Difference between revisions of "Team:Fudan-TSI/Composite Part"

(LC after)
m
 
Line 1: Line 1:
−
{{Fudan-TSI}}
+
{{Fudan-TSI}}<!-- jquery loaded by HQ 1.12.4 -->
 
<html></p></div></div></div><meta name="viewport" content="width=device-width, initial-scale=1"><meta charset="UTF-8">
 
<html></p></div></div></div><meta name="viewport" content="width=device-width, initial-scale=1"><meta charset="UTF-8">
 
   <link rel="stylesheet" href="https://2019.igem.org/wiki/index.php?title=Template:Fudan-TSI/materialize.css&action=raw&ctype=text/css">
 
   <link rel="stylesheet" href="https://2019.igem.org/wiki/index.php?title=Template:Fudan-TSI/materialize.css&action=raw&ctype=text/css">
Line 108: Line 108:
 
</div><!-- #pageCover -->
 
</div><!-- #pageCover -->
 
<script src="https://2019.igem.org/wiki/index.php?title=Template:Fudan-TSI/bkg&action=raw&ctype=text/javascript"></script>
 
<script src="https://2019.igem.org/wiki/index.php?title=Template:Fudan-TSI/bkg&action=raw&ctype=text/javascript"></script>
−
<script>function bkgFunction(e){if(e){var t=new Stats;t.domElement.style.position="absolute",t.domElement.style.left="0",t.domElement.style.top="0",document.body.appendChild(t.domElement),requestAnimationFrame(function e(){t.update(),requestAnimationFrame(e)})}var n=document.getElementById("demo");tesselation.setup(n),gradients.setup();var o,r=1e4,a=3e3;window.requestAnimationFrame(function e(t){(!o||r<t-o)&&(o=t,tesselation.next(a),gradients.next(a)),window.requestAnimationFrame(e)})}var calcDelaunayTriangulation=function(){var E=1/1048576;function p(e,t,n,o){var r,a,i,l,c,u,s,d,h,f,p=e[t][0],m=e[t][1],g=e[n][0],y=e[n][1],w=e[o][0],I=e[o][1],b=Math.abs(m-y),v=Math.abs(y-I);if(b<E&&v<E)throw new Error("Can't get circumcircle since all 3 points are y-aligned");return i=-(g-p)/(y-m),l=-(w-g)/(I-y),c=(p+g)/2,u=(g+w)/2,s=(m+y)/2,d=(y+I)/2,{i:t,j:n,k:o,x:r=b<E?c:v<E?u:(i*c-l*u+d-s)/(i-l),y:a=v<b?i*(r-c)+s:l*(r-u)+d,r:(h=g-r)*h+(f=y-a)*f}}function m(e){var t,n,o,r,a,i;for(n=e.length;n;)for(r=e[--n],o=e[--n],t=n;t;){if(i=e[--t],o===(a=e[--t])&&r===i){e.splice(n,2),e.splice(t,2);break}if(o===i&&r===a){e.splice(n,2),e.splice(t,2);break}}}return function(n){var e,t,o,r,a,i,l,c,u,s,d,h,f=n.length;if(f<3||2e3<f)return[];for(n=n.slice(0),o=new Array(f),e=f;e--;)o[e]=e;for(o.sort(function(e,t){return n[t][0]-n[e][0]}),r=function(e){var t,n,o,r,a,i,l=Number.POSITIVE_INFINITY,c=Number.POSITIVE_INFINITY,u=Number.NEGATIVE_INFINITY,s=Number.NEGATIVE_INFINITY;for(t=e.length;t--;)e[t][0]<l&&(l=e[t][0]),e[t][0]>u&&(u=e[t][0]),e[t][1]<c&&(c=e[t][1]),e[t][1]>s&&(s=e[t][1]);return o=s-c,[[(a=l+.5*(n=u-l))-20*(r=Math.max(n,o)),(i=c+.5*o)-r],[a,i+20*r],[a+20*r,i-r]]}(n),n.push(r[0],r[1],r[2]),a=[p(n,f+0,f+1,f+2)],i=[],l=[],e=o.length;e--;l.length=0){for(h=o[e],t=a.length;t--;)0<(c=n[h][0]-a[t].x)&&c*c>a[t].r?