Part:BBa_K2350017
J23118-B0034-NrtA-B0015
NrtA (a part of nitrate channel protein from Synechocystis sp. PCC 6803)
This part produces a part of nitrate channel protein from Synechocystis sp. PCC 6803 which plays the role of catching nitrate ion from the environment.
Usage and Biology
NrtA is a high-affinity nitrate/nitrite-binding lipoprotein .On Synechocystis sp. PCC 6803,it’s tethered to the cell membrane by a lipidated cysteine and a flexible linker rich in glycine and serine.The two domains of it are both composed of five-stranded mixed sheet surrounded by helices.When catch occur,the nitrate ion is on the middle region of two domains.The resonance state of the nitrate during binding distributes evenly among the three oxygen atoms. However, the second oxygen atom and its interactions with the affinity protein will be absent in the case of nitrite. The second oxygen atom in the bound nitrate molecule also has relatively few interactions.It’s the possible answer that nitrate and nitrite have almost the same affinity on NrtA protein. We arrange the coding region with a moderate constitutive promoter, and then nrtA will be all over the modified microbe’s cell membrane.
Analysis
To test whether NrtA protein could capture nitrate and nitrite, we used French Pressure Press to isolate NrtA protein, and used Cayman Nitrate/Nitrite Colorimetric Assay Kit to measure the nitrate and nitrite concentration in the medium with NrtA protein.
Figure 1. Nitrate absorbance of different cell lysate. Blank:nitrate concentration assay kit assay buffer CC:competent cell BG11:microalgae culture medium buffer NrtA:NrtA producing E.coli.
Table 1. Blank:nitrate concentration assay kit assay buffer CC:competent cell BG11:microalgae culture medium buffer NrtA:NrtA producing E.coli.
The nitrate and nitrite concentration of competent cell and NrtA was significant different. The results indicated that NrtA protein can capture nitrite and nitrate.
Then we wanted to know whether the engineered E.coli could decrease nitrate and nitrite in the environment. We used Cayman Nitrate/Nitrite Colorimetric Assay Kit to measure the nitrate and nitrite concentration of the supernatant of the engineered E.coli liquid culture.
Figure 2. Nitrate absorbance of cell. Blank:nitrate concentration assay kit assay buffer CC:competent cell BG11:microalgae culture medium buffer NrtA:NrtA producing E.coli.
Table 2. Dunnett 3. Blank:nitrate concentration assay kit assay buffer CC:competent cell BG11:microalgae culture medium buffer NrtA:NrtA producing E.coli
The nitrate and nitrite concentration of NrtA and competent cell had slight but not significant difference. The result implied that NrtA protein could capture nitrate and nitrite while the engineered E.coli with NrtA gene couldn’t. The engineered E.coli with NrtA gene could express NrtA protein, while NrtA protein might be inside the cell, so nitrate and nitrite concentration outside the cell didn’t change a lot.
References
1. Atomic structure of a nitrate-binding protein crucial for photosynthetic productivity Nicole M. Koropatkin, Himadri B. Pakrasi, and Thomas J. Smith Koropatkin et al.PNAS June 27, 2006 vol. 103 no. 26 9821
2. Substrate-binding Lipoprotein of the Cyanobacterium Synechococcus sp. Strain PCC 7942 Involved in the Transport of Nitrate and Nitrite* Shin-ichi Maeda and Tatsuo Omata. The Journal of Biological Chemistry Vol. 272, No. 5, Issue of January 31, pp. 3036 –3041, 1997
3. Genetically engineered mutant of the cyanobacterium Synechococcus PCC 7942 defective in nitrate transport Tatsuo Omata, Masayuki Ohmorit, Nobuyuki Arai, and Teruo Ogawa. Proc. Nati. Acad. Sci. USA Vol. 86, pp. 6612-6616, September 1989
J23118-B0034-NrtA-B0015
- 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 BglII site found at 1240
Illegal BamHI site found at 610 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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