Difference between revisions of "Part:BBa K3332047"

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The iNAP is fused at N-terminal with INPNC anchoring protein. We use K880005 to construct the expression system and anchor iNAP on the surface of ''E.coli''.
 
The iNAP is fused at N-terminal with INPNC anchoring protein. We use K880005 to construct the expression system and anchor iNAP on the surface of ''E.coli''.
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===Biology===
 
===Biology===
  
GRHPR, a glyoxylate reductase from human liver, can reduce glyoxylic acid when NADPH is used as cofactor. GRHPR converts glyoxylic acid while consuming NADPH. NADPH is a suitable target compound that can be detected by the signal of fluorescence or OD<sub>340</sub>.<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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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;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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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;iNap is a chimera of circularly permuted YFP (cpYFP) and the NADP(H) binding domain of Rex from Thermus aquaticus (T-Rex). Upon NADPH binding, iNap shows apparent fluorescence changes. iNap is fused at N terminal with INPNC so that iNap can be displayed on the surface of ''E.coli''.<ref>Van Bloois E, Winter R T, Kolmar H, et al. Decorating microbes: surface display of proteins on Escherichia coli[J]. Trends Biotechnol, 2011, 29(2): 79-86.</ref><ref>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</ref><ref>http://2016.igem.org/Team:TJUSLS_China</ref>
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     <figure>
 
     <figure>
         <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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         <img src="https://2020.igem.org/wiki/images/9/94/T--XMU-China--XMU-China_2020-iNAP_Mechanism.png" width="80%" style="float:center">
 
         <figcaption>
 
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:'''Fig 1.''' GRHPR mechanism.
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:'''Fig 1.''' Mechanism of iNap anchored on the ''E. coli'' surface
  
  
 
===Usage===
 
===Usage===
  
By codon optimization and adding a 6His-tag, the sequence suitable for expression in ''E. coli'' was constructed, and we hoped that it could reduce glyoxylic acid in ''E. coli'' to get fluorescence signal in the next processes we design.
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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 INPNC-iNap on the surface of ''E. coli''. We obtained the composite part <partinfo>BBa_K3332047</partinfo> and transformed the constructed plasmid into ''E. coli'' BL21 (DE3) to verify its expression and enzyme activity. The positive clones were cultivated.
 
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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_K3332056</partinfo>. We transformed the constructed plasmid into ''E. coli'' BL21 (DE3) to verify its successful heterologous expression.
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<html>
 
<html>
 
     <figure>
 
     <figure>
         <img src="https://2020.igem.org/wiki/images/2/2e/T--XMU-China--XMU-China_2020-J23100_B0034_grhpr-his-tag_B0015.png" height="150" style="float:center">
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         <img src="https://2020.igem.org/wiki/images/2/29/T--XMU-China--XMU-China_2020-J23100_B0034_inpnc-inap_B0015.png" width="80%" style="float:center">
 
         <figcaption>
 
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</html>
  
:'''Fig 2.''' Gene circuit of GRHPR.
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:'''Fig 2.''' Gene circuit of INPNC-iNap
  
  
 
===Characterization===
 
===Characterization===
  
'''1. Identification'''
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'''1.Identification'''
 
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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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&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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<html>
 
<html>
 
     <figure>
 
     <figure>
         <img src="https://2020.igem.org/wiki/images/e/e8/T--XMU-China--07181.png" width="65%" style="float:center">
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         <img src="https://2020.igem.org/wiki/images/a/a5/T--XMU-China--08101.png" width="80%" style="float:center">
 
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:'''Fig 3.''' DNA gel electrophoresis of restriction digest products of GRHPR-His-pSB1C3 (''Xbal'' I & ''Pst'' I sites)
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:'''Fig 3.''' DNA gel electrophoresis of restriction digest products of INPNC-iNap-pSB1C3 (''Xba'' I & ''Pst'' I sites)
  
'''2. Purification and Proof of the expression'''
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'''2. Ability of sensing NADPH'''
  
We used J23100 promoter to highly express GRHPR-Histag in ''E. coli'' in our composite part <partinfo>BBa_K3332056</partinfo>. Then, we used GE AKTA Prime Plus FPLC System to get purified GRHPR protein. We found an apparent protein peak in AKTA FPLC System and correct purified protein.  
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;After culturing our engineering bacteria to OD<sub>600</sub>=1.8~2.0, we obtained ''E. Coli'' with <partinfo>BBa_K3332047</partinfo> by centrifuging at 4000 rpm. Then, the cell precipitation was washed three times with PBS buffer (pH=8.0), and the final precipitation was resuspended in PBS buffer, which was equal to the volume of the original medium.
  
Then, our target bands are observed through SDS-PAGE and the result is shown in figure4.
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;We mixed NADPH solutions half-in-half with washed INPNC-iNap cell precipitation dissolved in PBS buffer (pH=8.0) and measured fluorescence changes in the presence of different concentrations of NADPH. TECAN<sup>®</sup> Infinite M200 Pro was used to detect fluorescence intensity. The samples were excited in 420 nm and the emission was measured at 528 nm.  
  
