Difference between revisions of "Part:BBa K2350017"
 
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<h3>J23118-B0034-NrtA-B0015</h3>
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__NOTOC__
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== Short Description ==
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<partinfo>BBa_K2350017 short</partinfo>
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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.
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== NrtA (a part of nitrate channel protein from Synechocystis sp. PCC 6803) ==
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This part codes a part of nitrate channel protein from cyanobacteria Synechocystis sp. PCC 6803 which plays the role of catching nitrate ion from the environment.
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== Usage and Biology ==  
 
== Usage and Biology ==  
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NrtA is a high-affinity periplasmic solute-binding lipoprotein .It is composed of two domains. Both domains consist of a five-stranded mixed -sheet surrounded by helices. Nitrate occupies the cleft between the two domains in catching phase. Like most bacterial periplasmic-binding proteins, NrtA is
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tethered to the membrane surface by a lipidated cysteine  and a very long flexible linker rich in glycine and serine. Therefore NrtA is akin to a ‘‘balloon on a string’’with its solute-binding domain capturing nitrate/nitrite in the periplasm and transporting it to the NrtB transmembrane permease.
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NrtA is a high-affinity nitrate/nitrite-binding lipoprotein .On Synechocystis sp. PCC 6803, it is 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 sheets surrounded by helices. When catch occurs, 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 is the possible answer that nitrate and nitrite have almost the same affinity on NrtA protein.
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The order of the strands within domain from some researchers’ point of view suggest that NrtA belongsto class II of the periplasmic-binding protein (PBP) superfamily (7) which also includes the oxyanion-binding proteins.
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These structures consist of two globular domains of mixed sheet flanked by helices, with the solute-binding cleft located between the two domains. However, NrtA has an extra 100aa extending from the C terminus. These residues comprise several -helices and a two-stranded antiparallel -sheet that
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cradle the back of the C-clamp. This architectural feature provides structural support for the nitrate-binding pocket or plays a role in the conformational changes associated with solute transport.
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The resonance state of the nitrate anion in NrtA binding pocket evenly distributes the negative charge among the three oxygen atoms. However, the environment surrounding the nitrate oxygens in the NrtA binding pocket is clearly asymmetric.
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It seems likely that the O1 and O3 interactions will remain the same but that the O2 atom and its interactions with the protein will be absent in the case of nitrite. O2 in the bound nitrate molecule has relatively few interactions and, hence, maybe the reason why NrtA binds nitrate and nitrite with approximately the
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same affinity.
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We arrange the coding region with a moderate constitutive promoter, and then nrtA will be all over the modified microbe’s cell membrane.
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== Analysis ==  
 
== Analysis ==  
  
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BG11, competent cell, NrtA have no significant difference.
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To test whether NrtA protein could capture nitrate and nitrite, we used French Pressure Press to lyse the cell. After dialysis of the cell lysates containing NrtA protein, we used Cayman Nitrate/Nitrite Colorimetric Assay Kit to measure the nitrate and nitrite concentration in the medium.
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Howere there is a significant difference between competent cell and NrtA.This represents that NrtA is useful.
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In addition, after the competent cell is milled , the E.coli itself will have the nitrate production be placed inside the medium to increase the amount of nitrate.
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In short, NrtA can also combine nitrates.
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We know that E.coli, which has not been shattered, has no significant difference in the case of containing NrtA and no NrtA. Both will reduce the nitrate, so we may be towards the NrtA into a secreted protein in the future and it can release into the medium and then absorb the nitrate.
 
  
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'''Figure 1.'''
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Nitrate absorbance of different cell lysate. <br>
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Blank: nitrate concentration assay kit assay buffer.
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CC: competent cell.
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BG11: microalgae culture medium buffer.
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NrtA: NrtA producing E.coli.                   
  
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[[File:T--NYMU-Taipei--NrtA-abc.jpeg|650px]]
  
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Figure 1
 
  
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'''Table 1.''' Dunnett t test.  Blank: nitrate concentration assay kit assay buffer. CC: competent cell. BG11: microalgae culture medium buffer. NrtA: NrtA producing E.coli.
  
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[[File:T--NYMU-Taipei--NrtA-Table-1.png]]
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The nitrate and nitrite concentrations of competent cell and NrtA were significantly different. The results indicated that NrtA protein can capture nitrite and/or nitrate.
  
