Difference between revisions of "Team:SDSZ China/Project"
| Line 6: | Line 6: | ||
<div class="col-xs-2 pull-left affix hidden-xs " id = "sidebar" style="margin-top:50px;"> | <div class="col-xs-2 pull-left affix hidden-xs " id = "sidebar" style="margin-top:50px;"> | ||
<p class="title" style="font-size: 30px;">Project</p> | <p class="title" style="font-size: 30px;">Project</p> | ||
| − | <ul class="nav nav-bar nav-stacked" style=""> | + | <ul class="nav nav-bar nav-stacked" style="text-align: right;"> |
<li class="sidebar-link"><a href="#1_0">1.0</a></li> | <li class="sidebar-link"><a href="#1_0">1.0</a></li> | ||
<li class="sidebar-link"><a href="#2_0">2.0</a></li> | <li class="sidebar-link"><a href="#2_0">2.0</a></li> | ||
| Line 18: | Line 18: | ||
<br/> | <br/> | ||
<p class="title">Principles：</p><br/><p> | <p class="title">Principles：</p><br/><p> | ||
| − | 结构图 | + | 结构图 |
| − | PBP are ubiquitous bacterial enzymes involved in cell wall biosynthesis. | + | PBP are ubiquitous bacterial enzymes involved in cell wall biosynthesis. |
| − | + | Penicillin-binding protein 5 (PBP 5) of Escherichia coli hydrolyzes the terminal D-Ala-D-Ala peptide bond of the stem precursor peptides of the cell wall peptidoglycan.(Investigation of the Mechanism of the Cell Wall DD-Carboxypeptidase Reaction of Penicillin-Binding Protein 5 of Escherichia coli by Quantum Mechanics/Molecular Mechanics Calculations | |
| − | Qicun Shi, Samy O. Meroueh, Jed F. Fisher, and Shahriar Mobashery) | + | Qicun Shi, Samy O. Meroueh, Jed F. Fisher, and Shahriar Mobashery) |
| − | We designed a part that generates Penicillin Binding Protein 5-Green Fluorescence Protein (PBP5-GFP), and bind penicillin to the coated wells on the ELISA PLATE. The protein is added to the sample, where the penicillin in the sample compete for PBP5-GFP with those on the plate, and proteins unbound with penicillin stays on the plane. So we measure the fluorescence generated by GFP in the liquid, which indicates the content of the protein PBP5. With more penicillin present, more PBP5 binds to them, and greater fluorescence intensity will be measured. (See protein purification protocol) | + | We designed a part that generates Penicillin Binding Protein 5-Green Fluorescence Protein (PBP5-GFP), and bind penicillin to the coated wells on the ELISA PLATE. The protein is added to the sample, where the penicillin in the sample compete for PBP5-GFP with those on the plate, and proteins unbound with penicillin stays on the plane. So we measure the fluorescence generated by GFP in the liquid, which indicates the content of the protein PBP5. With more penicillin present, more PBP5 binds to them, and greater fluorescence intensity will be measured. (See protein purification protocol) |
| − | </p><br/> | + | </p><br/> |
| − | <p class="title">Proof of concept:</p><br/> | + | <p class="title">Proof of concept:</p><br/> |
| − | <p>图：蛋白质电泳＋解析</p><br/> | + | <p>图：蛋白质电泳＋解析</p><br/> |
| − | <p class="title">Results:</p><br/> | + | <p class="title">Results:</p><br/> |
| − | <p>图：用青霉素溶液做的结果图＋解析 | + | <p>图：用青霉素溶液做的结果图＋解析 |
| − | See protocol | + | See protocol |
| − | Demonstration: | + | Demonstration: |
| − | 图：用牛奶测得结果图＋解析 | + | 图：用牛奶测得结果图＋解析 |
| − | See Protocol | + | See Protocol |
| − | Optimization: | + | Optimization: |
| − | 改分光光度计</p> | + | 改分光光度计</p> |
</div> | </div> | ||
</div> | </div> | ||
| Line 46: | Line 46: | ||
<p class="title">2.0</p> | <p class="title">2.0</p> | ||
<br/> | <br/> | ||
| − | <p></p> | + | <p class="title">Principles:</p><br/> |
| + | <p>Many poplypetides with a d-ala-d-ala end can be the substrate for PBP5, diacetyl-L-LysD-Ala-D-Ala (Ac2-L-Lys-D-Ala-D-Ala, Ac2-KAA) is the common substrate used to detect PBP5’s activity（pH, inhibitor, and substrate specificity studies on Escherichia coli penicillin-binding protein 5） . The PBP5 causes Ac2-KAA to hydrolyze, releasing one alanine and exposing one amino group. If bound with penicillin, PBP5 loses activity. And the protein is very sensitive to the penicillin in the liquid, so this mechanism is applicable as we try to determine the amount of penicillin in a low concentration system. the amino group, which turns Kaiser blue. (See protocol) | ||
| + | </p><br/><p class="title">Results: | ||
| + | </p> | ||
</div> | </div> | ||
</div> | </div> | ||
| Line 53: | Line 56: | ||
<p class="title">3.0</p> | <p class="title">3.0</p> | ||
<br/> | <br/> | ||
| − | <p></p> | + | <p>bb</p> |
</div> | </div> | ||
| − | |||
</div> | </div> | ||
</div> | </div> | ||
Revision as of 10:29, 14 October 2016
1.o
Principles：
结构图 PBP are ubiquitous bacterial enzymes involved in cell wall biosynthesis. Penicillin-binding protein 5 (PBP 5) of Escherichia coli hydrolyzes the terminal D-Ala-D-Ala peptide bond of the stem precursor peptides of the cell wall peptidoglycan.(Investigation of the Mechanism of the Cell Wall DD-Carboxypeptidase Reaction of Penicillin-Binding Protein 5 of Escherichia coli by Quantum Mechanics/Molecular Mechanics Calculations Qicun Shi, Samy O. Meroueh, Jed F. Fisher, and Shahriar Mobashery) We designed a part that generates Penicillin Binding Protein 5-Green Fluorescence Protein (PBP5-GFP), and bind penicillin to the coated wells on the ELISA PLATE. The protein is added to the sample, where the penicillin in the sample compete for PBP5-GFP with those on the plate, and proteins unbound with penicillin stays on the plane. So we measure the fluorescence generated by GFP in the liquid, which indicates the content of the protein PBP5. With more penicillin present, more PBP5 binds to them, and greater fluorescence intensity will be measured. (See protein purification protocol)
Proof of concept:
图：蛋白质电泳＋解析
Results:
图：用青霉素溶液做的结果图＋解析 See protocol Demonstration: 图：用牛奶测得结果图＋解析 See Protocol Optimization: 改分光光度计
2.0
Principles:
Many poplypetides with a d-ala-d-ala end can be the substrate for PBP5, diacetyl-L-LysD-Ala-D-Ala (Ac2-L-Lys-D-Ala-D-Ala, Ac2-KAA) is the common substrate used to detect PBP5’s activity（pH, inhibitor, and substrate specificity studies on Escherichia coli penicillin-binding protein 5） . The PBP5 causes Ac2-KAA to hydrolyze, releasing one alanine and exposing one amino group. If bound with penicillin, PBP5 loses activity. And the protein is very sensitive to the penicillin in the liquid, so this mechanism is applicable as we try to determine the amount of penicillin in a low concentration system. the amino group, which turns Kaiser blue. (See protocol)
Results:
3.0
bb