(i.push(a[t]),a.splice(t,1)):c*c+(u=n[h][1]-a[t].y)*u-a[t].r>E||(l.push(a[t].i,a[t].j,a[t].j,a[t].k,a[t].k,a[t].i),a.splice(t,1));for(m(l),t=l.length;t;)d=l[--t],s=l[--t],a.push(p(n,s,d,h))}for(e=a.length;e--;)i.push(a[e]);for(a.length=0,e=i.length;e--;)i[e].i<f&&i[e].j<f&&i[e].k<f&&a.push(i[e].i,i[e].j,i[e].k);return a}}(),tesselation=function(){var a,h,f,i;return{setup:function(e){var t=(a=e).getAttribute("viewBox").split(/\D/g);h=t[2],f=t[3]},next:function(e){var t,n,o;if(e/=1e3,i&&i.children&&i.children.length){for(n=o=(t=i).children.length;n--;)TweenMax.to(t.children[n],.4*e,{opacity:0,delay:e*(.3*n/o)});TweenMax.delayedCall(.75*e,function(e){a.removeChild(e)},[t],this)}var r=function(){var e,t,n,o,r=window.innerWidth,a=window.innerHeight,i=[],l=h%(e=h/f<r/a?250*h/r:250*f/a)/2,c=f%e/2;for(n=Math.floor(h/e)+1;-1<=n;n--)for(o=Math.floor(f/e)+1;-1<=o;o--)i.push([l+e*(n+.75*(Math.random()-.5)),c+e*(o+.75*(Math.random()-.5))]);var u,s=calcDelaunayTriangulation(i),d=document.createElementNS("http://www.w3.org/2000/svg","g");for(t=s.length;t;)(u=document.createElementNS("http://www.w3.org/2000/svg","polygon")).setAttribute("points",i[s[--t]][0]+","+i[s[t]][1]+" "+i[s[--t]][0]+","+i[s[t]][1]+" "+i[s[--t]][0]+","+i[s[t]][1]),d.appendChild(u);return d}();for(n=o=r.children.length;n--;)TweenMax.fromTo(r.children[n],.4*e,{opacity:0},{opacity:.3+.25*Math.random(),delay:e*(.3*n/o+.3),ease:Back.easeOut});a.appendChild(r),i=r}}}(),gradients=function(){var o,r,a,i=["#3c6df0","#12a3b4","#00a78f","#00aa5e","#81b532","#e3bc13","#ffb000","#fe8500","#fe6100","#e62325","#dc267f","#c22dd5","#9753e1","#5a3ec8"];return{setup:function(){a=!1,o={stopA:document.getElementById("stop1a"),stopB:document.getElementById("stop1b"),rect:document.getElementById("rect1")},r={stopA:document.getElementById("stop2a"),stopB:document.getElementById("stop2b"),rect:document.getElementById("rect2")},o.rect.style.opacity=0,r.rect.style.opacity=0},next:function(e){var t,n;e/=1e3,t=a?(n=o,r):(n=r,o),a=!a,TweenMax.to(n.rect,.55*e,{opacity:0,delay:.2*e,ease:Sine.easeOut}),function(e){var t=Math.floor(i.length*Math.random()),n=Math.floor(3*Math.random())+3;n=(t+n*(Math.random()<.5?-1:1)+i.length)%i.length,e.stopA.setAttribute("stop-color",i[t]),e.stopB.setAttribute("stop-color",i[n])}(t),TweenMax.to(t.rect,.65*e,{opacity:1,ease:Sine.easeIn})}}}();bkgFunction();
+
        <script>
−
</script>
+
      //////////////////////////////
 +
      // Demo Functions
 +
      //////////////////////////////
 +
      function bkgFunction(showStats) {
 +
        // stats
 +
        if (showStats) {
 +
        var stats = new Stats();
 +
        stats.domElement.style.position = 'absolute';
 +
