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| − | <div id="sidebar"> | + | <header class="header"> |
| − | <div id="nav-anchor"></div> | + | <div class=" unique_header"> |
| − | <nav> | + | <h2 class="header_title">PROJECT</h2> |
| − | <ul style="list-style-type: none">
| + | </div> |
| − | <li style="list-style-type: none"><a href="#abstract" style="text-decoration: none; color:red;">Abstract</a></li>
| + | </header> |
| − | <li><a href="#intro">Introduction</a></li>
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| − | <li><a href="#project">Project Description</a></li>
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| − | <li><a href="#applications">Implications and applications</a></li>
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| − | <li><a href="#ref">References</a></li>
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| − | <h2> Project description </h2> | + | <h2> Project Overview</h2> |
| − | <h5> Genscript grant application iGEM Groningen 2017 </h5> | + | <h5> </h5> |
| | + | <hr class="small"> |
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| − | <section id="abstract">
| + | <p class="left">This part of the WIKI is dedicated to the research part of the IMPACT project. We will start by giving an overview of CRISPR-Cas, because this system is the central part of our project. In this overview the most important features will be discussed. If you are not known with the basic principles of <a href="">CRISPR-Cas</a> or you think you will need to refresh your memory, we would advise to read that part before continuing with the <a href="https://2017.igem.org/Team:Groningen/Description">project description</a>. This page will describe how we want to build our IMPACT-system and to what extent we have succeeded in it.</p> |
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| − | <p class="left"><b>Abstract</b></p> | + | |
| − | <p class="left">Bacteriophages are a major challenge for the dairy industry, having the potential to result in huge financial losses. To combat this labor intensive and costly phage infection screenings are performed as standard practice in current production procedures. The 2017 Groningen iGEM team sees an opportunity to improve on this and aims to build a genetically engineered phage detecting bacteria. The detector will be constructed in Lactococcus lactis and use a CRISPR adaptive immune system to constantly survey, recognize, and report specific virus infections. Through this proof of concept, a modular system will be developed which can detect virtually any DNA signal by swapping of a preprogrammed reporter plasmid.</p>
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| − | </section>
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| − | <section id="intro">
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| − | <p class="left"><b>Introduction</b></p>
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| − | <p class="left">In the Netherlands, export of dairy products, such as cheese, buttermilk, and yogurt, generates a combined yearly income of billions of euros with the Netherlands responsible for 5% of the world’s dairy products trade1. However, the industry is plagued by the very nature of its required processes. The bacteria used for the fabrication of these commodities are severely susceptible to viral infections, which can disrupt many production lines of the product that are a matter of national pride2. The iGEM (international Genetically Engineered Machine) Groningen team is determined to improve the current quality control standard practices by developing an improved system for the detection of phage infections. By implementation of our system, the Groningen iGEM team hopes to contribute to the on-going attempt to safeguard the production and export of our world famous dairy products.
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| − | The goal of the Groningen iGEM 2017 project is to design a system that can be used to survey for multiple pre-programmed nucleotide sequences together with a convenient readout. To do so, a CRISPR-based detection system will be developed that will be utilized to detect bacteriophages, bacterial viruses that are common spoilers of dairy fermentation processes. This system will rely on a modified version the “adaptive immune system” of bacteria, CRISPR-Cas, which naturally protects them from invading DNA from viruses. Various readout modules will be implemented to allow for proactive, quick and cheap screening of high-risk bacteriophage infections in the industrial pipeline.
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| − | To cover the costs of this project we rely on sponsoring of companies. We think that this project is worthy of the Genscript grant because of our shared interest in expanding the Cas9 based genome editing tools in the bio-brick database and further characterizing them. In addition, to adapt our system to recognize specific phage sequences, we will use your excellent services to custom synthesis phage homologous sequences in the reporter plasmid. If successful, our system will demonstrate the value of your services without which our project would not be possible and it would potentially increase the demand if adopted by others.</p>
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| − | </section>
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| − | | + | |
| − | <section id="project">
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| − | | + | |
| − | <p class="left"><b>Project Description</b></p>
| + | |
| − | <p class="left">This section will give a step by step description of how the mechanism behind this detector is envisioned, which is also depicted in the image below. Our engineered Lactococcus lactis detector cells will be exposed to a sample from the main daisy production line potentially containing phages. If present the phages will infect and insert their DNA into the detector cells and the highly active CRISPR adaptation pathway3 we introduced that will mediate the acquisition of a virus-specific spacer into a CRISPR array (2). Since the detection cells are the same organism as used in the fermentation, the infection will occur naturally. Next, from this array, new guide RNAs (gRNAs) will be transcribed that target a phage specific sequence. These gRNAs are used by Cas9 to survey and protect the cell against additional infections (3).
