Difference between revisions of "Team:Waterloo/Results"

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{{Waterloo}}
 
{{Waterloo}}
 
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        /*
 +
          Industrious by TEMPLATED
 +
          templated.co @templatedco
 +
          Released for free under the Creative Commons Attribution 3.0 license (templated.co/license)
 +
        */
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        /* Basic */
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        @media screen and (max-width: 480px) {
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          html, body {
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        *, *:before, *:after {
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        pre, a, abbr, acronym, address, big, cite,
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          h2 {
 +
            font-size: 1.75rem;
 +
            line-height: 1.5; }
 +
       
 +
          h3 {
 +
            font-size: 1.25rem; } }
 +
        sub {
 +
          font-size: 0.8rem;
 +
          position: relative;
 +
          top: 0.5rem; }
 +
       
 +
        sup {
 +
          font-size: 0.8rem;
 +
          position: relative;
 +
          top: -0.5rem; }
 +
       
 +
        blockquote {
 +
          border-left: solid 0.5rem;
 +
          font-style: italic;
 +
          margin: 0 0 2rem 0;
 +
          padding: 1rem 0 1rem 2rem; }
 +
       
 +
        code {
 +
          border-radius: 4px;
 +
          border: solid 1px;
 +
          font-family: "Courier New", monospace;
 +
          font-size: 0.9rem;
 +
          margin: 0 0.25rem;
 +
          padding: 0.25rem 0.65rem; }
 +
       
 +
        pre {
 +
          -webkit-overflow-scrolling: touch;
 +
          font-family: "Courier New", monospace;
 +
          font-size: 0.9rem;
 +
          margin: 0 0 2rem 0; }
 +
          pre code {
 +
            display: block;
 +
            line-height: 1.75;
 +
            padding: 1rem 1.5rem;
 +
            overflow-x: auto; }
 +
       
 +
        hr {
 +
          border: 0;
 +
          border-bottom: solid 1px;
 +
          margin: 2rem 0; }
 +
          hr.major {
 +
            margin: 4rem 0; }
 +
       
 +
        input, select, textarea {
 +
          color: #555555; }
 +
       
 +
        a {
 +
          color: #B3DC77; }
 +
       
 +
        strong, b {
 +
          color: #555555; }
 +
       
 +
        h1, h2, h3, h4, h5, h6 {
 +
          color: #555555; }
 +
       
 +
        blockquote {
 +
          border-left-color: rgba(0, 0, 0, 0.25); }
 +
       
 +
        code {
 +
          background: rgba(0, 0, 0, 0.075);
 +
          border-color: rgba(0, 0, 0, 0.25); }
 +
       
 +
        hr {
 +
          border-bottom-color: rgba(0, 0, 0, 0.25); }
 +
       
 +
        /* Inner */
 +
        .inner {
 +
          margin: 0 auto;
 +
          width: 75rem;
 +
          max-width: calc(100% - 6rem); }
 +
          @media screen and (max-width: 480px) {
 +
            .inner {
 +
              max-width: calc(100% - 3rem); } }
 +
       
 +
        /* Button */
 +
        input[type="submit"],
 +
        input[type="reset"],
 +
        input[type="button"],
 +
        button,
 +
        .button {
 +
          -moz-appearance: none;
 +
          -webkit-appearance: none;
 +
          -ms-appearance: none;
 +
          appearance: none;
 +
          -moz-transition: background-color 0.2s ease-in-out, box-shadow 0.2s ease-in-out, color 0.2s ease-in-out;
 +
          -webkit-transition: background-color 0.2s ease-in-out, box-shadow 0.2s ease-in-out, color 0.2s ease-in-out;
 +
          -ms-transition: background-color 0.2s ease-in-out, box-shadow 0.2s ease-in-out, color 0.2s ease-in-out;
 +
          transition: background-color 0.2s ease-in-out, box-shadow 0.2s ease-in-out, color 0.2s ease-in-out;
 +
          border: 0;
 +
          border-radius: 4px;
 +
          cursor: pointer;
 +
          display: inline-block;
 +
          font-weight: 600;
 +
          height: 3.25rem;
 +
          line-height: 3.25rem;
 +
          padding: 0 1.75rem;
 +
          text-align: center;
 +
          text-decoration: none;
 +
          white-space: nowrap; }
 +
          input[type="submit"].small,
 +
          input[type="reset"].small,
 +
          input[type="button"].small,
 +
          button.small,
 +
          .button.small {
 +
            font-size: 0.8rem;
 +
            height: 2.4375rem;
 +
            line-height: 2.4375rem;
 +
            padding: 0 1.25rem; }
 +
          input[type="submit"].large,
 +
          input[type="reset"].large,
 +
          input[type="button"].large,
 +
          button.large,
 +
          .button.large {
 +
            font-size: 1.35rem;
 +
            height: 4.0625rem;
 +
            line-height: 4.0625rem;
 +
            padding: 0 2rem; }
 +
          input[type="submit"].wide,
 +
          input[type="reset"].wide,
 +
          input[type="button"].wide,
 +
          button.wide,
 +
          .button.wide {
 +
            min-width: 13rem; }
 +
          input[type="submit"].icon:before,
 +
          input[type="reset"].icon:before,
 +
          input[type="button"].icon:before,
 +
          button.icon:before,
 +
          .button.icon:before {
 +
            margin-right: 0.5rem; }
 +
          input[type="submit"].fit,
 +
          input[type="reset"].fit,
 +
          input[type="button"].fit,
 +
          button.fit,
 +
          .button.fit {
 +
            width: 100%; }
 +
          input[type="submit"].disabled, input[type="submit"]:disabled,
 +
          input[type="reset"].disabled,
 +
          input[type="reset"]:disabled,
 +
          input[type="button"].disabled,
 +
          input[type="button"]:disabled,
 +
          button.disabled,
 +
          button:disabled,
 +
          .button.disabled,
 +
          .button:disabled {
 +
            pointer-events: none;
 +
            opacity: 0.25; }
 +
       
 +
        input[type="submit"],
 +
        input[type="reset"],
 +
        input[type="button"],
 +
        button,
 +
        .button {
 +
          background-color: transparent;
 +
          box-shadow: inset 0 0 0 1px #555555;
 +
          color: #555555 !important; }
 +
          input[type="submit"]:hover,
 +
          input[type="reset"]:hover,
 +
          input[type="button"]:hover,
 +
          button:hover,
 +
          .button:hover {
 +
            box-shadow: inset 0 0 0 1px #B3DC77;
 +
            color: #B3DC77 !important; }
 +
            input[type="submit"]:hover:active,
 +
            input[type="reset"]:hover:active,
 +
            input[type="button"]:hover:active,
 +
            button:hover:active,
 +
            .button:hover:active {
 +
              background-color: rgba(206, 27, 40, 0.25); }
 +
          input[type="submit"].primary,
 +
          input[type="reset"].primary,
 +
          input[type="button"].primary,
 +
          button.primary,
 +
          .button.primary {
 +
            box-shadow: none;
 +
            background-color: #B3DC77;
 +
            color: #ffffff !important; }
 +
            input[type="submit"].primary:hover,
 +
            input[type="reset"].primary:hover,
 +
            input[type="button"].primary:hover,
 +
            button.primary:hover,
 +
            .button.primary:hover {
 +
              background-color: #B3DC77;
 +
              box-shadow: none; }
 +
              input[type="submit"].primary:hover:active,
 +
              input[type="reset"].primary:hover:active,
 +
              input[type="button"].primary:hover:active,
 +
              button.primary:hover:active,
 +
              .button.primary:hover:active {
 +
                background-color: #B3DC77; }
 +
       