        stats.domElement.style.left = '0';
 +
        stats.domElement.style.top = '0';
 +
        document.body.appendChild(stats.domElement);
 +
        requestAnimationFrame(function updateStats(){
 +
          stats.update();
 +
          requestAnimationFrame(updateStats);
 +
        });
 +
        }
 +
        // init
 +
        var svg = document.getElementById('demo');
 +
        tesselation.setup(svg);
 +
        gradients.setup();
 +
        var lastTransitionAt, transitionDelay = 10000, transitionDuration = 3000;
 +
        function playNextTransition() {
 +
        tesselation.next(transitionDuration);
 +
        gradients.next(transitionDuration);
 +
        };
 +
        function tick(time) {
 +
        if (!lastTransitionAt || time - lastTransitionAt > transitionDelay) {
 +
          lastTransitionAt = time;
 +
          playNextTransition();
 +
        }
 +
        window.requestAnimationFrame(tick);
 +
        }
 +
        window.requestAnimationFrame(tick);
 +
      }
 +
      //////////////////////////////
 +
      // Delaunay Triangulation
 +
      //////////////////////////////
 +
      var calcDelaunayTriangulation = (function() {
 +
        var EPSILON = 1.0 / 1048576.0;
 +
        function getSuperT(vertices) {
 +
        var xMin = Number.POSITIVE_INFINITY, yMin = Number.POSITIVE_INFINITY,
 +
          xMax = Number.NEGATIVE_INFINITY, yMax = Number.NEGATIVE_INFINITY,
 +
          i, xDiff, yDiff, maxDiff, xCenter, yCenter;
 +
        for(i = vertices.length; i--; ) {
 +
          if(vertices[i][0] < xMin) xMin = vertices[i][0];
 +
          if(vertices[i][0] > xMax) xMax = vertices[i][0];
 +
          if(vertices[i][1] < yMin) yMin = vertices[i][1];
 +
          if(vertices[i][1] > yMax) yMax = vertices[i][1];
 +
        }
 +
        xDiff = xMax - xMin;
 +
        yDiff = yMax - yMin;
 +
        maxDiff = Math.max(xDiff, yDiff);
 +
        xCenter = xMin + xDiff * 0.5;
 +
        yCenter = yMin + yDiff * 0.5;
 +
        return [
 +
          [xCenter - 20 * maxDiff, yCenter - maxDiff],
 +
          [xCenter, yCenter + 20 * maxDiff],
 +
          [xCenter + 20 * maxDiff, yCenter - maxDiff]
 +
        ];
 +
        }
 +
        function circumcircle(vertices, i, j, k) {
 +
        var xI = vertices[i][0], yI = vertices[i][1],
 +
          xJ = vertices[j][0], yJ = vertices[j][1],
 +
          xK = vertices[k][0], yK = vertices[k][1],
 +
          yDiffIJ = Math.abs(yI - yJ), yDiffJK = Math.abs(yJ - yK),
 +
          xCenter, yCenter, m1, m2, xMidIJ, xMidJK, yMidIJ, yMidJK, xDiff, yDiff;
 +
        // bail condition
 +
        if(yDiffIJ < EPSILON){
 +
          if (yDiffJK < EPSILON){
 +
            throw new Error("Can't get circumcircle since all 3 points are y-aligned");
 +
          }
 +
        }
 +
 
