Difference between revisions of "Part:BBa K4623009"
(→‎Cultivation, Purification and SDS-PAGE)
Line 23: Line 23:
  
  
−
===Cultivation, Purification and SDS-PAGE===
+
==Cultivation, Purification and SDS-PAGE==
−
<partinfo>BBa_K4623009 parameters</partinfo>
+
=== induction condition===
−
<!-- -->
+
<html><!-- 前缀：HTML元素的开始标签 -->
 +
 
 +
<head> <!-- HTML头部开始标签 -->
 +
  <style>
 +
    .image-container {
 +
      float: left; /* 图像容器向左浮动 */
 +
      width: 50%; /* 图像容器宽度占据页面的二分之一 */
 +
      text-align: center; /* 图像容器中的内容居中对齐 */
 +
    }
 +
 
 +
    .image-container img {
 +
      display: block; /* 图像显示为块级元素 */
 +
      margin: 0 auto; /* 上下居中，左右自动居中 */
 +
      border: 1px solid black;
 +
      max-width: 100%; /* 图像最大宽度为容器宽度 */
 +
    }
 +
 
 +
    .image-container figcaption {
 +
      text-align: center; /* 图注居中对齐 */
 +
    }
 +
 
 +
    .text-container {
 +
      float: left; /* 文字容器向左浮动 */
 +
      width: 50%; /* 文字容器宽度占据页面的二分之一 */
 +
    }
 +
 
 +
    /* 清除浮动 */
 +
    .clearfix::after {
 +
      content: "";
 +
      display: table;
 +
      clear: both;
 +
    }
 +
  </style>
 +
</head> <!-- HTML头部结束标签 -->
 +
 
 +
<body> <!-- HTML主体开始标签 -->
 +
    <!-- 在这里添加网页内容，包括文本、图片、链接等 -->
 +
 
 +
   
 +
    In order to visualize the protein function of Twisted Silinker, we replaced the linker portion with a GFP sequence in subsequent experiments. This way, when CBP and calmodulin successfully bind together, the GFP protein will be activated, emitting green fluorescence.
 +
   
 +
    The presence of mSA monomers can easily lead to the formation of inclusion bodies, increasing the difficulty of purification. To achieve efficient expression of our Twisted Silinker and reduce the formation of inclusion bodies, we screened the IPTG induction conditions. We tested five different IPTG concentrations: 0 mM, 0.1 mM, 0.25 mM, and 0.5 mM. The results showed that the optimal concentration for protein expression was 0.1 mM.
 +
   
 +
    To ensure proper folding of mSA and minimize inclusion body formation, we modified the protein buffer by adding biotin. The binding of biotin to mSA can help facilitate proper folding of the Twisted Silinker protein, reducing the formation of inclusion bodies resulting from misfolding. As a result, we obtained soluble protein extract in the supernatant. The formulation of the buffer and experimental procedures can be found in **(protocol)** for reference.
 +
</body> <!-- HTML主体结束标签 -->
 +
 
 +
</html> <!-- 后缀：HTML元素的结束标签 -->

Revision as of 08:40, 10 October 2023


Twisted Silinker,Silica-protein junctions that curving in response to calcium ions


Usage and Biology

Twisted Silinker (TS) is an intelligent recombinant protein that efficiently connects to the surface of silicon dioxide while undergoing conformational changes in response to environmental stimuli.

The sequence in the FASTA file has a His tag added, allowing purification of TS protein using a nickel column. An upstream TrxA fusion tag (part number) is added to aid in protein folding and reduce the formation of inclusion bodies in the bacterial host. After protein expression, thrombin (part number) cleavage exposes the mSA (part number) site, allowing the binding of biotinylated functional proteins. CBP (part number) and calmodulin (part number) undergo conformational changes in the presence of calcium ions, tightly folding together to achieve the desired conformation. The SBP (part number) sequence can bind to the silicon dioxide surface, facilitating the modification of functional proteins onto the surface.

We transferred the pET-DUT1 plasmid into our engineered strain BL21(DE3) and performed small-scale expression to determine the production conditions for His-tagged Twisted Silinker. The purified Twisted Silinker was detected using SDS-PAGE and Western Blot, with a molecular weight of 53 kDa. To improve the purification strategy, we have also developed corresponding hardware for protein purification using the binding affinity between SBP and silicon dioxide, significantly enhancing the efficiency of protein production and purification.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 955
    Illegal EcoRI site found at 1123
    Illegal EcoRI site found at 1345
    Illegal PstI site found at 1066
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 955
    Illegal EcoRI site found at 1123
    Illegal EcoRI site found at 1345
    Illegal PstI site found at 1066
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 955
    Illegal EcoRI site found at 1123
    Illegal EcoRI site found at 1345
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 955
    Illegal EcoRI site found at 1123
    Illegal EcoRI site found at 1345
    Illegal PstI site found at 1066
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 955
    Illegal EcoRI site found at 1123
    Illegal EcoRI site found at 1345
    Illegal PstI site found at 1066
    Illegal AgeI site found at 445
    Illegal AgeI site found at 505
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 940


Cultivation, Purification and SDS-PAGE

induction condition

In order to visualize the protein function of Twisted Silinker, we replaced the linker portion with a GFP sequence in subsequent experiments. This way, when CBP and calmodulin successfully bind together, the GFP protein will be activated, emitting green fluorescence. The presence of mSA monomers can easily lead to the formation of inclusion bodies, increasing the difficulty of purification. To achieve efficient expression of our Twisted Silinker and reduce the formation of inclusion bodies, we screened the IPTG induction conditions. We tested five different IPTG concentrations: 0 mM, 0.1 mM, 0.25 mM, and 0.5 mM. The results showed that the optimal concentration for protein expression was 0.1 mM. To ensure proper folding of mSA and minimize inclusion body formation, we modified the protein buffer by adding biotin. The binding of biotin to mSA can help facilitate proper folding of the Twisted Silinker protein, reducing the formation of inclusion bodies resulting from misfolding. As a result, we obtained soluble protein extract in the supernatant. The formulation of the buffer and experimental procedures can be found in **(protocol)** for reference.