Difference between revisions of "Part:BBa K3332051"
 
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===Biology===
 
===Biology===
  
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;As massive glyphosate has been over used all over the world, a world-wide evolution of glyphosate resistant (GR) weed species happens. An economically damaging grass weed called ''Echinochloa colona'' being an example. GOX, also known as EcAKR4-1, is found in ''Echinochloa colona''. It can decompose glyphosate into AMPA and glyoxylic acid.  <ref>Pan L, Yu Q, Han H, et al. Aldo-keto Reductase Metabolizes Glyphosate and Confers Glyphosate Resistance in Echinochloa colona[J]. Plant Physiol, 2019, 181(4): 1519-1534.</ref> <ref>Rumsby G, Cregeen D P. Identification and expression of a cDNA for human hydroxypyruvate/glyoxylate reductase[J]. Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1999, 1446(3): 383-388.</ref>
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Ice nucleoprotein is an anchor protein from ''Pseudomonas syringae''. It can anchor its passenger protein to the cell membrane. N and C terminal of Ice nucleoprotein, which is named after INPNC, can also anchor passenger protein fused with it to the cell membrane.  
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GRHPR, a glyoxylate reductase from human liver, can reduce glyoxylic acid when NADPH is used as cofactor. GRHPR is fused at N terminal with INPNC so that GRHPR can be displayed on the surface of ''E. coli''.<ref>Rumsby G, Cregeen D P. Identification and expression of a cDNA for human hydroxypyruvate/glyoxylate reductase[J]. Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1999, 1446(3): 383-388.</ref><ref>http://2016.igem.org/Team:TJUSLS_China</ref>
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         <img src="https://2020.igem.org/wiki/images/f/f1/T--XMU-China--XMU-China_2020-Mechanism_of_GOX_and_GRHPR.png" width="100%" style="float:center">
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         <img src="https://2020.igem.org/wiki/images/8/82/T--XMU-China--XMU-China_2020-GRHPR%E9%94%9A%E5%AE%9A.png" width="40%" style="float:center">
 
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:'''Fig 1.''' Mechanism of GRHPR on the surface of ''E. coli''.
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:'''Fig 1.''' Mechanism of GOX
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===Usage===
 
===Usage===
  
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;By codon optimization and adding a 6His-tag, the sequence suitable for expression in ''E. coli'' was constructed. And we hoped that it could transform glyphosate into glyoxylic acid in ''E. coli'' to get fluorescence signal in the next processes we design.
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Here, we used <partinfo>BBa_K880005</partinfo> to construct the expression system and demonstrated the effect of INPNC-GRHPR on the surface of ''E. coli''. We obtained the composite part <partinfo>BBa_K3332057</partinfo> and transformed the constructed plasmid into ''E. coli'' BL21 (DE3) to verify its expression. The positive clones were cultivated.
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;The coding sequence of target gene was inserted into an expression vectors with <partinfo>BBa_K880005</partinfo>(<partinfo>BBa_J23100 </partinfo>& <partinfo>BBa_B0034</partinfo>) to obtain <partinfo>BBa_K3332051</partinfo>. We transformed the constructed plasmid into ''E. coli'' BL21 (DE3) to verify its successful heterologous expression.
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         <img src="https://2020.igem.org/wiki/images/b/b6/T--XMU-China--XMU-China_2020-J23100_B0034_gox-his-tag_B0015.png" width="50%" style="float:center">
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         <img src="https://2020.igem.org/wiki/images/d/d2/T--XMU-China--XMU-China_2020-J23100_B0034_inpnc-grhpr_B0015.png" width="40%" style="float:center">
 
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:'''Fig 2.''' Gene circuit of INPNC-GRHPR.
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:'''Fig 2.''' Gene circuit of GOX
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===Characterization===
 
===Characterization===
  
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'''1. Identification'''
 
  
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;After receiving the synthesized DNA, restriction digestion was done to certify that the plasmid was correct, and the experimental results were shown in figure3.
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'''1. Identification'''
  
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After receiving the synthesized DNA, PCR was done to certify that the plasmid was correct, and the experimental results were shown in figure 3.
 
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         <img src="https://2020.igem.org/wiki/images/e/e2/T--XMU-China--07172.png" width="90%" style="float:center">
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:'''Fig 3.''' DNA gel electrophoresis of PCR products of INPNC-GRHPR-pSB1C3 (''Xba''l I & ''Pst'' I sites)
  
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:'''Fig 3.''' DNA gel electrophoresis of restriction digest products of GOX-Histag-pSB1C3 (''Xba''l I & ''Pst'' I sites)
 
  
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'''2. Purification and Proof of the expression'''
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'''2. Ability of consuming NADPH'''
  
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;We used J23100 promoter to highly express GOX-Histag in ''E. coli'' in our composite part <partinfo>BBa_K3332051</partinfo>. Then, we used GE AKTA Prime Plus FPLC System to get purified GOX protein. We found an apparent protein peak in AKTA FPLC System and correct purified protein.  
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We mixed glyoxylic acid solution, NADPH solution and bacteria solution carrying INPNC-GRHPR. Then, we immediately measured OD<sub>340</sub> changes. TECAN<sup>®</sup> Infinite M200 Pro was used to detect OD<sub>340</sub>. And when NADPH is consumed, OD<sub>340</sub> declines.  
  