 +
 
 +
        // calc circumcircle center x/y, radius
 +
        m1  = -((xJ - xI) / (yJ - yI));
 +
        m2  = -((xK - xJ) / (yK - yJ));
 +
        xMidIJ = (xI + xJ) / 2.0;
 +
        xMidJK = (xJ + xK) / 2.0;
 +
        yMidIJ = (yI + yJ) / 2.0;
 +
        yMidJK = (yJ + yK) / 2.0;
 +
        xCenter = (yDiffIJ < EPSILON) ? xMidIJ :
 +
          (yDiffJK < EPSILON) ? xMidJK :
 +
          (m1 * xMidIJ - m2 * xMidJK + yMidJK - yMidIJ) / (m1 - m2);
 +
        yCenter  = (yDiffIJ > yDiffJK) ?
 +
          m1 * (xCenter - xMidIJ) + yMidIJ :
 +
          m2 * (xCenter - xMidJK) + yMidJK;
 +
        xDiff = xJ - xCenter;
 +
        yDiff = yJ - yCenter;
 +
        // return
 +
        return {i: i, j: j, k: k, x: xCenter, y: yCenter, r: xDiff * xDiff + yDiff * yDiff};
 +
        }
 +
        function dedupeEdges(edges) {
 +
        var i, j, a, b, m, n;
 +
        for(j = edges.length; j; ) {
 +
          b = edges[--j]; a = edges[--j];
 +
          for(i = j; i; ) {
 +
          n = edges[--i]; m = edges[--i];
 +
          if(a === m){
 +
            if (b===n){
 +
              edges.splice(j, 2); edges.splice(i, 2);
 +
              break;
 +
            }
 +
          }
 +
          if(a === n){
 +
            if (b===m){
 +
              edges.splice(j, 2); edges.splice(i, 2);
 +
              break;
 +
            }
 +
          }
 +
          }
 +
        }
 +
        }
 +
        return function(vertices) {
 +
        var n = vertices.length,
 +
          i, j, indices, st, candidates, locked, edges, dx, dy, a, b, c;
 +
        // bail if too few / too many verts
 +
        if(n < 3 || n > 2000)
 +
          return [];
 +
        // copy verts and sort indices by x-position
 +
        vertices = vertices.slice(0);
 +
        indices = new Array(n);
 +
        for(i = n; i--; )
 +
          indices[i] = i;
 +
        indices.sort(function(i, j) {
 +
          return vertices[j][0] - vertices[i][0];
 +
        });
 +
        // supertriangle
 +
        st = getSuperT(vertices);
 +
        vertices.push(st[0], st[1], st[2]);
 +
        // init candidates/locked tris list
 +
        candidates = [circumcircle(vertices, n + 0, n + 1, n + 2)];
 +
        locked = [];
 +
        edges = [];
 +
        // scan left to right
 +
        for(i = indices.length; i--; edges.length = 0) {
 +
          c = indices[i];
 +
          // check candidates tris against point
 +
          for(j = candidates.length; j--; ) {
 +
          // lock tri if point to right of circumcirc
 +
          dx = vertices[c][0] - candidates[j].x;
 +
          if (dx > 0.0){
 +
            if(dx * dx > candidates[j].r){
 +
              locked.push(candidates[j]);
 +
            candidates.splice(j, 1);
 +
            continue;
 +
            }
 +
          }
 +
 
 +
 
 +
          // point outside circumcirc = leave candidates
 +
          dy = vertices[c][1] - candidates[j].y;
 +
          if(dx * dx + dy * dy - candidates[j].r > EPSILON)
 +
            continue;
 +
          // point inside circumcirc = break apart, save edges
 +
          edges.push(
 +
            candidates[j].i, candidates[j].j,
 +
            candidates[j].j, candidates[j].k,
 +
            candidates[j].k, candidates[j].i
 +
          );
 +
          candidates.splice(j, 1);
 +
          }
 +
          // new candidates from broken edges
 +
          dedupeEdges(edges);
 +
          for(j = edges.length; j; ) {
 +
          b = edges[--j];
 +
          a = edges[--j];
 +
          candidates.push(circumcircle(vertices, a, b, c));
 +
          }
 +
        }
 +
        // close candidates tris, remove tris touching supertri verts
 +
        for(i = candidates.length; i--; )
 +
          locked.push(candidates[i]);
 +
        candidates.length = 0;
 +
        for(i = locked.length; i--; )
 +
          if(locked[i].i < n){
 +
            if(locked[i].j < n){
 +
              if(locked[i].k < n){
 +
                candidates.push(locked[i].i, locked[i].j, locked[i].k);
 +
              }
 +
            }
 +
          }
 +
 