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| − | One of the many orthologous Cas9 systems that use the same gRNA structure will also complex and use this guide RNA to try and find matching sequences4. However, its nuclease domains will be inactivated, and its PAM will not match the viral DNA. Instead, it will match a specifically designed region of our reporter plasmid that matches the viral spacer and altered adjacent PAM sequence. The nuclease active Cas9 can never bind and cut here due to the altered PAM. Binding of the nuclease inactive orthologous Cas9 will lead to the reduction or increase in expression of a nearby gene on the reporter plasmid. This in turn will activate a signalling cascade that can spread from cell to cell eventually alerting the dairy workers a phage has been detected (4a). Additional modules can be added that allow for virus-specific readout by modification of restriction enzyme recognition sites (4b).
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| − | The first sub-project is the equipment of L. lactis cells with an adaptive CRISPR-Cas system. Heler et al. have succeeded in transferring a working CRISPR system of Staphylococcus pyogenes to Staphylococcus aureus5. A comparable approach will be used to transform the CRISPR system of S. pyogenes into L. lactis.
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| − | The Cas9 protein of S. pyogenes is well studied, and many variants of the Cas9 protein from the S. pyogenes system are known, such as dCas9 and Cas9 fusion proteins. The second sub-project is to test the working of these different Cas9 variants in L. lactis and identify which is most effective at inducing a reporter pathway. The Cas9 variants can alter gene expression via various mechanisms. The simplest is by for example cutting a GFP-expressing plasmid which will result in the loss of signal of a reporter gene, located on the plasmid. dCas9 can be used to repress gene expression using CRISPR interference. This gene can either be a repressor of a signalling gene (CRISPR interference results in the signal) or a signalling gene itself (CRISPR interference results in loss of signal). The last Cas9 derivative that will be tested is the dCas9-Omega fusion protein. This variant can either activate a gene, if it binds on the intended position (sweet spot) in the promoter region, or repress expression if it is targeted outside the sweet spot. Besides testing their activity separately, additional tests will be performed to confirm that these Cas9 proteins do not interfere with the functioning of the entire CRISPR system.
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| − | The third sub-project will be the amplification of the initial signal. Since only a small percentage of detection cells will be infected, this needs to be amplified to enable detection without single cell analysis. This will be done by using the other uninfected cells, and different mechanisms will be explored to achieve this. Currently, the primary candidate mechanism is the expression and secretion of nisin by the infected cells to trigger the expression of additional reporter signal in uninfected L. lactis cells. The nisin inducible expression system is a well-characterized positive feedback system in L. lactis and works with low concentrations of nisin6.
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| − | If the sub-goals mentioned above can rapidly be achieved, we will move on developing a system that can detect and differentiate between multiple pre-programed sequences. One way this could be achieved is by using a Cas9 variant to make mutations in regions recognized by restriction enzymes. Alteration of the sequences of these restriction sites would result in them no longer be recognized by the intended restriction enzymes. This way a restriction analysis would result in a different band pattern dependent on which restriction site was targeted by Cas9 variant and could give us sgRNA specific readouts.</p>
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| − | </section>
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| − | | + | |
| − | <section id="applications">
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| − | <p class="left"><b>Implications and applications</b></p>
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| − | <p class="left">If the envisioned detection system is constructed successfully, it will be a great help to the dairy industry. In this case, it would offer a cheap and quick way to detect phages, which would allow for a timely response to an infection. Moreover, the system could also be used in other industries which use L. lactis as fermentation organism. Also, due to the bio brick standard, the system should theoretically be transferable to other organisms. Another application for this detection system would be for phage research because it would offer a quick way to determine whether the organism is infected and by which phage. In conclusion, the envisioned detection system has the potential to be of great benefit for both phage detection and phage research.
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| − | Once the system is placed in the detector organism, it would be quite easy to switch the target sequence. If you would like to target another phage or sequence the only thing that has to be done is change the part of the reporter plasmids (4a and 4b) that are homologous to the phage-specific spacer.
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| − | In the unfortunate event that not all the main goals are completed, each of the sub-projects could be of value for other applications. Transforming a CRISPR-Cas system capable of spacer acquisition into L. lactis is a challenge, which has to our knowledge not been achieved before. This alone would make the bacteria more resistant to phages, which would be a huge benefit for fermentation cultures. Unfortunately, this holds no value for the primarily targeted dairy industry since they cannot use GMO’s directly in their fermentation processes. This may change in the future and for other industries, in which GMO’s can be used, it would directly be beneficial.