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Then, our target bands are observed through SDS-PAGE and the result is shown in figure4.
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We successfully got OD<sub>340</sub>-Time curves of GRHPR fused with 4 types of anchor protein. When using INPNC-GRHPR, we could see OD<sub>340</sub> decreased as the reaction went on. And by using J23100-RBS (<partinfo>BBa_K880005</partinfo>) and GRHPR-Histag as control, we could find that the slopes of experimental and control curves have a significant difference, which means that our fusion protein INPNC-GRHPR is displayed on the surface and works very well.  
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        <img src="https://2020.igem.org/wiki/images/b/b1/T--XMU-China--XMU-China_2020-GRHPR_and_GOX.png" width="90%" style="float:center">
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:Fig 4. SDS-PAGE of purification products of GOX-Histag-pSB1C3
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'''3. Ability of degrading glyphosate'''
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;After transforming glyphosate into glyoxylic acid with the function of GOX, we use GRHPR, a Glyoxylate reductase from human liver, to reduce glyoxylic acid. GRHPR can convert glyoxylic acid when NADPH is consumed as cofactor. NADPH is a suitable target compound that can be detected by the signal of OD<sub>340</sub>. And when NADPH is consumed, OD<sub>340</sub> declines.
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;We mixed glyphosate solution, NADPH solution, GOX and GRHPR purified protein dissolved in Tris-HCl(pH=7.5). Then, we immediately detected OD<sub>340</sub> by using TECAN<sup>®</sup> Infinite M200 Pro to see the effect of GOX.  
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;When using GOX & GRHPR, we successfully found OD<sub>340</sub> decrease as time went on. And in negative control sample (only using GRHPR), we cannot get any decrease. The results prove that GOX can convert glyphosate, which is shown in figure 5.
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The result is shown in figure 4.
  
 
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         <img src="https://2020.igem.org/wiki/images/c/cf/T--XMU-China--XMU-China_2020-GOX_OD340.png" width="50%" style="float:center">
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         <img src="https://2020.igem.org/wiki/images/a/a5/T--XMU-China--XMU-China_2020-GRHPR%E9%94%9A%E5%AE%9A%E9%85%B6%E6%B4%BB.png" width="40%" style="float:center">
 
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:'''Fig 4.''' OD<sub>340</sub>-Time curves of GRHPR fused with 4 types of anchor protein.
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:Fig 5. OD<sub>340</sub>-Time curve of GOX & GRHPR and negative control
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===References===
 
===References===

Latest revision as of 16:21, 27 October 2020


J23100-RBS-GOX-his-tag-terminator

The enzyme catalyzes the reaction of degradating glyphosate to form glyoxalic acid and AMPA and add his-tag to purify the protein. We use K880005 to construct the expression system and to express and to purify the protein.


Biology

Ice nucleoprotein is an anchor protein from Pseudomonas syringae. It can anchor its passenger protein to the cell membrane. N and C terminal of Ice nucleoprotein, which is named after INPNC, can also anchor passenger protein fused with it to the cell membrane. GRHPR, a glyoxylate reductase from human liver, can reduce glyoxylic acid when NADPH is used as cofactor. GRHPR is fused at N terminal with INPNC so that GRHPR can be displayed on the surface of E. coli.[1][2]

Fig 1. Mechanism of GRHPR on the surface of E. coli.

Usage

Here, we used BBa_K880005 to construct the expression system and demonstrated the effect of INPNC-GRHPR on the surface of E. coli. We obtained the composite part BBa_K3332057 and transformed the constructed plasmid into E. coli BL21 (DE3) to verify its expression. The positive clones were cultivated.

Fig 2. Gene circuit of INPNC-GRHPR.


Characterization

1. Identification

After receiving the synthesized DNA, PCR was done to certify that the plasmid was correct, and the experimental results were shown in figure 3.

Fig 3. DNA gel electrophoresis of PCR products of INPNC-GRHPR-pSB1C3 (Xbal I & Pst I sites)


2. Ability of consuming NADPH

We mixed glyoxylic acid solution, NADPH solution and bacteria solution carrying INPNC-GRHPR. Then, we immediately measured OD340 changes. TECAN® Infinite M200 Pro was used to detect OD340. And when NADPH is consumed, OD340 declines.

We successfully got OD340-Time curves of GRHPR fused with 4 types of anchor protein. When using INPNC-GRHPR, we could see OD340 decreased as the reaction went on. And by using J23100-RBS (BBa_K880005) and GRHPR-Histag as control, we could find that the slopes of experimental and control curves have a significant difference, which means that our fusion protein INPNC-GRHPR is displayed on the surface and works very well.

The result is shown in figure 4.

Fig 4. OD340-Time curves of GRHPR fused with 4 types of anchor protein.

References

  1. ↑ Rumsby G, Cregeen D P. Identification and expression of a cDNA for human hydroxypyruvate/glyoxylate reductase[J]. Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1999, 1446(3): 383-388.
  2. ↑ http://2016.igem.org/Team:TJUSLS_China



Sequence and Features


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 355
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 574
    Illegal AgeI site found at 644
    Illegal AgeI site found at 721
  • 1000
    COMPATIBLE WITH RFC[1000]