Difference between revisions of "Part:BBa K3332014"

 
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<partinfo>BBa_K3332014 short</partinfo>
 
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We anchor GRHPR protein onto membranes through AIDA to catalyze the reaction of reducing glyoxalic acid and consuming NADPH. We use K880005 to construct the expression system and anchor GRHPR on the surface of E.coli.
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;We anchor GRHPR protein onto membranes through AIDA to catalyze the reaction of reducing glyoxalic acid and consuming NADPH. We use <partinfo>BBa_K880005</partinfo> to construct the expression system and anchor GRHPR on the surface of ''E.coli''.
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===Biology===
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;AIDA is an anchor protein from ''E. coli'', which has been widely used in cell-surface display. GRHPR, a glyoxylate reductase from human liver, can reduce glyoxylic acid when NADPH is used as cofactor.
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;GRHPR is fused at C terminal with AIDA 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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<html>
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    <figure>
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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="45%" style="float:center">
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        <figcaption>
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        <p style="font-size:1rem">
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        </p>
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        </figcaption>
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    </figure>
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</html>
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:'''Fig 1'''. mechanism
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===Usage===
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Here, we used <partinfo>BBa_K880005</partinfo> to construct the expression system and demonstrated the effect of GRHPR-AIDA on the surface of ''E. coli''. We obtained the composite part <partinfo>BBa_K3332059</partinfo> and transformed the constructed plasmid into ''E. coli'' BL21 (DE3) to verify its expression. The positive clones were cultivated.
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<html>
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    <figure>
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        <img src="https://2020.igem.org/wiki/images/2/21/T--XMU-China--XMU-China_2020-J23100_B0034_grhpr-aidA_B0015.png" width="35%" style="float:center">
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        <figcaption>
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        <p style="font-size:1rem">
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        </p>
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        </figcaption>
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    </figure>
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</html>
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:'''Fig 2'''. Gene circuit of GRHPR-AIDA.
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===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 figure 3.
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<html>
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    <figure>
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        <img src="https://2020.igem.org/wiki/images/2/20/T--XMU-China--09031.png" width="60%" style="float:center">
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        <figcaption>
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        <p style="font-size:1rem">
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        </p>
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        </figcaption>
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    </figure>
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</html>
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:'''Fig 3'''.DNA gel electrophoresis of restriction digest products of GRHPR-AIDA-pSB1C3 (''Xba''l I & ''Pst'' I sites)
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'''2.Ability of consuming NADPH'''
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;We mixed glyoxylic acid solution, NADPH solution and bacteria solution carrying GRHPR-AIDA. 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;We successfully got OD340-Time curves of GRHPR fused with 4 types of anchor protein. When using bacteria carrying GRHPR-AIDA, we could see OD<sub>340</sub> decreased as the reaction went on. However, by using bacteria carrying J23100-RBS (<partinfo>BBa_K880005</partinfo>) and GRHPR-Histag as control, we could also find that the slopes of these three curves are similar. The results show that our fusion protein GRHPR-AIDA do not work well. The result is shown in figure 4.
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<html>
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    <figure>
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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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        <figcaption>
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        <p style="font-size:1rem">
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        </p>
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        </figcaption>
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    </figure>
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</html>
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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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===References===
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<references/>
  
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===Usage and Biology===
 
  
 
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Latest revision as of 22:58, 27 October 2020


GRHPR-AIDA

        We anchor GRHPR protein onto membranes through AIDA to catalyze the reaction of reducing glyoxalic acid and consuming NADPH. We use BBa_K880005 to construct the expression system and anchor GRHPR on the surface of E.coli.

Biology

        AIDA is an anchor protein from E. coli, which has been widely used in cell-surface display. GRHPR, a glyoxylate reductase from human liver, can reduce glyoxylic acid when NADPH is used as cofactor.

        GRHPR is fused at C terminal with AIDA so that GRHPR can be displayed on the surface of E. coli.[1][2]

Fig 1. mechanism

Usage

        Here, we used BBa_K880005 to construct the expression system and demonstrated the effect of GRHPR-AIDA on the surface of E. coli. We obtained the composite part BBa_K3332059 and transformed the constructed plasmid into E. coli BL21 (DE3) to verify its expression. The positive clones were cultivated.

Fig 2. Gene circuit of GRHPR-AIDA.

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 GRHPR-AIDA-pSB1C3 (Xbal I & Pst I sites)

2.Ability of consuming NADPH

        We mixed glyoxylic acid solution, NADPH solution and bacteria solution carrying GRHPR-AIDA. 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 bacteria carrying GRHPR-AIDA, we could see OD340 decreased as the reaction went on. However, by using bacteria carrying J23100-RBS (BBa_K880005) and GRHPR-Histag as control, we could also find that the slopes of these three curves are similar. The results show that our fusion protein GRHPR-AIDA do not work 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
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 2264
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 547
  • 1000
    COMPATIBLE WITH RFC[1000]