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Team:Heidelberg/Toolbox Guide
From 2014.igem.org
(Difference between revisions)
| Line 38: | Line 38: | ||
/* Enlarge Icons on hover */ | /* Enlarge Icons on hover */ | ||
.toolbox-icons-bar a:hover img { | .toolbox-icons-bar a:hover img { | ||
| - | + | transform: scale(1.15); | |
| - | + | -webkit-transform: scale(1.15); | |
| - | + | -moz-transform: scale(1.15); | |
| - | + | -o-transform: scale(1.15); | |
| - | + | -ms-transform: scale(1.15); | |
} | } | ||
| Line 54: | Line 54: | ||
</style> | </style> | ||
| - | + | <div id="myContainer" class="container"> | |
</html> | </html> | ||
| - | + | {{:Team:Heidelberg/Templates/BootstrapNav| | |
| - | + | red=| | |
| - | + | white=true| | |
| - | + | red-logo=true| | |
| - | + | white-logo=| | |
| - | + | header-bg=| | |
| - | + | header-img=| | |
| - | + | title= | |
| - | + | }} | |
| - | + | <html> | |
| - | + | <div class="container main" style="color: white;"> | |
| - | + | <div class="row"> | |
| - | + | <div class="col-lg-12"> | |
| - | + | <span class="middle">use the <span class="red-text">intein</span></span> | |
| - | + | </div> | |
| - | + | </div> | |
| - | + | <div class="row"> | |
| - | + | <div class="col-lg-12" style="position: relative; top: -20px;"> | |
| - | + | <span class="large">TOOLBOX </span><span class="middle" >to</span> | |
| - | + | </div> | |
| - | + | </div> | |
| - | + | <div class="row toolbox-icons-bar"> | |
| - | + | <div class="col-lg-2 col-lg-offset-1 col-md-offset-1 col-md-2 col-sm-4 col-xs-6"> | |
| - | + | <a href="https://2014.igem.org/Team:Heidelberg/Toolbox/Circularization"><span class="col-lg-offset-3 col-lg-6 col-md-offset-2 col-md-8 col-sm-12 nopadding nofloat"><img src="/wiki/images/5/58/Heidelberg_Toolbox_Circularization.png" class="img-responsive" /></span>CIRCULARIZE</a> | |
| - | + | </div> | |
| - | + | <div class="col-lg-2 col-md-2 col-sm-4 col-xs-6"> | |
| - | + | <a href="https://2014.igem.org/Team:Heidelberg/Toolbox/Oligomerization"><span class="col-lg-offset-3 col-lg-6 col-md-offset-2 col-md-8 col-sm-12 nopadding nofloat"><img src="/wiki/images/4/40/Oligomerization.png" class="img-responsive" /></span>OLIGOMERIZE</a> | |
| - | + | </div> | |
| - | + | <div class="col-lg-2 col-md-2 col-sm-4 col-xs-6"> | |
| - | + | <a href="https://2014.igem.org/Team:Heidelberg/Toolbox/Fusion"><span class="col-lg-offset-3 col-lg-6 col-md-offset-2 col-md-8 col-sm-12 nopadding nofloat"><img src="/wiki/images/8/87/Heidelberg_Toolbox_Fusion.png" class="img-responsive" /></span>FUSE & TAG</a> | |
| - | + | </div> | |
| - | + | <div class="col-lg-2 col-md-2 col-sm-4 col-xs-6"> | |
| - | + | <a href="https://2014.igem.org/Team:Heidelberg/Toolbox/Purification"><span class="col-lg-offset-3 col-lg-6 col-md-offset-2 col-md-8 col-sm-12 nopadding nofloat"><img src="/wiki/images/0/04/Heidelberg_Toolbox_Purification.png" class="img-responsive" /></span>PURIFY</a> | |
| - | + | </div> | |
| - | + | <div class="col-lg-2 col-md-2 col-sm-4 col-xs-6"> | |
| - | + | <a href="https://2014.igem.org/Team:Heidelberg/Toolbox/OnOff"><span class="col-lg-offset-3 col-lg-6 col-md-offset-2 col-md-8 col-sm-12 nopadding nofloat"><img src="/wiki/images/c/c2/Heidelberg_Toolbox_On-Off.png" class="img-responsive" /></span>ACTIVATE &<br />DEACTIVATE</a> | |
| - | + | </div> | |
| - | + | </div> | |
<div class="col-lg-12" style="color:#333;"> | <div class="col-lg-12" style="color:#333;"> | ||
<div class="panel panel-default"> | <div class="panel panel-default"> | ||