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| − | The characterization of the Cas9 derivatives in L. lactis will expand the genome editing or gene expression alteration tools available for researchers working with those organisms. In addition, these systems will be fabricated as a new bio brick making them convenient to adopt. Finally, the signal amplification system will be widely applicable as a convenient and modular tool for any detection system.
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| − | In summary, our proposed project will provide a valuable tool for the dairy industry and further expand and characterize genome editing told that will be widely applicable.</p>
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| − | </section>
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| − | <section id="ref">
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| − | <p class="left"><b>Refernces</b></p>
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| − | <ol type="1" style="text-align: left;">
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| − | <li>CBS (Central Bureau of Statistics). (08-06-2016). The Netherlands earns billions in agro-sector exports. Retrieved from: https://www.cbs.nl/en-gb/news/2016/23/the-netherlands-earns-billions-in-agro-sector-exports</li>
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| − | <li>Garneau, J.E. and Moineau S. (2011). Bacteriophages of lactic acid bacteria and their impact on milk fermentations. Microb Cell Fact. 10 Suppl 1:S20. doi: 10.1186/1475-2859-10-S1-S20.</li>
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| − | <li>Heler, R. et al. Mutations in Cas9 Enhance the Rate of Acquisition of Viral Spacer Sequences during the CRISPR-Cas Immune Response. Mol. Cell 65, 168–175 (2017).</li>
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| − | <li>Braff, J. L., Yaung, S. J., Esvelt, K. M. & Church, G. M. Characterization of Cas9–guide RNA orthologs. Cold Spring Harb. Protoc. 2016, 422–425 (2016).</li>
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| − | <li>Heler, R. et al. Cas9 specifies functional viral targets during CRISPR–Cas adaptation. Nature 519, 199–202 (2015).</li>
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| − | <li>Kuipers, O. P., De Ruyter, P. G. G. A., Kleerebezem, M. & De Vos, W. M. Quorum sensing-controlled gene expression in lactic acid bacteria. J. Biotechnol. 64, 15–21 (1998).</li>
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| − | </ol>
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| − | | + | |
| − | </section>
| + | |
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| | + | <p class="left">After discussing the project overview we will introduce you to our <a href="https://2017.igem.org/Team:Groningen/Model">model</a>, which was used for the constructions of our parts. How we have constructed the different parts and the reasons why we have made certain choices will be discussed in the <a href="https://2017.igem.org/Team:Groningen/Design">experimental plan.</a> Experimental details unique to the experiment, such as PCR conditions, and the results of each construction step will be discussed in the <a href="https://2017.igem.org/Team:Groningen/Results">result subpage.</a> The general protocols of all lab techniques are discussed on the <a href="https://2017.igem.org/Team:Groningen/Protocols">protocol page.</a></p> |
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| − | </div> | + | <div class="footer"> |
| − | | + | <img style="width: 100%;" src="https://static.igem.org/mediawiki/2017/b/b5/Banner.jpg" alt="Sponsors"> |
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b.isWindow(e)?e:9===e.nodeType?e.defaultView||e.parentWindow:!1}b.each({Height:"height",Width:"width"},function(e,n){b.each({padding:"inner"+e,content:n,"":"outer"+e},function(r,i){b.fn[i]=function(i,o){var a=arguments.length&&(r||"boolean"!=typeof i),s=r||(i===!0||o===!0?"margin":"border");return b.access(this,function(n,r,i){var o;return b.isWindow(n)?n.document.documentElement["client"+e]:9===n.nodeType?(o=n.documentElement,Math.max(n.body["scroll"+e],o["scroll"+e],n.body["offset"+e],o["offset"+e],o["client"+e])):i===t?b.css(n,r,s):b.style(n,r,i,s)},n,a?i:t,a,null)}})}),e.jQuery=e.$=b,"function"==typeof define&&define.amd&&define.amd.jQuery&&define("jquery",[],function(){return b})})(window);
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| − |
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| − | </script>
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| − |
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| − | <script>
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| − |
| |
| − | $(document).ready(function(){
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| − |
| |
| − | /**
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| − | * This part does the "fixed navigation after scroll" functionality
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| − | * We use the jQuery function scroll() to recalculate our variables as the
| |
| − | * page is scrolled/
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| − | */
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| − | $(window).scroll(function(){
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| − | var window_top = $(window).scrollTop() + 12; // the "12" should equal the margin-top value for nav.stick
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| − | var div_top = $('#nav-anchor').offset().top;
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| − | if (window_top > div_top) {
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| − | $('nav').addClass('stick');
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| − | } else {
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| − | $('nav').removeClass('stick');
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| − | }
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| − | });
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| − |
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| − |
| |
| − | /**
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| − | * This part causes smooth scrolling using scrollto.js
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| − | * We target all a tags inside the nav, and apply the scrollto.js to it.