 +
        /* Form */
 +
        form {
 +
          margin: 0 0 2rem 0; }
 +
       
 +
        input[type="text"],
 +
        input[type="password"],
 +
        input[type="email"],
 +
        input[type="tel"],
 +
        input[type="search"],
 +
        input[type="url"],
 +
        select,
 +
        textarea {
 +
          -moz-appearance: none;
 +
          -webkit-appearance: none;
 +
          -ms-appearance: none;
 +
          appearance: none;
 +
          border-radius: 4px;
 +
          border: none;
 +
          border: solid 1px;
 +
          color: inherit;
 +
          display: block;
 +
          outline: 0;
 +
          padding: 0 1rem;
 +
          text-decoration: none;
 +
          width: 100%; }
 +
          input[type="text"]:invalid,
 +
          input[type="password"]:invalid,
 +
          input[type="email"]:invalid,
 +
          input[type="tel"]:invalid,
 +
          input[type="search"]:invalid,
 +
          input[type="url"]:invalid,
 +
          select:invalid,
 +
          textarea:invalid {
 +
            box-shadow: none; }
 +
       
 +
        label {
 +
          display: block;
 +
          font-size: 1rem;
 +
          font-weight: 600;
 +
          margin: 0 0 1rem 0; }
 +
       
 +
        input[type="text"],
 +
        input[type="password"],
 +
        input[type="email"],
 +
        input[type="tel"],
 +
        input[type="search"],
 +
        input[type="url"] {
 +
          height: 3.25rem; }
 +
       
 +
        select {
 +
          background-size: 1.25rem;
 +
          background-repeat: no-repeat;
 +
          background-position: calc(100% - 1rem) center;
 +
          height: 3.25rem;
 +
          padding-right: 3.25rem;
 +
          text-overflow: ellipsis; }
 +
          select:focus::-ms-value {
 +
            background-color: transparent; }
 +
          select::-ms-expand {
 +
            display: none; }
 +
       
 +
        textarea {
 +
          padding: 0.75rem 1rem; }
 +
       
 +
        input[type="checkbox"],
 +
        input[type="radio"] {
 +
          -moz-appearance: none;
 +
          -webkit-appearance: none;
 +
          -ms-appearance: none;
 +
          appearance: none;
 +
          display: block;
 +
          float: left;
 +
          margin-right: -2rem;
 +
          opacity: 0;
 +
          width: 1rem;
 +
          z-index: -1; }
 +
          input[type="checkbox"] + label,
 +
          input[type="radio"] + label {
 +
            text-decoration: none;
 +
            cursor: pointer;
 +
            display: inline-block;
 +
            font-size: 1rem;
 +
            font-weight: 400;
 +
            padding-left: 2.825rem;
 +
            padding-right: 0.875rem;
 +
            position: relative; }
 +
            input[type="checkbox"] + label:before,
 +
            input[type="radio"] + label:before {
 +
              -moz-osx-font-smoothing: grayscale;
 +
              -webkit-font-smoothing: antialiased;
 +
              font-family: FontAwesome;
 +
              font-style: normal;
 +
              font-weight: normal;
 +
              text-transform: none !important; }
 +
            input[type="checkbox"] + label:before,
 +
            input[type="radio"] + label:before {
 +
              border-radius: 4px;
 +
              border: solid 1px;
 +
              content: '';
 +
              display: inline-block;
 +
              height: 1.95rem;
 +
              left: 0;
 +
              line-height: 1.86875rem;
 +
              position: absolute;
 +
              text-align: center;
 +
              top: -0.1625rem;
 +
              width: 1.95rem; }
 +
          input[type="checkbox"]:checked + label:before,
 +
          input[type="radio"]:checked + label:before {
 +
            content: '\f00c'; }
 +
       
 +
        input[type="checkbox"] + label:before {
 +
          border-radius: 4px; }
 +
       
 +
        input[type="radio"] + label:before {
 +
          border-radius: 100%; }
 +
       
 +
        ::-webkit-input-placeholder {
 +
          opacity: 1.0; }
 +
       
 +
        :-moz-placeholder {
 +
          opacity: 1.0; }
 +
       
 +
        ::-moz-placeholder {
 +
          opacity: 1.0; }
 +
       
 +
        :-ms-input-placeholder {
 +
          opacity: 1.0; }
 +
       
 +
        label {
 +
          color: #555555; }
 +
       
 +
        input[type="text"],
 +
        input[type="password"],
 +
        input[type="email"],
 +
        input[type="tel"],
 +
        input[type="search"],
 +
        input[type="url"],
 +
        select,
 +
        textarea {
 +
          background-color: rgba(0, 0, 0, 0.075);
 +
          border-color: rgba(0, 0, 0, 0.25); }
 +
          input[type="text"]:focus,
 +
          input[type="password"]:focus,
 +
          input[type="email"]:focus,
 +
          input[type="tel"]:focus,
 +
          input[type="search"]:focus,
 +
          input[type="url"]:focus,
 +
          select:focus,
 +
          textarea:focus {
 +
            border-color: #B3DC77;
 +
            box-shadow: 0 0 0 1px #B3DC77; }
 +
       
 +
        select {
 +
          background-image: url("data:image/svg+xml;charset=utf8,%3Csvg xmlns='http://www.w3.org/2000/svg' width='40' height='40' preserveAspectRatio='none' viewBox='0 0 40 40'%3E%3Cpath d='M9.4,12.3l10.4,10.4l10.4-10.4c0.2-0.2,0.5-0.4,0.9-0.4c0.3,0,0.6,0.1,0.9,0.4l3.3,3.3c0.2,0.2,0.4,0.5,0.4,0.9 c0,0.4-0.1,0.6-0.4,0.9L20.7,31.9c-0.2,0.2-0.5,0.4-0.9,0.4c-0.3,0-0.6-0.1-0.9-0.4L4.3,17.3c-0.2-0.2-0.4-0.5-0.4-0.9 c0-0.4,0.1-0.6,0.4-0.9l3.3-3.3c0.2-0.2,0.5-0.4,0.9-0.4S9.1,12.1,9.4,12.3z' fill='rgba(0, 0, 0, 0.25)' /%3E%3C/svg%3E"); }
 +
          select option {
 +
            color: #444444;
 +
            background-color: #ffffff; }
 +
       
