gene circuit of iNap with Histag |
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After receiving the synthesized DNA, restriction digestion was done to certify that the plasmid was correct, and the experimental results were shown in figure 3. | After receiving the synthesized DNA, restriction digestion was done to certify that the plasmid was correct, and the experimental results were shown in figure 3. | ||
| − | + | <html> | |
| − | :Fig 3.DNA gel electrophoresis of restriction digest products of iNap-His-pSB1C3 (''Xba'' I & ''Pst'' I sites) | + | <figure> |
| + | <img src="https://2020.igem.org/wiki/images/e/ea/T--XMU-China--07161.png" width="90%" style="float:center"> | ||
| + | <figcaption> | ||
| + | <p style="font-size:1rem"> | ||
| + | </p> | ||
| + | </figcaption> | ||
| + | </figure> | ||
| + | </html> | ||
| + | :'''Fig 3.'''DNA gel electrophoresis of restriction digest products of iNap-His-pSB1C3 (''Xba'' I & ''Pst'' I sites) | ||
'''2. Purification and Proof of expression''' | '''2. Purification and Proof of expression''' | ||
| − | We used J23100 promoter and RBS (<partinfo>BBa_K880005</partinfo>) to highly express iNap-Histag in E. coli in our composite part <partinfo>BBa_K3332045</partinfo>. Then, we used GE AKTA Prime Plus FPLC System to get purified iNap protein. We found no apparent protein peak in AKTA FPLC System and cannot correct purified protein. | + | We used J23100 promoter and RBS (<partinfo>BBa_K880005</partinfo>) to highly express iNap-Histag in ''E. coli'' in our composite part <partinfo>BBa_K3332045</partinfo>. Then, we used GE AKTA Prime Plus FPLC System to get purified iNap protein. We found no apparent protein peak in AKTA FPLC System and cannot correct purified protein. |
It is supposed that iNap cannot work normally after fused with 6His-Tag in C-Terminal. | It is supposed that iNap cannot work normally after fused with 6His-Tag in C-Terminal. | ||
Latest revision as of 13:00, 27 October 2020
iNAP-his-tag
It is a fusion protein of TEX and cpYFP that can be purified with the function of His-Tag on the N-terminal. We use it to construct a new part that can be purified with the function of His-Tag on the N-terminal and detect NADPH.
Biology
iNap is a chimera of circularly permuted YFP (cpYFP) and the NADP(H) binding domain of Rex from Thermus aquaticus (T-Rex). iNap has an excitation peak around 420 nm and one emission peak near 528 nm. Upon NADPH binding, iNap show apparent fluorescence changes. [1][2][3]
- Fig 1. iNap mechanism
Usage
Here, 6His-Tag is fused on the C-Terminal of iNap to help purify it, which can be used in vitro characterization of iNap sensors. Then, coding sequence of target gene iNap-Histag was inserted into an expression vectors with J23100 and RBS(BBa_K880005) to obtain BBa_K3332045. We transformed the constructed plasmid into E. coli BL21 (DE3) to verify its expression.
- Fig 2. Gene circuit of iNap with Histag
Characterization
1. Identification
After receiving the synthesized DNA, restriction digestion was done to certify that the plasmid was correct, and the experimental results were shown in figure 3.
- Fig 3.DNA gel electrophoresis of restriction digest products of iNap-His-pSB1C3 (Xba I & Pst I sites)
2. Purification and Proof of expression
We used J23100 promoter and RBS (BBa_K880005) to highly express iNap-Histag in E. coli in our composite part BBa_K3332045. Then, we used GE AKTA Prime Plus FPLC System to get purified iNap protein. We found no apparent protein peak in AKTA FPLC System and cannot correct purified protein.
It is supposed that iNap cannot work normally after fused with 6His-Tag in C-Terminal.
Reference
- ↑ Zou Y, Wang A, Shi M, et al. Analysis of redox landscapes and dynamics in living cells and in vivo using genetically encoded fluorescent sensors[J]. Nat Protoc, 2018, 13(10): 2362-2386.
- ↑ Zhao Y, Hu Q, Cheng F, et al. SoNar, a Highly Responsive NAD+/NADH Sensor, Allows High-Throughput Metabolic Screening of Anti-tumor Agents[J]. Cell Metabolism, 2015, 21(5): 777-789.
- ↑ Tao R, Zhao Y, Chu H, et al. Genetically encoded fluorescent sensors reveal dynamic regulation of NADPH metabolism[J]. Nat Methods, 2017, 14(7): 720-728.
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 272
Illegal XhoI site found at 298 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 38
- 1000COMPATIBLE WITH RFC[1000]