| - | + | <div class="panel-heading"> | |
| - | + | <h3 class="panel-title">1. Please go to <a href="http://www.rcsb.org/">www.rcsb.org</a> and get a pdb file of your protein. If you cannot find one, we will not be able to assist you circularizing it.</h3> | |
| - | + | </div> | |
| - | + | <div class="panel-body"> | |
| - | + | Additionally, check the DNA sequence of your proteins for EcoRI, XbaI, SpeI, PstI and BsaI recognition sites. If there are E/X/S/P sites, you might have problems to change your backbone or add a promotor. If there is a BsaI recognition site, the cloning will be more difficult. | |
| - | + | <div class="radio"> | |
| - | + | <label> | |
| - | + | <input type="radio" data-bind="checked: data.hasBsaI, checkedValue: false, click: data.q0A.bind(null, true)" /> | |
| - | + | There is no BsaI site. | |
| - | + | </label> | |
| - | + | </div> | |
| - | + | <div class="radio"> | |
| - | + | <label> | |
| - | + | <input type="radio" data-bind="checked: data.hasBsaI, checkedValue: true, click: data.q0A.bind(null, true)" /> | |
| - | + | There is a BsaI site. | |
| - | + | </label> | |
| - | + | </div> | |
| - | + | </div> | |
</div> | </div> | ||
<!-- ko if: data.q0A --> | <!-- ko if: data.q0A --> | ||
<div class="panel panel-default"> | <div class="panel panel-default"> | ||
| - | + | <div class="panel-heading"> | |
| - | + | <h3 class="panel-title">2. Do you want to use split inteins or sortase to circularize your protein?</h3> | |
| - | + | </div> | |
| - | + | <div class="panel-body"> | |
| - | + | <div class="radio"> | |
| - | + | <label> | |
| - | + | <input type="radio" data-bind="checked: data.useSortase, checkedValue: false, click: data.q1A.bind(null, true)" /> | |
| - | + | NpuDnaE intein RFC <span data-bind="value: RFCnumber"></span> circularization construct | |
| - | + | </label> | |
| - | + | <ul> | |
| - | + | <li>Successfully used in our project</li> | |
| - | + | <li>High efficiency</li> | |
| - | + | <li>In vivo circularization</li> | |
| - | + | </ul> | |
| - | + | </div> | |
| - | + | <div class="radio"> | |
| - | + | <label> | |
| - | + | <input type="radio" data-bind="checked: data.useSortase, checkedValue: true, click: data.q1A.bind(null, true)" /> | |
| - | + | Sortase A circularization construct | |
| - | + | </label> | |
| - | + | <ul> | |
| - | + | <li>In vitro only</li> | |
| - | + | <li>Well-purified protein required</li> | |
| - | + | <li>Not successfully tested yet</li> | |
| - | + | </ul> | |
| - | + | </div> | |
| - | + | </div> | |
</div> | </div> | ||
<!-- /ko --> | <!-- /ko --> | ||
| Line 150: | Line 150: | ||
<!-- ko if: data.q1A --> | <!-- ko if: data.q1A --> | ||
<div class="panel panel-default"> | <div class="panel panel-default"> | ||
| - | + | <div class="panel-heading"> | |
| - | + | <h3 class="panel-title">3. Can your protein be easily expressed in <i>E. coli</i>?</h3> | |
| - | + | </div> | |
| - | + | <div class="panel-body"> | |
| - | + | <div class="radio"> | |
| - | + | <label> | |
| - | + | <input type="radio" data-bind="checked: data.needsSmt3, checkedValue: false, click: data.q2A.bind(null, true)" /> | |
| - | + | Yes, no problem. | |
| - | + | </label> | |
| - | + | </div> | |
| - | + | <div class="radio"> | |
| - | + | <label> | |
| - | + | <input type="radio" data-bind="checked: data.needsSmt3, checkedValue: true, click: data.q2A.bind(null, true)" /> | |
| - | + | No, I will have to add <a href="http://parts.igem.org/Part:BBa_K1362429">Smt3</a>. | |