 +
        input[type="checkbox"] + label,
 +
        input[type="radio"] + label {
 +
          color: #444444; }
 +
          input[type="checkbox"] + label:before,
 +
          input[type="radio"] + label:before {
 +
            background: rgba(0, 0, 0, 0.075);
 +
            border-color: rgba(0, 0, 0, 0.25); }
 +
        input[type="checkbox"]:checked + label:before,
 +
        input[type="radio"]:checked + label:before {
 +
          background-color: #B3DC77;
 +
          border-color: #B3DC77;
 +
          color: #ffffff; }
 +
        input[type="checkbox"]:focus + label:before,
 +
        input[type="radio"]:focus + label:before {
 +
          border-color: #B3DC77;
 +
          box-shadow: 0 0 0 1px #B3DC77; }
 +
       
 +
        ::-webkit-input-placeholder {
 +
          color: #bbbbbb !important; }
 +
       
 +
        :-moz-placeholder {
 +
          color: #bbbbbb !important; }
 +
       
 +
        ::-moz-placeholder {
 +
          color: #bbbbbb !important; }
 +
       
 +
        :-ms-input-placeholder {
 +
          color: #bbbbbb !important; }
 +
       
 +
        /* List */
 +
        ol {
 +
          list-style: decimal;
 +
          margin: 0 0 2rem 0;
 +
          padding-left: 1.25rem; }
 +
          ol li {
 +
            padding-left: 0.25rem; }
 +
       
 +
        ul {
 +
          list-style: disc;
 +
          margin: 0 0 2rem 0;
 +
          padding-left: 1rem; }
 +
          ul li {
 +
            padding-left: 0.325rem; }
 +
          ul.plain {
 +
            list-style: none;
 +
            padding-left: 0; }
 +
            ul.plain li {
 +
              margin-bottom: 1rem; }
 +
              ul.plain li .icon {
 +
                border-radius: 4px;
 +
                color: #ffffff;
 +
                display: inline-block;
 +
                margin-right: 1rem;
 +
                text-align: center;
 +
                width: 2rem;
 +
                height: 2rem;
 +
                line-height: 2rem;
 +
                background: rgba(0, 0, 0, 0.5); }
 +
          ul.alt {
 +
            list-style: none;
 +
            padding-left: 0; }
 +
            ul.alt li {
 +
              border-top: solid 1px;
 +
          }
 +
              ul.alt li:first-child {
 +
                border-top: 0;
 +
                padding-top: 0; }
 +
              ul.alt li:last-child {
 +
                padding-bottom: 0; }
 +
       
 +
        dl {
 +
          margin: 0 0 2rem 0; }
 +
          dl dt {
 +
            display: block;
 +
            font-weight: 600;
 +
            margin: 0 0 1rem 0; }
 +
          dl dd {
 +
            margin-left: 1.5rem; }
 +
       
 +
        ul.alt li {
 +
          border-top-color: rgba(0, 0, 0, 0.25); }
 +
       
 +
        /* Table */
 +
        .table-wrapper {
 +
          -webkit-overflow-scrolling: touch;
 +
          overflow-x: auto; }
 +
       
 +
        table {
 +
          margin: 0 0 2rem 0;
 +
          width: 100%; }
 +
          table tbody tr {
 +
            border: solid 1px;
 +
            border-left: 0;
 +
            border-right: 0; }
 +
          table td {
 +
            padding: 0.75rem 0.75rem; }
 +
          table th {
 +
            font-size: 0.9rem;
 +
            font-weight: 600;
 +
            padding: 0 0.75rem 0.75rem 0.75rem;
 +
            text-align: left; }
 +
          table thead {
 +
            border-bottom: solid 2px; }
 +
          table tfoot {
 +
            border-top: solid 2px; }
 +
          table.alt {
 +
            border-collapse: separate; }
 +
            table.alt tbody tr td {
 +
              border: solid 1px;
 +
              border-left-width: 0;
 +
              border-top-width: 0; }
 +
              table.alt tbody tr td:first-child {
 +
                border-left-width: 1px; }
 +
            table.alt tbody tr:first-child td {
 +
              border-top-width: 1px; }
 +
            table.alt thead {
 +
              border-bottom: 0; }
 +
            table.alt tfoot {
 +
              border-top: 0; }
 +
       
 +
        table tbody tr {
 +
          border-color: rgba(0, 0, 0, 0.25); }
 +
          table tbody tr:nth-child(2n + 1) {
 +
            background-color: rgba(0, 0, 0, 0.075); }
 +
        table th {
 +
          color: #555555; }
 +
        table thead {
 +
          border-bottom-color: rgba(0, 0, 0, 0.25); }
 +
        table tfoot {
 +
          border-top-color: rgba(0, 0, 0, 0.25); }
 +
        table.alt tbody tr td {
 +
          border-color: rgba(0, 0, 0, 0.25); }
 +
       