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[[Image:S_6878288491476.png]]
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Then we wanted to know whether the engineered E.coli could decrease nitrate and nitrite in the medium. We used Cayman Nitrate/Nitrite Colorimetric Assay Kit to measure the nitrate and nitrite concentration of the supernatant of the engineered and native E.coli liquid culture.
  
  
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Figure 2
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'''Figure 2.''' Nitrate absorbance of cell.<br>
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Blank: nitrate concentration assay kit assay buffer. CC: competent cell. BG11: microalgae culture medium buffer. NrtA: NrtA producing E.coli.                       
  
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[[File:T--NYMU-Taipei--NrtA-ab.jpeg|650px]]
  
  
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[[Image:S_6878288739507.png]]
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'''Table 2.''' Dunnett t test. Blank: nitrate concentration assay kit assay buffer. CC: competent cell. BG11: microalgae culture medium buffer. NrtA: NrtA producing E.coli.
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[[Image:S_NRTABBB.jpg|650px]]
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The nitrate and nitrite concentrations 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 could not. The engineered E.coli with NrtA gene could express NrtA protein, and the NrtA protein might be inside the cell, so nitrate and nitrite concentration outside the cell did not change.
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== References ==
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1. Atomic structure of a nitrate-binding protein crucial for photosynthetic productivity
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Nicole M. Koropatkin, Himadri B. Pakrasi, and Thomas J. Smith
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Koropatkin et al.PNAS  June 27, 2006  vol. 103  no. 26  9821
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2. Substrate-binding Lipoprotein of the Cyanobacterium Synechococcus sp. Strain PCC 7942 Involved in the Transport of Nitrate and Nitrite*
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Shin-ichi Maeda and Tatsuo Omata.
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The Journal of Biological Chemistry Vol. 272, No. 5, Issue of January 31, pp. 3036 –3041, 1997
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3. Genetically engineered mutant of the cyanobacterium Synechococcus PCC 7942 defective in nitrate transport
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Tatsuo Omata, Masayuki Ohmorit, Nobuyuki Arai, and Teruo Ogawa.
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Proc. Nati. Acad. Sci. USA Vol. 86, pp. 6612-6616, September 1989
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__NOTOC__
 
 
<partinfo>BBa_K2350017 short</partinfo>
 
<partinfo>BBa_K2350017 short</partinfo>
  
 
<partinfo>BBa_K2350017 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2350017 SequenceAndFeatures</partinfo>

Latest revision as of 13:56, 1 November 2017

J23118-B0034-NrtA-B0015

NrtA (a part of nitrate channel protein from Synechocystis sp. PCC 6803)

This part codes a part of nitrate channel protein from cyanobacteria 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 is 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 sheets surrounded by helices. When catch occurs, 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 is the possible answer that nitrate and nitrite have almost the same affinity on NrtA protein.

Analysis

To test whether NrtA protein could capture nitrate and nitrite, we used French Pressure Press to lyse the cell. After dialysis of the cell lysates containing NrtA protein, we used Cayman Nitrate/Nitrite Colorimetric Assay Kit to measure the nitrate and nitrite concentration in the medium.


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.

T--NYMU-Taipei--NrtA-abc.jpeg


Table 1. Dunnett t test. Blank: nitrate concentration assay kit assay buffer. CC: competent cell. BG11: microalgae culture medium buffer. NrtA: NrtA producing E.coli.

T--NYMU-Taipei--NrtA-Table-1.png

The nitrate and nitrite concentrations of competent cell and NrtA were significantly different. The results indicated that NrtA protein can capture nitrite and/or nitrate.

Then we wanted to know whether the engineered E.coli could decrease nitrate and nitrite in the medium. We used Cayman Nitrate/Nitrite Colorimetric Assay Kit to measure the nitrate and nitrite concentration of the supernatant of the engineered and native 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.

T--NYMU-Taipei--NrtA-ab.jpeg


Table 2. Dunnett t test. Blank: nitrate concentration assay kit assay buffer. CC: competent cell. BG11: microalgae culture medium buffer. NrtA: NrtA producing E.coli.

S NRTABBB.jpg

The nitrate and nitrite concentrations 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 could not. The engineered E.coli with NrtA gene could express NrtA protein, and the NrtA protein might be inside the cell, so nitrate and nitrite concentration outside the cell did not change.

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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1240
    Illegal BamHI site found at 610
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]