Difference between revisions of "Part:BBa K1405002"
(→Background) |
|||
| Line 3: | Line 3: | ||
<html> | <html> | ||
<body> | <body> | ||
| − | modA a periplasmic binding protein | + | modA is a periplasmic binding protein. It has 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. |
| − | + | <br/><br/><br/><br/><br/><br/> | |
| − | + | ||
| − | < | + | |
| − | + | ||
| − | <br/> | + | |
| − | < | + | |
| − | + | ||
| − | + | ||
| − | <br/> | + | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | <br/><br/> | + | |
</body> | </body> | ||
</html> | </html> | ||
| − | |||
<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
===Usage and Biology=== | ===Usage and Biology=== | ||
| Line 32: | Line 19: | ||
<partinfo>BBa_K1405002 parameters</partinfo> | <partinfo>BBa_K1405002 parameters</partinfo> | ||
<!-- --> | <!-- --> | ||
| + | |||
| + | |||
| + | <h2>Result</h2> | ||
| + | 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. | ||
| + | <br/> | ||
| + | <a title="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." href="https://static.igem.org/mediawiki/2014/c/c6/Bnu_moda3.png" rel="prettyPhoto"> <span class="overlay zoom" style="display: none;"></span><img width="60%" style="margin-left:180px" src="https://static.igem.org/mediawiki/2014/c/c6/Bnu_moda3.png"> </a> | ||
| + | |||
| + | <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> | ||
| + | <br/> | ||
| + | |||
| + | <a title="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." href="https://static.igem.org/mediawiki/2014/9/90/Bnu_moda4.jpg" rel="prettyPhoto"> <span class="overlay zoom" style="display: none;"></span><img width="45%" style="margin-left:250px" src="https://static.igem.org/mediawiki/2014/e/e5/Bnu_moda4_01.jpg"> </a> | ||
| + | <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> | ||
| + | <br/><br/> | ||
Revision as of 14:31, 17 October 2014
Background
modA is a periplasmic binding protein. It has 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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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.
<a title="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." href="
" rel="prettyPhoto"> <img width="60%" style="margin-left:180px" src=""> </a>
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.
<a title="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." href="
" rel="prettyPhoto"> <img width="45%" style="margin-left:250px" src="
"> </a>
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.