 +
        /* Highlights */
 +
        .highlights {
 +
          width: 100%;
 +
          margin: 2.5rem 0;
 +
          display: -moz-flex;
 +
          display: -webkit-flex;
 +
          display: -ms-flex;
 +
          display: flex;
 +
          -moz-flex-wrap: wrap;
 +
          -webkit-flex-wrap: wrap;
 +
          -ms-flex-wrap: wrap;
 +
          flex-wrap: wrap;
 +
          -moz-align-items: -moz-stretch;
 +
          -webkit-align-items: -webkit-stretch;
 +
          -ms-align-items: -ms-stretch;
 +
          align-items: stretch; }
 +
          .highlights > * {
 +
            -moz-flex-shrink: 1;
 +
            -webkit-flex-shrink: 1;
 +
            -ms-flex-shrink: 1;
 +
            flex-shrink: 1;
 +
            -moz-flex-grow: 0;
 +
            -webkit-flex-grow: 0;
 +
            -ms-flex-grow: 0;
 +
            flex-grow: 0; }
 +
          .highlights > * {
 +
            width: 33.3333333333%; }
 +
          .highlights > * {
 +
            padding: 1.5rem;
 +
            width: calc(33.3333333333% + 1rem); }
 +
          .highlights > :nth-child(-n + 3) {
 +
            padding-top: 0; }
 +
          .highlights > :nth-last-child(-n + 3) {
 +
            padding-bottom: 0; }
 +
          .highlights > :nth-child(3n + 1) {
 +
            padding-left: 0; }
 +
          .highlights > :nth-child(3n) {
 +
            padding-right: 0; }
 +
          .highlights > :nth-child(3n + 1),
 +
          .highlights > :nth-child(3n) {
 +
            width: calc(33.3333333333% + -0.5rem); }
 +
          .highlights .content {
 +
            border-radius: 4px !important;
 +
            height: 100% !important;
 +
            padding: 3rem !important;
 +
            text-align: center; }
 +
            .highlights .content .icon {
 +
              font-size: 5rem; }
 +
          .highlights > div > :last-child {
 +
            margin-bottom: 0; }
 +
          @media screen and (max-width: 980px) {
 +
            .highlights > * {
 +
              width: 50%; }
 +
            .highlights > * {
 +
              padding: 1rem;
 +
              width: calc(33.3333333333% + 0.6666666667rem); }
 +
            .highlights > :nth-child(-n + 3) {
 +
              padding-top: 1rem; }
 +
            .highlights > :nth-last-child(-n + 3) {
 +
              padding-bottom: 1rem; }
 +
            .highlights > :nth-child(3n + 1) {
 +
              padding-left: 1rem; }
 +
            .highlights > :nth-child(3n) {
 +
              padding-right: 1rem; }
 +
            .highlights > :nth-child(3n + 1),
 +
            .highlights > :nth-child(3n) {
 +
              padding: 1rem;
 +
              width: calc(50% + 1rem); }
 +
            .highlights > * {
 +
              padding: 1rem;
 +
              width: calc(50% + 1rem); }
 +
            .highlights > :nth-child(-n + 2) {
 +
              padding-top: 0; }
 +
            .highlights > :nth-last-child(-n + 2) {
 +
              padding-bottom: 0; }
 +
            .highlights > :nth-child(2n + 1) {
 +
              padding-left: 0; }
 +
            .highlights > :nth-child(2n) {
 +
              padding-right: 0; }
 +
            .highlights > :nth-child(2n + 1),
 +
            .highlights > :nth-child(2n) {
 +
              width: calc(50% + 0rem); }
 +
            .highlights .content {
 +
              padding: 2rem; } }
 +
          @media screen and (max-width: 736px) {
 +
            .highlights > * {
 +
              width: 100%; }
 +
            .highlights > * {
 +
              padding: 1rem;
 +
              width: calc(33.3333333333% + 0.6666666667rem); }
 +
            .highlights > :nth-child(-n + 3) {
 +
              padding-top: 1rem; }
 +
            .highlights > :nth-last-child(-n + 3) {
 +
              padding-bottom: 1rem; }
 +
            .highlights > :nth-child(3n + 1) {
 +
              padding-left: 1rem; }
 +
            .highlights > :nth-child(3n) {
 +
              padding-right: 1rem; }
 +
            .highlights > :nth-child(3n + 1),
 +
            .highlights > :nth-child(3n) {
 +
              padding: 1rem;
 +
              width: calc(100% + 2rem); }
 +
            .highlights > * {
 +
              padding: 1rem;
 +
              width: calc(50% + 1rem); }
 +
            .highlights > :nth-child(-n + 2) {
 +
              padding-top: 1rem; }
 +
            .highlights > :nth-last-child(-n + 2) {
 +
              padding-bottom: 1rem; }
 +
            .highlights > :nth-child(2n + 1) {
 +
              padding-left: 1rem; }
 +
            .highlights > :nth-child(2n) {
 +
              padding-right: 1rem; }
 +
            .highlights > :nth-child(2n + 1),
 +
            .highlights > :nth-child(2n) {
 +
              padding: 1rem;
 +
              width: calc(100% + 2rem); }
 +
            .highlights > * {
 +
              padding: 1rem;
 +
              width: calc(100% + 2rem); }
 +
            .highlights > :nth-child(-n + 1) {
 +
              padding-top: 0; }
 +
            .highlights > :nth-last-child(-n + 1) {
 +
              padding-bottom: 0; }
 +
            .highlights > :nth-child(1n + 1) {
 +
              padding-left: 0; }
 +
            .highlights > :nth-child(1n) {
 +
              padding-right: 0; }
 +
            .highlights > :nth-child(1n + 1),
 +
            .highlights > :nth-child(1n) {
 +
              width: calc(100% + 1rem); } }
 +
       
 +
        .highlights .content {
 +
          background: #ffffff;
 +
          box-shadow: 0px 0px 4px 1px rgba(0, 0, 0, 0.025); }
 +
       
 +
        /* Testimonials */
 +
        .testimonials {
 +
          margin: 2.5rem 0;
 +
          width: 100%;
 +
          display: -moz-flex;
 +
          display: -webkit-flex;
 +
          display: -ms-flex;
 +
          display: flex;
 +
          -moz-flex-wrap: wrap;
 +
          -webkit-flex-wrap: wrap;
 +
          -ms-flex-wrap: wrap;
 +
          flex-wrap: wrap;
 +
          -moz-align-items: -moz-stretch;
 +
          -webkit-align-items: -webkit-stretch;
 +
          -ms-align-items: -ms-stretch;
 +
          align-items: stretch; }
 +
          .testimonials > * {
 +
            -moz-flex-shrink: 1;
 +
            -webkit-flex-shrink: 1;
 +
            -ms-flex-shrink: 1;
 +
            flex-shrink: 1;
 +
            -moz-flex-grow: 0;
 +
            -webkit-flex-grow: 0;
 +
            -ms-flex-grow: 0;
 +
            flex-grow: 0; }
 +
          .testimonials > * {
 +
            width: 33.3333333333%; }
 +
          .testimonials > * {
 +
            padding: 1.5rem;
 +
            width: calc(33.3333333333% + 1rem); }
 +
          .testimonials > :nth-child(-n + 3) {
 +
            padding-top: 0; }
 +
          .testimonials > :nth-last-child(-n + 3) {
 +
            padding-bottom: 0; }
 +
          .testimonials > :nth-child(3n + 1) {
 +
            padding-left: 0; }
 +
          .testimonials > :nth-child(3n) {
 +
            padding-right: 0; }
 +
          .testimonials > :nth-child(3n + 1),
 +
          .testimonials > :nth-child(3n) {
 +
            width: calc(33.3333333333% + -0.5rem); }
 +
          .testimonials .content {
 +
            border-radius: 4px;
 +
            height: 100%;
 +
            padding: 3rem; }
 +
            .testimonials .content .author {
 +
              display: -moz-flex;
 +
              display: -webkit-flex;
 +
              display: -ms-flex;
 +
              display: flex;
 +
              -moz-align-items: center;
 +
              -webkit-align-items: center;
 +
              -ms-align-items: center;
 +
              align-items: center; }
 +
              .testimonials .content .author blockquote {
 +
                margin: 0; }
 +
              .testimonials .content .author .image {
 +
                margin-right: 2rem;
 +
                width: 20%; }
 +
                .testimonials .content .author .image img {
 +
                  border-radius: 100%;
 +
                  max-width: 100%; }
 +
            .testimonials .content .credit {
 +
              font-size: 0.75rem; }
 +
            .testimonials .content p:last-child {
 +
              margin: 0; }
 +
          .testimonials > div > :last-child {
 +
            margin-bottom: 0; }
 +
          @media screen and (max-width: 980px) {
 +
            .testimonials > * {
 +
              width: 50%; }
 +
            .testimonials > * {
 +
              padding: 1rem;
 +
              width: calc(33.3333333333% + 0.6666666667rem); }
 +
            .testimonials > :nth-child(-n + 3) {
 +
              padding-top: 1rem; }
 +
            .testimonials > :nth-last-child(-n + 3) {
 +
              padding-bottom: 1rem; }
 +
            .testimonials > :nth-child(3n + 1) {
 +
              padding-left: 1rem; }
 +
            .testimonials > :nth-child(3n) {
 +
              padding-right: 1rem; }
 +
            .testimonials > :nth-child(3n + 1),
 +
            .testimonials > :nth-child(3n) {
 +
              padding: 1rem;
 +
              width: calc(50% + 1rem); }
 +
            .testimonials > * {
 +
              padding: 1rem;
 +
              width: calc(50% + 1rem); }
 +
            .testimonials > :nth-child(-n + 2) {
 +
              padding-top: 0; }
 +
            .testimonials > :nth-last-child(-n + 2) {
 +
              padding-bottom: 0; }
 +
            .testimonials > :nth-child(2n + 1) {
 +
              padding-left: 0; }
 +
            .testimonials > :nth-child(2n) {
 +
              padding-right: 0; }
 +
            .testimonials > :nth-child(2n + 1),
 +
            .testimonials > :nth-child(2n) {
 +
              width: calc(50% + 0rem); }
 +
            .testimonials .content {
 +
              padding: 2rem; } }
 +
          @media screen and (max-width: 736px) {
 +
            .testimonials > * {
 +
              width: 100%; }
 +
            .testimonials > * {
 +
              padding: 1rem;
 +
              width: calc(33.3333333333% + 0.6666666667rem); }
 +
            .testimonials > :nth-child(-n + 3) {
 +
              padding-top: 1rem; }
 +
            .testimonials > :nth-last-child(-n + 3) {
 +
              padding-bottom: 1rem; }
 +
            .testimonials > :nth-child(3n + 1) {
 +
              padding-left: 1rem; }
 +
            .testimonials > :nth-child(3n) {
 +
              padding-right: 1rem; }
 +
            .testimonials > :nth-child(3n + 1),
 +
            .testimonials > :nth-child(3n) {
 +
              padding: 1rem;
 +
              width: calc(100% + 2rem); }
 +
            .testimonials > * {
 +
              padding: 1rem;
 +
              width: calc(50% + 1rem); }
 +
            .testimonials > :nth-child(-n + 2) {
 +
              padding-top: 1rem; }
 +
            .testimonials > :nth-last-child(-n + 2) {
 +
              padding-bottom: 1rem; }
 +
            .testimonials > :nth-child(2n + 1) {
 +
              padding-left: 1rem; }
 +
            .testimonials > :nth-child(2n) {
 +
              padding-right: 1rem; }
 +
            .testimonials > :nth-child(2n + 1),
 +
            .testimonials > :nth-child(2n) {
 +
              padding: 1rem;
 +
              width: calc(100% + 2rem); }
 +
            .testimonials > * {
 +
              padding: 1rem;
 +
              width: calc(100% + 2rem); }
 +
            .testimonials > :nth-child(-n + 1) {
 +
              padding-top: 0; }
 +
            .testimonials > :nth-last-child(-n + 1) {
 +
              padding-bottom: 0; }
 +
            .testimonials > :nth-child(1n + 1) {
 +
              padding-left: 0; }
 +
            .testimonials > :nth-child(1n) {
 +
              padding-right: 0; }
 +
            .testimonials > :nth-child(1n + 1),
 +
            .testimonials > :nth-child(1n) {
 +
              width: calc(100% + 1rem); } }
 +
       
