Part:BBa_K3332046
J23100-RBS-his-tag-iNAP-terminator
This is a fusion protein of REX 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.
His-tag-iNAP
A fusion protein of REX and cpYFP that can be purified with the function of His-Tag on the C-terminal.We ues it to construct a new part that can be purified with the function of His-Tag on the C-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 shows apparent fluorescence changes. [1][2][3]
- Fig 1. iNap mechanism
Usage
Here, 6His-Tag is fused on the N-Terminal of iNap to help purify it, which can be used in vitro characterization of iNap sensors. Then, coding sequence of target gene Histag-iNap was inserted into an expression vectors with J23100 and RBS(BBa_K880005) to obtain BBa_K3332046. We transformed the constructed plasmid into E. coli BL21 (DE3) to verify its successful 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 figure3.
- Fig 3. DNA gel electrophoresis of restriction digest products of His-iNap-pSB1C3 (Xba I & Pst I sites)
2. Purification and Proof of the expression
We used J23100 promoter and RBS (BBa_K880005) to highly express Histag-iNap in E. coli in our composite part BBa_K3332046. Then, we used GE AKTA Prime Plus FPLC System to get purified iNap protein. We found an apparent protein peak in AKTA FPLC System and corrected purified protein.
Then, our target bands are observed through SDS-PAGE and the experimental results are shown in figure 4.
- Fig 4. SDS-PAGE of purification products of Histag-iNap-pSB1C3
3. Ability of sensing NADPH
We mixed NADPH solutions half-in-half with purified iNap protein dissolved in Tris-HCl (pH=7.5) and measure fluorescence changes in the presence of different concentrations of NADPH. TECAN® Infinite M200 Pro was used to detect fluorescence intensity. The samples were excited in 420 nm and the emission was measured at 528 nm.
We successfully got a working curve of iNap in the presence of different concentrations of NADPH. The result is shown in figure 5.
- Fig 5. iNap fluorescence intensity with excitation at 420 nm in the presence of different concentrations of NADPH. Emission was measured at 528 nm.
References
- ↑ 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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 351
Illegal XhoI site found at 377 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 117
- 1000COMPATIBLE WITH RFC[1000]
| None |