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| − | */
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| − | $("nav a").click(function(evn){
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| − | evn.preventDefault();
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| − | $('html,body').scrollTo(this.hash, this.hash);
| |
| − | });
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| − |
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| − |
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| − |
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| − | /**
| |
| − | * This part handles the highlighting functionality.
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| − | * We use the scroll functionality again, some array creation and
| |
| − | * manipulation, class adding and class removing, and conditional testing
| |
| − | */
| |
| − | var aChildren = $("nav li").children(); // find the a children of the list items
| |
| − | var aArray = []; // create the empty aArray
| |
| − | for (var i=0; i < aChildren.length; i++) {
| |
| − | var aChild = aChildren[i];
| |
| − | var ahref = $(aChild).attr('href');
| |
| − | aArray.push(ahref);
| |
| − | } // this for loop fills the aArray with attribute href values
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| − |
| |
| − | $(window).scroll(function(){
| |
| − | var windowPos = $(window).scrollTop(); // get the offset of the window from the top of page
| |
| − | var windowHeight = $(window).height(); // get the height of the window
| |
| − | var docHeight = $(document).height();
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| − |
| |
| − | for (var i=0; i < aArray.length; i++) {
| |
| − | var theID = aArray[i];
| |
| − | var divPos = $(theID).offset().top; // get the offset of the div from the top of page
| |
| − | var divHeight = $(theID).height(); // get the height of the div in question
| |
| − | if (windowPos >= divPos && windowPos < (divPos + divHeight)) {
| |
| − | $("a[href='" + theID + "']").addClass("nav-active");
| |
| − | } else {
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| − | $("a[href='" + theID + "']").removeClass("nav-active");
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| − | }
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| − | }
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| − |
| |
| − | if(windowPos + windowHeight == docHeight) {
| |
| − | if (!$("nav li:last-child a").hasClass("nav-active")) {
| |
| − | var navActiveCurrent = $(".nav-active").attr("href");
| |
| − | $("a[href='" + navActiveCurrent + "']").removeClass("nav-active");
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| − | $("nav li:last-child a").addClass("nav-active");
| |
| − | }
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| − | }
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| − | });
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| − | });
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| − |
| |
| − | </script>
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| − |
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| − | <script>
| |
| − | /*!
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| − | * jquery.scrollto.js 0.0.1 - https://github.com/yckart/jquery.scrollto.js
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| − | * Scroll smooth to any element in your DOM.
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| − | *
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| − | * Copyright (c) 2012 Yannick Albert (http://yckart.com)
| |
| − | * Licensed under the MIT license (http://www.opensource.org/licenses/mit-license.php).
| |
| − | * 2013/02/17
| |
| − | **/
| |
| − | $.scrollTo = $.fn.scrollTo = function(x, y, options){
| |
| − | if (!(this instanceof $)) return $.fn.scrollTo.apply($('html, body'), arguments);
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| − |
| |
| − | options = $.extend({}, {
| |
| − | gap: {
| |
| − | x: 0,
| |
| − | y: 0
| |
| − | },
| |
| − | animation: {
| |
| − | easing: 'swing',
| |
| − | duration: 600,
| |
| − | complete: $.noop,
| |
| − | step: $.noop
| |
| − | }
| |
| − | }, options);
| |
| − |
| |
| − | return this.each(function(){
| |
| − | var elem = $(this);
| |
| − | elem.stop().animate({
| |
| − | scrollLeft: !isNaN(Number(x)) ? x : $(y).offset().left + options.gap.x,
| |
| − | scrollTop: !isNaN(Number(y)) ? y : $(y).offset().top + options.gap.y
| |
| − | }, options.animation);
| |
| − | });
| |
| − | };
| |
| − | </script>
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| − |
| |
| − | <script>
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| − |
| |
| − | var windw = this;
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| − |
| |
| − | $.fn.followTo = function ( pos ) {
| |
| − | var $this = this,
| |
| − | $window = $(windw);
| |
| − |
| |
| − | $window.scroll(function(e){
| |
| − | if ($window.scrollTop() < pos) {
| |
| − | $this.css({
| |
| − | position: 'absolute',
| |
| − | top: pos
| |
| − | });
| |
| − | } else {
| |
| − | $this.css({
| |
| − | position: 'fixed',
| |
| − | top: 0
| |
| − | });
| |
| − | }
| |
| − | });
| |
| − | };
| |
| − |
| |
| − | $('#sidebar').followTo(750);
| |
| − |
| |
| − | </script>
| |
| − |
| |
| | | | |
| | </html> | | </html> |