 +
        .testimonials .content {
 +
          background: #ffffff;
 +
          box-shadow: 0px 0px 4px 1px rgba(0, 0, 0, 0.025); }
 +
          .testimonials .content .credit strong {
 +
            color: #B3DC77; }
 +
       
 +
        /* Actions */
 +
        ul.actions {
 +
          display: -moz-flex;
 +
          display: -webkit-flex;
 +
          display: -ms-flex;
 +
          display: flex;
 +
          cursor: default;
 +
          list-style: none;
 +
          margin-left: -1rem;
 +
          padding-left: 0; }
 +
          ul.actions li {
 +
            padding: 0 0 0 1rem;
 +
            vertical-align: middle; }
 +
          ul.actions.special {
 +
            -moz-justify-content: center;
 +
            -webkit-justify-content: center;
 +
            -ms-justify-content: center;
 +
            justify-content: center;
 +
            width: calc(100% + 1rem); }
 +
          ul.actions.stacked {
 +
            -moz-flex-direction: column;
 +
            -webkit-flex-direction: column;
 +
            -ms-flex-direction: column;
 +
            flex-direction: column;
 +
            margin-left: 0; }
 +
            ul.actions.stacked li {
 +
              padding: 1.3rem 0 0 0; }
 +
              ul.actions.stacked li:first-child {
 +
                padding-top: 0; }
 +
          ul.actions.fit {
 +
            width: calc(100% + 1rem); }
 +
            ul.actions.fit li {
 +
              -moz-flex-grow: 1;
 +
              -webkit-flex-grow: 1;
 +
              -ms-flex-grow: 1;
 +
              flex-grow: 1;
 +
              -moz-flex-shrink: 1;
 +
              -webkit-flex-shrink: 1;
 +
              -ms-flex-shrink: 1;
 +
              flex-shrink: 1;
 +
              width: 100%; }
 +
              ul.actions.fit li > * {
 +
                width: 100%; }
 +
            ul.actions.fit.stacked {
 +
              width: 100%; }
 +
          @media screen and (max-width: 480px) {
 +
            ul.actions:not(.fixed) {
 +
              -moz-flex-direction: column;
 +
              -webkit-flex-direction: column;
 +
              -ms-flex-direction: column;
 +
              flex-direction: column;
 +
              margin-left: 0;
 +
              width: 100% !important; }
 +
              ul.actions:not(.fixed) li {
 +
                -moz-flex-grow: 1;
 +
                -webkit-flex-grow: 1;
 +
                -ms-flex-grow: 1;
 +
                flex-grow: 1;
 +
                -moz-flex-shrink: 1;
 +
                -webkit-flex-shrink: 1;
 +
                -ms-flex-shrink: 1;
 +
                flex-shrink: 1;
 +
                padding: 1rem 0 0 0;
 +
                text-align: center;
 +
                width: 100%; }
 +
                ul.actions:not(.fixed) li > * {
 +
                  width: 100%; }
 +
                ul.actions:not(.fixed) li:first-child {
 +
                  padding-top: 0; }
 +
                ul.actions:not(.fixed) li input[type="submit"],
 +
                ul.actions:not(.fixed) li input[type="reset"],
 +
                ul.actions:not(.fixed) li input[type="button"],
 +
                ul.actions:not(.fixed) li button,
 +
                ul.actions:not(.fixed) li .button {
 +
                  width: 100%; }
 +
                  ul.actions:not(.fixed) li input[type="submit"].icon:before,
 +
                  ul.actions:not(.fixed) li input[type="reset"].icon:before,
 +
                  ul.actions:not(.fixed) li input[type="button"].icon:before,
 +
                  ul.actions:not(.fixed) li button.icon:before,
 +
                  ul.actions:not(.fixed) li .button.icon:before {
 +
                    margin-left: -0.5rem; } }
 +
       