| - | + | </label> | |
| - | + | </div> | |
| - | + | </div> | |
</div> | </div> | ||
| Line 172: | Line 172: | ||
<div data-bind="if: data.q2A() && !data.useSortase()"> | <div data-bind="if: data.q2A() && !data.useSortase()"> | ||
<div class="panel panel-default"> | <div class="panel panel-default"> | ||
| - | + | <div class="panel-heading"> | |
| - | + | <h3 class="panel-title">4. Which exteins do you want to use? They will remain as scars in your circular protein.</h3> | |
| - | + | </div> | |
| - | + | <div class="panel-body"> | |
| - | + | <!-- ko foreach: availableExteins --> | |
| - | + | <div class="radio"> | |
| - | + | <label> | |
| - | + | <input type="radio" data-bind="checked: $parent.data.exteins, checkedValue: $data, click: $parent.data.q3A.bind(null, true)" /> | |
| - | + | <span data-bind="text: N"></span>/<span data-bind="text: C"></span> | |
| - | + | <!-- ko if: comment --> | |
| - | + | (<span data-bind="text: comment"></span>) | |
| - | + | <!-- /ko --> | |
| - | + | <ul data-bind="foreach: notes"> | |
| - | + | <li data-bind="text: $data"></li> | |
| - | + | </ul> | |
| - | + | </label> | |
| - | + | </div> | |
| - | + | <!-- /ko --> | |
| - | + | </div> | |
</div> | </div> | ||
</div> | </div> | ||
<div data-bind="if: data.q3A() || (data.q2A() && data.useSortase())"> | <div data-bind="if: data.q3A() || (data.q2A() && data.useSortase())"> | ||
<div class="panel panel-default" > | <div class="panel panel-default" > | ||
| - | + | <div class="panel-heading"> | |
| - | + | <h3 class="panel-title"><span data-bind="text: data.useSortase() ? 4 : 5"></span>. If you want to save time, check manually whether the ends are close together (approx. <span data-bind="text: !data.useSortase() && data.exteins().N == 'XXX' ? 5 : 15"></span> Å or closer). For example, you can use the <a href="http://spdbv.vital-it.ch/">Swiss-PdbViewer</a> or <a href="http://www.pymol.org/">PyMOL</a>.</h3> | |
| - | + | </div> | |
| - | + | <div class="panel-body"> | |
| - | + | <div class="radio"> | |
| - | + | <label> | |
| - | + | <input type="radio" data-bind="checked: data.endsAreClose, checkedValue: true, click: data.q4A.bind(null, true)" /> | |
| - | + | They are close together. | |
| - | + | </label> | |
| - | + | </div> | |
| - | + | <div class="radio"> | |
| - | + | <label> | |
| - | + | <input type="radio" data-bind="checked: data.endsAreClose, checkedValue: false, click: data.q4A.bind(null, true)" /> | |
| - | + | I want/have to use <span data-bind="text: softwareName"></span>. | |
| - | + | </label> | |
| - | + | </div> | |
| - | + | </div> | |
</div> | </div> | ||
</div> | </div> | ||
| Line 240: | Line 240: | ||
<!-- ko if: data.comingBack --> | <!-- ko if: data.comingBack --> | ||
<div class="panel panel-default"> | <div class="panel panel-default"> | ||
| - | + | <div class="panel-heading"> | |
| - | + | <h3 class="panel-title"><span data-bind="text: data.useSortase() ? 5 : 6"></span>. Hello again. What is the result of <span data-bind="text: softwareName"></span>?</h3> | |
| - | + | </div> | |
| - | + | <div class="radio"> | |
| - | + | <label> | |
| - | + | <input type="radio" data-bind="checked: data.linkerLength, checkedValue: 0, click: data.q5A.bind(null, true)" /> | |
| - | + | I do not need a linker. | |
| - | + | </label> | |
| - | + | </div> | |
| - | + | <div class="radio"> | |
| - | + | <label> | |
| - | + | <input type="radio" data-bind="checked: data.linkerLength, checkedValue: 1, click: data.q5A.bind(null, true)" /> | |