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;When using bacteria carrying INPNC-iNap, we successfully got fluorescent gradient in the presence of different concentrations of NADPH. And when using ''E. coli'' with <partinfo>BBa_K880005</partinfo>, we cannot get any gradient. The results prove that our anchor protein works and iNap can sensing NADPH on the surface of bacteria, which is shown in figure 4.
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<html>
 
<html>
 
     <figure>
 
     <figure>
         <img src="https://2020.igem.org/wiki/images/b/b1/T--XMU-China--XMU-China_2020-GRHPR_and_GOX.png" width="80%" style="float:center">
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         <img src="https://2020.igem.org/File:T--XMU-China--XMU-China_2020-iNAP%E9%94%9A%E5%AE%9A%E9%85%B6%E6%B4%BB.png" width="80%" style="float:center">
 
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</html>
:'''Fig 4.''' SDS-PAGE of purification products of GRHPR-Histag-pSB1C3
 
  
'''3. Ability of consuming NADPH'''  
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:'''Fig 4.''' (a)Fluorescent-Time curve of INPNC-iNap and negative control in the presence of different NADPH concentrations;(b) Fluorescent-Time curve of iNap-AIDA and negative control in the presence of different NADPH concentrations.
  
We mixed glyoxylic acid solution, NADPH solution and purified GRHPR protein dissolved in Tris-HCl(pH=7.5). Then, we immediately measured OD<sub>340</sub> changes of our samples. And when NADPH is consumed, OD<sub>340</sub> declines.
 
  
The experimental result is shown on Figure 5. We can see the OD<sub>340</sub> of samples adding GRHPR decrease very quickly while the OD<sub>340</sub> of control stay almost the same.
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===Reference===
 +
<references/>
  
<html>
 
    <figure>
 
        <img src="https://2020.igem.org/wiki/images/d/d4/T--XMU-China--XMU-China_2020-GRHPR酶活.png" width="45%" style="float:center">
 
        <figcaption>
 
        <p style="font-size:1rem">
 
        </p>
 
        </figcaption>
 
    </figure>
 
</html>
 
:'''Fig 5.'''' Enzyme activity of GRHPR.
 
 
'''4. Kinetic parameter determination'''
 
 
We successfully got OD<sub>340</sub>-Time curves of GRHPR in the presence of NADPH concentration and OD<sub>340</sub>-Time curves of GRHPR in the presence of glyoxylic acid concentration. Then we calculated relevant enzyme activity and drew 1/V-1/[NADPH] and 1/V-1/[glyoxylic acid] curves, from which we can obtain relevant Km and Vmax.
 
 
The result is shown in figure 6 and figure 7.
 
 
<html>
 
    <figure>
 
        <img src="https://2020.igem.org/wiki/images/4/4c/T--XMU-China--XMU-China_2020-GRHPR_动力学参数1-NADPH底物.png" width="50%" style="float:center">
 
        <figcaption>
 
        <p style="font-size:1rem">
 
        </p>
 
        </figcaption>
 
    </figure>
 
</html>
 
:'''Fig 6.''' 1/V-1/[NADPH] curve of purified GRHPR reacting with NADPH and glyoxylic acid
 
 
<html>
 
    <figure>
 
        <img src="https://2020.igem.org/wiki/images/3/3d/T--XMU-China--XMU-China_2020-GRHPR动力学参数-2-glyoxycolic_底物.png"width="50%" style="float:center">
 
        <figcaption>
 
        <p style="font-size:1rem">
 
        </p>
 
        </figcaption>
 
    </figure>
 
</html>
 
:'''Fig 7.''' 1/V-1/[glyoxylic acid] curve of purified GRHPR reacting with NADPH and glyoxylic acid
 
 
 
===References===
 
<references/>
 
  
  

Revision as of 23:29, 27 October 2020


J23100-RBS-INPNC-iNAP-terminator

The iNAP is fused at N-terminal with INPNC anchoring protein. We use K880005 to construct the expression system and anchor iNAP on the surface of E.coli.


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.

        iNap is a chimera of circularly permuted YFP (cpYFP) and the NADP(H) binding domain of Rex from Thermus aquaticus (T-Rex). Upon NADPH binding, iNap shows apparent fluorescence changes. iNap is fused at N terminal with INPNC so that iNap can be displayed on the surface of E.coli.[1][2][3]

Fig 1. Mechanism of iNap anchored on the E. coli surface


Usage

        Here, we used BBa_K880005 to construct the expression system and demonstrated the effect of INPNC-iNap on the surface of E. coli. We obtained the composite part BBa_K3332047 and transformed the constructed plasmid into E. coli BL21 (DE3) to verify its expression and enzyme activity. The positive clones were cultivated.

Fig 2. Gene circuit of INPNC-iNap


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 INPNC-iNap-pSB1C3 (Xba I & Pst I sites)

2. Ability of sensing NADPH

        After culturing our engineering bacteria to OD600=1.8~2.0, we obtained E. Coli with BBa_K3332047 by centrifuging at 4000 rpm. Then, the cell precipitation was washed three times with PBS buffer (pH=8.0), and the final precipitation was resuspended in PBS buffer, which was equal to the volume of the original medium.

        We mixed NADPH solutions half-in-half with washed INPNC-iNap cell precipitation dissolved in PBS buffer (pH=8.0) and measured 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.

        When using bacteria carrying INPNC-iNap, we successfully got fluorescent gradient in the presence of different concentrations of NADPH. And when using E. coli with BBa_K880005, we cannot get any gradient. The results prove that our anchor protein works and iNap can sensing NADPH on the surface of bacteria, which is shown in figure 4.

Fig 4. (a)Fluorescent-Time curve of INPNC-iNap and negative control in the presence of different NADPH concentrations;(b) Fluorescent-Time curve of iNap-AIDA and negative control in the presence of different NADPH concentrations.


Reference

  1. Van Bloois E, Winter R T, Kolmar H, et al. Decorating microbes: surface display of proteins on Escherichia coli[J]. Trends Biotechnol, 2011, 29(2): 79-86.
  2. 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
  3. 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 62
    Illegal BamHI site found at 1314
    Illegal XhoI site found at 1340
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 472
    Illegal NgoMIV site found at 1080
  • 1000
    COMPATIBLE WITH RFC[1000]