 +
        /* Grid */
 +
        .row {
 +
          display: flex;
 +
          flex-wrap: wrap;
 +
          box-sizing: border-box; }
 +
          .row > * {
 +
            box-sizing: border-box; }
 +
          .row.gtr-uniform > * > :last-child {
 +
            margin-bottom: 0; }
 +
       
 +
        .row > .imp {
 +
          order: -1; }
 +
        .row > .col-1 {
 +
          width: 8.3333333333%; }
 +
        .row > .off-1 {
 +
          margin-left: 8.3333333333%; }
 +
        .row > .col-2 {
 +
          width: 16.6666666667%; }
 +
        .row > .off-2 {
 +
          margin-left: 16.6666666667%; }
 +
        .row > .col-3 {
 +
          width: 25%; }
 +
        .row > .off-3 {
 +
          margin-left: 25%; }
 +
        .row > .col-4 {
 +
          width: 33.3333333333%; }
 +
        .row > .off-4 {
 +
          margin-left: 33.3333333333%; }
 +
        .row > .col-5 {
 +
          width: 41.6666666667%; }
 +
        .row > .off-5 {
 +
          margin-left: 41.6666666667%; }
 +
        .row > .col-6 {
 +
          width: 50%; }
 +
        .row > .off-6 {
 +
          margin-left: 50%; }
 +
        .row > .col-7 {
 +
          width: 58.3333333333%; }
 +
        .row > .off-7 {
 +
          margin-left: 58.3333333333%; }
 +
        .row > .col-8 {
 +
          width: 66.6666666667%; }
 +
        .row > .off-8 {
 +
          margin-left: 66.6666666667%; }
 +
        .row > .col-9 {
 +
          width: 75%; }
 +
        .row > .off-9 {
 +
          margin-left: 75%; }
 +
        .row > .col-10 {
 +
          width: 83.3333333333%; }
 +
        .row > .off-10 {
 +
          margin-left: 83.3333333333%; }
 +
        .row > .col-11 {
 +
          width: 91.6666666667%; }
 +
        .row > .off-11 {
 +
          margin-left: 91.6666666667%; }
 +
        .row > .col-12 {
 +
          width: 100%; }
 +
        .row > .off-12 {
 +
          margin-left: 100%; }
 +
        .row.gtr-0 {
 +
          margin-top: 0;
 +
          margin-left: 0rem; }
 +
          .row.gtr-0 > * {
 +
            padding: 0 0 0 0rem; }
 +
          .row.gtr-0.gtr-uniform {
 +
            margin-top: 0rem; }
 +
            .row.gtr-0.gtr-uniform > * {
 +
              padding-top: 0rem; }
 +
        .row.gtr-25 {
 +
          margin-top: 0;
 +
          margin-left: -0.75rem; }
 +
          .row.gtr-25 > * {
 +
            padding: 0 0 0 0.75rem; }
 +
          .row.gtr-25.gtr-uniform {
 +
            margin-top: -0.75rem; }
 +
            .row.gtr-25.gtr-uniform > * {
 +
              padding-top: 0.75rem; }
 +
        .row.gtr-50 {
 +
          margin-top: 0;
 +
          margin-left: -1.5rem; }
 +
          .row.gtr-50 > * {
 +
            padding: 0 0 0 1.5rem; }
 +
          .row.gtr-50.gtr-uniform {
 +
            margin-top: -1.5rem; }
 +
            .row.gtr-50.gtr-uniform > * {
 +
              padding-top: 1.5rem; }
 +
        .row {
 +
          margin-top: 0;
 +
          margin-left: -3rem; }
 +
          .row > * {
 +
            padding: 0 0 0 3rem; }
 +
          .row.gtr-uniform {
 +
            margin-top: -3rem; }
 +
            .row.gtr-uniform > * {
 +
              padding-top: 3rem; }
 +
        .row.gtr-150 {
 +
          margin-top: 0;
 +
          margin-left: -4.5rem; }
 +
          .row.gtr-150 > * {
 +
            padding: 0 0 0 4.5rem; }
 +
          .row.gtr-150.gtr-uniform {
 +
            margin-top: -4.5rem; }
 +
            .row.gtr-150.gtr-uniform > * {
 +
              padding-top: 4.5rem; }
 +
        .row.gtr-200 {
 +
          margin-top: 0;
 +
          margin-left: -6rem; }
 +
          .row.gtr-200 > * {
 +
            padding: 0 0 0 6rem; }
 +
          .row.gtr-200.gtr-uniform {
 +
            margin-top: -6rem; }
 +
            .row.gtr-200.gtr-uniform > * {
 +
              padding-top: 6rem; }
 +
       
 +
        @media screen and (max-width: 1680px) {
 +
          .row {
 +
            display: flex;
 +
            flex-wrap: wrap;
 +
            box-sizing: border-box; }
 +
            .row > * {
 +
              box-sizing: border-box; }
 +
            .row.gtr-uniform > * > :last-child {
 +
              margin-bottom: 0; }
 +
       