| - | + | The linker + scars are < 15 amino acids in length. | |
| - | + | </label> | |
| - | + | </div> | |
| - | + | <div class="radio"> | |
| - | + | <label> | |
| - | + | <input type="radio" data-bind="checked: data.linkerLength, checkedValue: 2, click: data.q5A.bind(null, true)" /> | |
| - | + | The linker + scars are betweeen 16 and 30 amino acids in length. | |
| - | + | </label> | |
| - | + | </div> | |
| - | + | <div class="radio"> | |
| - | + | <label> | |
| - | + | <input type="radio" data-bind="checked: data.linkerLength, checkedValue: 3, click: data.q5A.bind(null, true)" /> | |
| - | + | The linker + scars are > 30 amino acids in length. | |
| - | + | </label> | |
| - | + | </div> | |
</div> | </div> | ||
<!-- /ko --> | <!-- /ko --> | ||
<!-- ko if: data.q5A --> | <!-- ko if: data.q5A --> | ||
<div class="panel panel-default"> | <div class="panel panel-default"> | ||
| - | + | <div class="panel-heading"> | |
| - | + | <h3 class="panel-title"><span data-bind="text: data.useSortase() ? 6 : 7"></span>. Have you decided to use one linker or try different linkers?</h3> | |
| - | + | </div> | |
| - | + | <div class="radio"> | |
| - | + | <label> | |
| - | + | <input type="radio" data-bind="checked: data.testSeveral, checkedValue: false, click: data.q6A.bind(null, true)" /> | |
| - | + | I trust <span data-bind="text: softwareName"></span>. One should be enough. | |
| - | + | </label> | |
| - | + | </div> | |
| - | + | <div class="radio"> | |
| - | + | <label> | |
| - | + | <input type="radio" data-bind="checked: data.testSeveral, checkedValue: true, click: data.q6A.bind(null, true)" /> | |
| - | + | I want to test several linkers. | |
| - | + | </label> | |
| - | + | </div> | |
</div> | </div> | ||
<!-- /ko --> | <!-- /ko --> | ||
</div> | </div> | ||
| - | + | </div> | |
</div> | </div> | ||
</html> | </html> | ||
{{:Team:Heidelberg/Templates/IncludeJS|:Team:Heidelberg/js/toolboxguide}} | {{:Team:Heidelberg/Templates/IncludeJS|:Team:Heidelberg/js/toolboxguide}} | ||
Revision as of 04:42, 15 October 2014
use the intein
TOOLBOX to
1. Please go to www.rcsb.org and get a pdb file of your protein. If you cannot find one, we will not be able to assist you circularizing it.
Additionally, check the DNA sequence of your proteins for EcoRI, XbaI, SpeI, PstI and BsaI recognition sites. If there are E/X/S/P sites, you might have problems to change your backbone or add a promotor. If there is a BsaI recognition site, the cloning will be more difficult.
2. Do you want to use split inteins or sortase to circularize your protein?
- Successfully used in our project
- High efficiency
- In vivo circularization
- In vitro only
- Well-purified protein required
- Not successfully tested yet
3. Can your protein be easily expressed in E. coli?
4. Which exteins do you want to use? They will remain as scars in your circular protein.
. If you want to save time, check manually whether the ends are close together (approx. Å or closer). For example, you can use the Swiss-PdbViewer or PyMOL.
Please use to generate a linker for your circular protein. [LINK]
This step might take up to 11 days.
NILS – hier könnte dein instruction-file-ersatz stehen
NILS – hier auch
NILS – hier immernoch
NILS – hier ebnfalls und auch gerne noch umfangreicher
Please save this link:
https://2014.igem.org/{{PAGENAME}}#
In order to generate your linker, needs to know the scar amino acid sequence that is caused by circularization. In your case, it is .