 +
 
 +
        // done
 +
        return candidates;
 +
        };
 +
      })();
 +
      var tesselation = (function() {
 +
        var svg, svgW, svgH, prevGroup;
 +
        function createRandomTesselation() {
 +
        var wW = window.innerWidth;
 +
        var wH = window.innerHeight;
 +
        var gridSpacing = 250, scatterAmount = 0.75;
 +
        var gridSize, i, x, y;
 +
        if (wW / wH > svgW / svgH) { // window wider than svg = use width for gridSize
 +
          gridSize = gridSpacing * svgW / wW;
 +
        } else { // window taller than svg = use height for gridSize
 +
          gridSize = gridSpacing * svgH / wH;
 +
        }
 +
        var vertices = [];
 +
        var xOffset = (svgW % gridSize) / 2, yOffset = (svgH % gridSize) / 2;
 +
        for (x = Math.floor(svgW/gridSize) + 1; x >= -1; x--) {
 +
          for (y = Math.floor(svgH/gridSize) + 1; y >= -1; y--) {
 +
          vertices.push(
 +
            [
 +
            xOffset + gridSize * (x + scatterAmount * (Math.random() - 0.5)),
 +
            yOffset + gridSize * (y + scatterAmount * (Math.random() - 0.5))
 +
            ]
 +
          );
 +
          }
 +
        }
 +
        var triangles = calcDelaunayTriangulation(vertices);
 +
        var group = document.createElementNS('http://www.w3.org/2000/svg','g');
 +
        var polygon;
 +
        for(i = triangles.length; i; ) {
 +
          polygon = document.createElementNS('http://www.w3.org/2000/svg','polygon');
 +
          polygon.setAttribute('points',
 +
          vertices[triangles[--i]][0] + ',' + vertices[triangles[i]][1] + ' ' +
 +
          vertices[triangles[--i]][0] + ',' + vertices[triangles[i]][1] + ' ' +
 +
          vertices[triangles[--i]][0] + ',' + vertices[triangles[i]][1]
 +
          );
 +
          group.appendChild(polygon);
 +
        }
 +
        return group;
 +
        }
 +
        return {
 +
        setup: function(svgElement) {
 +
          svg = svgElement;
 +
          var vb = svg.getAttribute('viewBox').split(/\D/g);
 +
          svgW = vb[2];
 +
          svgH = vb[3];
 +
        },
 +
        next: function(t) {
 +
          var toRemove, i, n;
 +
          t /= 1000;
 +
          if(prevGroup){
 +
            if(prevGroup.children){
 +
              if(prevGroup.children.length){
 +
                toRemove = prevGroup;
 +
                n = toRemove.children.length;
 +
                for (i = n; i--; ) {
 +
                  TweenMax.to(toRemove.children[i], t*0.4, {opacity: 0, delay: t*(0.3*i/n)});
 +
                }
 +
                TweenMax.delayedCall(t * (0.7 + 0.05), function(group) { svg.removeChild(group); }, [toRemove], this);
 +
              }
 +
            }
 +
          }
 +
 
 +
          var g = createRandomTesselation();
 +
          n = g.children.length;
 +
          for (i = n; i--; ) {
 +
          TweenMax.fromTo(g.children[i], t*0.4, {opacity: 0}, {opacity: 0.3 + 0.25 * Math.random(), delay: t*(0.3*i/n + 0.3), ease: Back.easeOut});
 +
          }
 +
          svg.appendChild(g);
 +
          prevGroup = g;
 +
        }
 +
        }
 +
      })();
 +
      //////////////////////////////
 +
      // Gradients
 +
      //////////////////////////////
 +
      var gradients = (function() {
 +
        var grad1, grad2, showingGrad1;
 +
        // using colors from IBM Design Colors this time
 +
        var colors = [ // 14 colors - use 3-5 span
 +
        '#3c6df0', // ultramarine50
 +
        '#12a3b4', // aqua40
 +
        '#00a78f', // teal40
 +
        '#00aa5e', // green40
 +
        '#81b532', // lime30
 +
        '#e3bc13', // yellow20
 +
        '#ffb000', // gold20
 +
        '#fe8500', // orange30
 +
        '#fe6100', // peach40
 +
        '#e62325', // red50
 +
        '#dc267f', // magenta50
 +
        '#c22dd5', // purple50
 +
        '#9753e1', // violet50
 +
        '#5a3ec8'  // indigo60
 +
        ];
 +
        function assignRandomColors(gradObj) {
 +
        var rA = Math.floor(colors.length * Math.random());
 +
        var rB = Math.floor(Math.random() * 3) + 3; // [3 - 5]
 +
        rB = (rA + (rB * (Math.random() < 0.5 ? -1 : 1)) + colors.length) % colors.length;
 +
        gradObj.stopA.setAttribute('stop-color', colors[rA]);
 +
        gradObj.stopB.setAttribute('stop-color', colors[rB]);
 +
        }
 +
        return {
 +
        setup: function() {
 +
          showingGrad1 = false;
 +
          grad1 = {
 +
          stopA: document.getElementById('stop1a'),
 +
          stopB: document.getElementById('stop1b'),
 +
          rect: document.getElementById('rect1')
 +
          };
 +
          grad2 = {
 +
          stopA: document.getElementById('stop2a'),
 +
          stopB: document.getElementById('stop2b'),
 +
          rect: document.getElementById('rect2')
 +
          };
 +
          grad1.rect.style.opacity = 0;
 +
          grad2.rect.style.opacity = 0;
 +
        },
 +
        next: function(t) {
 +
          t /= 1000;
 +
          var show, hide;
 +
          if (showingGrad1) {
 +
          hide = grad1;
 +
          show = grad2;
 +
          } else {
 +
          hide = grad2;
 +
          show = grad1;
 +
          }
 +
          showingGrad1 = !showingGrad1;
 +
          TweenMax.to(hide.rect, 0.55*t, {opacity: 0, delay: 0.2*t, ease: Sine.easeOut});
 +
          assignRandomColors(show);
 +
          TweenMax.to(show.rect, 0.65*t, {opacity: 1, ease: Sine.easeIn});
 +
        }
 +
        };
 +
      })();
 +
      //////////////////////////////
 +
      // Start
 +
      //////////////////////////////
 +
      bkgFunction();
 +
    </script>
 