 +
          .row > .imp-xlarge {
 +
            order: -1; }
 +
          .row > .col-1-xlarge {
 +
            width: 8.3333333333%; }
 +
          .row > .off-1-xlarge {
 +
            margin-left: 8.3333333333%; }
 +
          .row > .col-2-xlarge {
 +
            width: 16.6666666667%; }
 +
          .row > .off-2-xlarge {
 +
            margin-left: 16.6666666667%; }
 +
          .row > .col-3-xlarge {
 +
            width: 25%; }
 +
          .row > .off-3-xlarge {
 +
            margin-left: 25%; }
 +
          .row > .col-4-xlarge {
 +
            width: 33.3333333333%; }
 +
          .row > .off-4-xlarge {
 +
            margin-left: 33.3333333333%; }
 +
          .row > .col-5-xlarge {
 +
            width: 41.6666666667%; }
 +
          .row > .off-5-xlarge {
 +
            margin-left: 41.6666666667%; }
 +
          .row > .col-6-xlarge {
 +
            width: 50%; }
 +
          .row > .off-6-xlarge {
 +
            margin-left: 50%; }
 +
          .row > .col-7-xlarge {
 +
            width: 58.3333333333%; }
 +
          .row > .off-7-xlarge {
 +
            margin-left: 58.3333333333%; }
 +
          .row > .col-8-xlarge {
 +
            width: 66.6666666667%; }
 +
          .row > .off-8-xlarge {
 +
            margin-left: 66.6666666667%; }
 +
          .row > .col-9-xlarge {
 +
            width: 75%; }
 +
          .row > .off-9-xlarge {
 +
            margin-left: 75%; }
 +
          .row > .col-10-xlarge {
 +
            width: 83.3333333333%; }
 +
          .row > .off-10-xlarge {
 +
            margin-left: 83.3333333333%; }
 +
          .row > .col-11-xlarge {
 +
            width: 91.6666666667%; }
 +
          .row > .off-11-xlarge {
 +
            margin-left: 91.6666666667%; }
 +
          .row > .col-12-xlarge {
 +
            width: 100%; }
 +
          .row > .off-12-xlarge {
 +
            margin-left: 100%; }
 +
          .row.gtr-0 {
 +
            margin-top: 0;
 +
            margin-left: 0rem; }
 +
            .row.gtr-0 > * {
 +
              padding: 0 0 0 0rem; }
 +
            .row.gtr-0.gtr-uniform {
 +
              margin-top: 0rem; }
 +
              .row.gtr-0.gtr-uniform > * {
 +
                padding-top: 0rem; }
 +
          .row.gtr-25 {
 +
            margin-top: 0;
 +
            margin-left: -0.75rem; }
 +
            .row.gtr-25 > * {
 +
              padding: 0 0 0 0.75rem; }
 +
            .row.gtr-25.gtr-uniform {
 +
              margin-top: -0.75rem; }
 +
              .row.gtr-25.gtr-uniform > * {
 +
                padding-top: 0.75rem; }
 +
          .row.gtr-50 {
 +
            margin-top: 0;
 +
            margin-left: -1.5rem; }
 +
            .row.gtr-50 > * {
 +
              padding: 0 0 0 1.5rem; }
 +
            .row.gtr-50.gtr-uniform {
 +
              margin-top: -1.5rem; }
 +
              .row.gtr-50.gtr-uniform > * {
 +
                padding-top: 1.5rem; }
 +
          .row {
 +
            margin-top: 0;
 +
            margin-left: -3rem; }
 +
            .row > * {
 +
              padding: 0 0 0 3rem; }
 +
            .row.gtr-uniform {
 +
              margin-top: -3rem; }
 +
              .row.gtr-uniform > * {
 +
                padding-top: 3rem; }
 +
          .row.gtr-150 {
 +
            margin-top: 0;
 +
            margin-left: -4.5rem; }
 +
            .row.gtr-150 > * {
 +
              padding: 0 0 0 4.5rem; }
 +
            .row.gtr-150.gtr-uniform {
 +
              margin-top: -4.5rem; }
 +
              .row.gtr-150.gtr-uniform > * {
 +
                padding-top: 4.5rem; }
 +
          .row.gtr-200 {
 +
            margin-top: 0;
 +
            margin-left: -6rem; }
 +
            .row.gtr-200 > * {
 +
              padding: 0 0 0 6rem; }
 +
            .row.gtr-200.gtr-uniform {
 +
              margin-top: -6rem; }
 +
              .row.gtr-200.gtr-uniform > * {
 +
                padding-top: 6rem; } }
 +
        @media screen and (max-width: 1280px) {
 +
          .row {
 +
            display: flex;
 +
            flex-wrap: wrap;
 +
            box-sizing: border-box; }
 +
            .row > * {
 +
              box-sizing: border-box; }
 +
            .row.gtr-uniform > * > :last-child {
 +
              margin-bottom: 0; }
 +
       
 +
          .row > .imp-large {
 +
            order: -1; }
 +
          .row > .col-1-large {
 +
            width: 8.3333333333%; }
 +
          .row > .off-1-large {
 +
            margin-left: 8.3333333333%; }
 +
          .row > .col-2-large {
 +
            width: 16.6666666667%; }
 +
          .row > .off-2-large {
 +
            margin-left: 16.6666666667%; }
 +
          .row > .col-3-large {
 +
            width: 25%; }
 +
          .row > .off-3-large {
 +
            margin-left: 25%; }
 +
          .row > .col-4-large {
 +
            width: 33.3333333333%; }
 +
          .row > .off-4-large {
 +
            margin-left: 33.3333333333%; }
 +
          .row > .col-5-large {
 +
            width: 41.6666666667%; }
 +
          .row > .off-5-large {
 +
            margin-left: 41.6666666667%; }
 +
          .row > .col-6-large {
 +
            width: 50%; }
 +
          .row > .off-6-large {
 +
            margin-left: 50%; }
 +
          .row > .col-7-large {
 +
            width: 58.3333333333%; }
 +
          .row > .off-7-large {
 +
            margin-left: 58.3333333333%; }
 +
          .row > .col-8-large {
 +
            width: 66.6666666667%; }
 +
          .row > .off-8-large {
 +
            margin-left: 66.6666666667%; }
 +
          .row > .col-9-large {
 +
            width: 75%; }
 +
          .row > .off-9-large {
 +
            margin-left: 75%; }
 +
          .row > .col-10-large {
 +
            width: 83.3333333333%; }
 +
          .row > .off-10-large {
 +
            margin-left: 83.3333333333%; }
 +
          .row > .col-11-large {
 +
            width: 91.6666666667%; }
 +
          .row > .off-11-large {
 +
            margin-left: 91.6666666667%; }
 +
          .row > .col-12-large {
 +
            width: 100%; }
 +
          .row > .off-12-large {
 +
            margin-left: 100%; }
 +
          .row.gtr-0 {
 +
            margin-top: 0;
 +
            margin-left: 0rem; }
 +
            .row.gtr-0 > * {
 +
              padding: 0 0 0 0rem; }
 +
            .row.gtr-0.gtr-uniform {
 +
              margin-top: 0rem; }
 +
              .row.gtr-0.gtr-uniform > * {
 +
                padding-top: 0rem; }
 +
          .row.gtr-25 {
 +
            margin-top: 0;
 +
            margin-left: -0.375rem; }
 +
            .row.gtr-25 > * {
 +
              padding: 0 0 0 0.375rem; }
 +
            .row.gtr-25.gtr-uniform {
 +
              margin-top: -0.375rem; }
 +
              .row.gtr-25.gtr-uniform > * {
 +
                padding-top: 0.375rem; }
 +
          .row.gtr-50 {
 +
            margin-top: 0;
 +
            margin-left: -0.75rem; }
 +
            .row.gtr-50 > * {
 +
              padding: 0 0 0 0.75rem; }
 +
            .row.gtr-50.gtr-uniform {
 +
              margin-top: -0.75rem; }
 +
              .row.gtr-50.gtr-uniform > * {
 +
                padding-top: 0.75rem; }
 +
          .row {
 +
            margin-top: 0;
 +
            margin-left: -1.5rem; }
 +
            .row > * {
 +
              padding: 0 0 0 1.5rem; }
 +
            .row.gtr-uniform {
 +
              margin-top: -1.5rem; }
 +
              .row.gtr-uniform > * {
 +
                padding-top: 1.5rem; }
 +
          .row.gtr-150 {
 +
            margin-top: 0;
 +
            margin-left: -2.25rem; }
 +
            .row.gtr-150 > * {
 +
              padding: 0 0 0 2.25rem; }
 +
            .row.gtr-150.gtr-uniform {
 +
              margin-top: -2.25rem; }
 +
              .row.gtr-150.gtr-uniform > * {
 +
                padding-top: 2.25rem; }
 +
          .row.gtr-200 {
 +
            margin-top: 0;
 +
            margin-left: -3rem; }
 +
            .row.gtr-200 > * {
 +
              padding: 0 0 0 3rem; }
 +
            .row.gtr-200.gtr-uniform {
 +
              margin-top: -3rem; }
 +
              .row.gtr-200.gtr-uniform > * {
 +
                padding-top: 3rem; } }
 +
        @media screen and (max-width: 980px) {
 +
          .row {
 +
            display: flex;
 +
            flex-wrap: wrap;
 +
            box-sizing: border-box; }
 +
            .row > * {
 +
              box-sizing: border-box; }
 +
            .row.gtr-uniform > * > :last-child {
 +
              margin-bottom: 0; }
 +
       
