Difference between revisions of "Part:BBa K1405002"

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ModA
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=='''Introduction'''==
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Background
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modA a periplasmic binding protein, . had a affinity (Km) for molybdate of 3–5 mM, this is one of the highest substrate Kd values reported for any periplasmic binding protein, the BBa_K1405002 contains the ModA coding sequence. And we used pET-21a BL21 to express and use His-chelating chromatography to purify.
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Results
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Expression and purification of ModA. Expression of ModA and its purification by His-chelating chromatography. Lane 1, molecular weight standards (kDa); lane 2, total bacterial proteins before IPTG induction; lane 3, total bacterial proteins after 0.5 mM IPTG induction; lane 4, total bacterial lysate after 0.5 mM IPTG induction; lane 5,the sediment after 16,000 rpm 30 min; lane 6 the flow through of column loading; lane 7,the flow of buffer B .lane 8 The purification of ModA by size-elution chromatography.  
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<p>modA is a periplasmic binding protein. It has a affinity (Km) for molybdate of 3–5 mM. ModA is one of the highest substrate Kd values reported for any periplasmic binding protein, the BBa_K1405002 contains the ModA coding sequence. And we used pET-21a BL21 to express and use His-chelating chromatography to purify.</p>
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===Usage and Biology===
  
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Native-PAGE to provide the molybdate lane 1, the modA(acetic buffer PH 5.0); lane 2, the modA incubate with 20mM molybdate.
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1405002 SequenceAndFeatures</partinfo>
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===Functional Parameters===
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<partinfo>BBa_K1405002 parameters</partinfo>
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<h2>Result</h2>
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Figure 1 Expression and purification of ModA. Expression of ModA and its purification by His-chelating chromatography. Lane 1, molecular weight standards (kDa); lane 2, total bacterial proteins before IPTG induction; lane 3, total bacterial proteins after 0.5 mM IPTG induction; lane 4, total bacterial lysate after 0.5 mM IPTG induction; lane 5,the sediment after 16,000 rpm 30 min; lane 6 the flow through of column loading; lane 7,the flow of buffer B .lane 8 The purification of ModA by size-elution chromatography.
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<img width="60%" style="margin-left:180px" src="https://static.igem.org/mediawiki/2014/c/c6/Bnu_moda3.png">
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<p class="fig">Figure 1 Expression and purification of ModA. Expression of ModA and its purification by His-chelating chromatography. Lane 1, molecular weight standards (kDa); lane 2, total bacterial proteins before IPTG induction; lane 3, total bacterial proteins after 0.5 mM IPTG induction; lane 4, total bacterial lysate after 0.5 mM IPTG induction; lane 5,the sediment after 16,000 rpm 30 min; lane 6 the flow through of column loading; lane 7,the flow of buffer B .lane 8 The purification of ModA by size-elution chromatography.</p>
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<img width="45%" style="margin-left:250px" src="https://static.igem.org/mediawiki/2014/e/e5/Bnu_moda4_01.jpg">
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<p class="fig" style="width:85%; margin-left:50px">Figure 2 Native-PAGE to provide the molybdate lane 1, the modA(acetic buffer PH 5.0); lane 2, the modA incubate with 20mM molybdate.</p>
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Latest revision as of 14:40, 17 October 2014

Introduction

modA is a periplasmic binding protein. It has a affinity (Km) for molybdate of 3–5 mM. ModA is one of the highest substrate Kd values reported for any periplasmic binding protein, the BBa_K1405002 contains the ModA coding sequence. And we used pET-21a BL21 to express and use His-chelating chromatography to purify.





Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Result

Figure 1 Expression and purification of ModA. Expression of ModA and its purification by His-chelating chromatography. Lane 1, molecular weight standards (kDa); lane 2, total bacterial proteins before IPTG induction; lane 3, total bacterial proteins after 0.5 mM IPTG induction; lane 4, total bacterial lysate after 0.5 mM IPTG induction; lane 5,the sediment after 16,000 rpm 30 min; lane 6 the flow through of column loading; lane 7,the flow of buffer B .lane 8 The purification of ModA by size-elution chromatography.

Figure 1 Expression and purification of ModA. Expression of ModA and its purification by His-chelating chromatography. Lane 1, molecular weight standards (kDa); lane 2, total bacterial proteins before IPTG induction; lane 3, total bacterial proteins after 0.5 mM IPTG induction; lane 4, total bacterial lysate after 0.5 mM IPTG induction; lane 5,the sediment after 16,000 rpm 30 min; lane 6 the flow through of column loading; lane 7,the flow of buffer B .lane 8 The purification of ModA by size-elution chromatography.


Figure 2 Native-PAGE to provide the molybdate lane 1, the modA(acetic buffer PH 5.0); lane 2, the modA incubate with 20mM molybdate.