               <div style="position:absolute;top:100px;left:9%"><center><img style="height:120px;width:auto" alt="cover gif 1st added" src="https://static.igem.org/mediawiki/2019/e/ee/T--Fudan-TSI--coverCompositeParts.gif" /></center></div>
 
               <div style="position:absolute;top:100px;left:9%"><center><img style="height:120px;width:auto" alt="cover gif 1st added" src="https://static.igem.org/mediawiki/2019/e/ee/T--Fudan-TSI--coverCompositeParts.gif" /></center></div>
 
           </div>
 
           </div>

Latest revision as of 05:57, 16 November 2019

Composite Parts | 2019 iGEM Team:Fudan-TSI


Composite Parts

cover gif 1st added

For best composite part, we would like to present you with our Cre recombinase together with degradation tag. Cre recombinase (BBa_K3257044) is responsible for the recombination process between the reverse transcribed cDNA and the target Gene of Interest (GOI), which is rather one of the most important processes in our system.

Because of the leakage of uninduced Cre expression, we have spent much effort in lowering the steady-state expression level of Cre. An important approach is to attach 5 different types of degradation tags after the CDS of Cre. Using EGFP (BBa_E0040) as a reporter, we can see that the degradation rate of degradation tag with amino acid residual sequence of AANDENWVLAA (BBa_K3257073) is moderate for Cre recombinase to reach a steady but relatively low level of expression (Figure 1).

Figure 1. Degradation tag greatly reduces the protein level at stable state.
WT represents the positive control of EGFP without any tag attachment. The five degradation tags are represented by their last five amino acid sequence. The vertical axe shows the quantitative analysis of EGFP fluorescence (excitation wavelength: 485 nm; detection wavelength: 528 nm), normalized by cell amount (OD600). The fluorescence is quantified by the concentration of green fluorescein, cell number is quantified by the number of silicon beads, both are from the distributed measurement kit. Fluorescence below detection level are eliminated. Error bar stands for the SEM of 3 replicates. t-test is performed between WT and each degradation tag, P<0.0001 (****).

Transformed into BL21(DE3) with another plasmid containing lox sites and analyzed by colony PCR, we can see that DNA cleavage between identical-oriented loxP sites happens indicating the normal function of Cre recombinase (Figure 2). For more information of other composite parts, please refer to the part collection page and the iGEM Part Registry ranging from BBa_K3257100 to BBa_K3257118.

Figure 2. The verification of DNA cleavage between LoxP sites.
Plasmids containing the Cre gene were co-transformed with plasmids containing mCherry flanked by LoxP sites. A pair of sequencing primers were used to amplify the mCherry gene. PCR product would be around 1000 bp while fragments cleaved by Cre recombinase would be approximately 250 bp. The positive control group is the PCR product of plasmids containing the original mCherry gene. The negative control group is the PCR product of plasmids containing the Cre gene only.

Combining these two results, we show that this composite part is able to express Cre recombinase which functions well in an E. coli cell at a relatively low expression level, which is we need for new recombinants accumulating during the R-Evolution process.

In the future, iGEMers and synthetic biologists may use this part for homologous recombination with different degradation tags and promoters based on their own requirements.

For more information of other composite parts, please refer to the part collection page and the iGEM Part Registry ranging from BBa_K3257100 to BBa_K3257118.