 +
          .row > .imp-medium {
 +
            order: -1; }
 +
          .row > .col-1-medium {
 +
            width: 8.3333333333%; }
 +
          .row > .off-1-medium {
 +
            margin-left: 8.3333333333%; }
 +
          .row > .col-2-medium {
 +
            width: 16.6666666667%; }
 +
          .row > .off-2-medium {
 +
            margin-left: 16.6666666667%; }
 +
          .row > .col-3-medium {
 +
            width: 25%; }
 +
          .row > .off-3-medium {
 +
            margin-left: 25%; }
 +
          .row > .col-4-medium {
 +
            width: 33.3333333333%; }
 +
          .row > .off-4-medium {
 +
            margin-left: 33.3333333333%; }
 +
          .row > .col-5-medium {
 +
            width: 41.6666666667%; }
 +
          .row > .off-5-medium {
 +
            margin-left: 41.6666666667%; }
 +
          .row > .col-6-medium {
 +
            width: 50%; }
 +
          .row > .off-6-medium {
 +
            margin-left: 50%; }
 +
          .row > .col-7-medium {
 +
            width: 58.3333333333%; }
 +
          .row > .off-7-medium {
 +
            margin-left: 58.3333333333%; }
 +
          .row > .col-8-medium {
 +
            width: 66.6666666667%; }
 +
          .row > .off-8-medium {
 +
            margin-left: 66.6666666667%; }
 +
          .row > .col-9-medium {
 +
            width: 75%; }
 +
          .row > .off-9-medium {
 +
            margin-left: 75%; }
 +
          .row > .col-10-medium {
 +
            width: 83.3333333333%; }
 +
          .row > .off-10-medium {
 +
            margin-left: 83.3333333333%; }
 +
          .row > .col-11-medium {
 +
            width: 91.6666666667%; }
 +
          .row > .off-11-medium {
 +
            margin-left: 91.6666666667%; }
 +
          .row > .col-12-medium {
 +
            width: 100%; }
 +
          .row > .off-12-medium {
 +
            margin-left: 100%; }
 +
          .row.gtr-0 {
 +
            margin-top: 0;
 +
            margin-left: 0rem; }
 +
            .row.gtr-0 > * {
 +
              padding: 0 0 0 0rem; }
 +
            .row.gtr-0.gtr-uniform {
 +
              margin-top: 0rem; }
 +
              .row.gtr-0.gtr-uniform > * {
 +
                padding-top: 0rem; }
 +
          .row.gtr-25 {
 +
            margin-top: 0;
 +
            margin-left: -0.375rem; }
 +
            .row.gtr-25 > * {
 +
              padding: 0 0 0 0.375rem; }
 +
            .row.gtr-25.gtr-uniform {
 +
              margin-top: -0.375rem; }
 +
              .row.gtr-25.gtr-uniform > * {
 +
                padding-top: 0.375rem; }
 +
          .row.gtr-50 {
 +
            margin-top: 0;
 +
            margin-left: -0.75rem; }
 +
            .row.gtr-50 > * {
 +
              padding: 0 0 0 0.75rem; }
 +
            .row.gtr-50.gtr-uniform {
 +
              margin-top: -0.75rem; }
 +
              .row.gtr-50.gtr-uniform > * {
 +
                padding-top: 0.75rem; }
 +
          .row {
 +
            margin-top: 0;
 +
            margin-left: -1.5rem; }
 +
            .row > * {
 +
              padding: 0 0 0 1.5rem; }
 +
            .row.gtr-uniform {
 +
              margin-top: -1.5rem; }
 +
              .row.gtr-uniform > * {
 +
                padding-top: 1.5rem; }
 +
          .row.gtr-150 {
 +
            margin-top: 0;
 +
            margin-left: -2.25rem; }
 +
            .row.gtr-150 > * {
 +
              padding: 0 0 0 2.25rem; }
 +
            .row.gtr-150.gtr-uniform {
 +
              margin-top: -2.25rem; }
 +
              .row.gtr-150.gtr-uniform > * {
 +
                padding-top: 2.25rem; }
 +
          .row.gtr-200 {
 +
            margin-top: 0;
 +
            margin-left: -3rem; }
 +
            .row.gtr-200 > * {
 +
              padding: 0 0 0 3rem; }
 +
            .row.gtr-200.gtr-uniform {
 +
              margin-top: -3rem; }
 +
              .row.gtr-200.gtr-uniform > * {
 +
                padding-top: 3rem; } }
 +
        @media screen and (max-width: 736px) {
 +
          .row {
 +
            display: flex;
 +
            flex-wrap: wrap;
 +
            box-sizing: border-box; }
 +
            .row > * {
 +
              box-sizing: border-box; }
 +
            .row.gtr-uniform > * > :last-child {
 +
              margin-bottom: 0; }
 +
       
 +
          .row > .imp-small {
 +
            order: -1; }
 +
          .row > .col-1-small {
 +
            width: 8.3333333333%; }
 +
          .row > .off-1-small {
 +
            margin-left: 8.3333333333%; }
 +
          .row > .col-2-small {
 +
            width: 16.6666666667%; }
 +
          .row > .off-2-small {
 +
            margin-left: 16.6666666667%; }
 +
          .row > .col-3-small {
 +
            width: 25%; }
 +
          .row > .off-3-small {
 +
            margin-left: 25%; }
 +
          .row > .col-4-small {
 +
            width: 33.3333333333%; }
 +
          .row > .off-4-small {
 +
            margin-left: 33.3333333333%; }
 +
          .row > .col-5-small {
 +
            width: 41.6666666667%; }
 +
          .row > .off-5-small {
 +
            margin-left: 41.6666666667%; }
 +
          .row > .col-6-small {
 +
            width: 50%; }
 +
          .row > .off-6-small {
 +
            margin-left: 50%; }
 +
          .row > .col-7-small {
 +
            width: 58.3333333333%; }
 +
          .row > .off-7-small {
 +
            margin-left: 58.3333333333%; }
 +
          .row > .col-8-small {
 +
            width: 66.6666666667%; }
 +
          .row > .off-8-small {
 +
            margin-left: 66.6666666667%; }
 +
          .row > .col-9-small {
 +
            width: 75%; }
 +
          .row > .off-9-small {
 +
            margin-left: 75%; }
 +
          .row > .col-10-small {
 +
            width: 83.3333333333%; }
 +
          .row > .off-10-small {
 +
            margin-left: 83.3333333333%; }
 +
          .row > .col-11-small {
 +
            width: 91.6666666667%; }
 +
          .row > .off-11-small {
 +
            margin-left: 91.6666666667%; }
 +
          .row > .col-12-small {
 +
            width: 100%; }
 +
          .row > .off-12-small {
 +
            margin-left: 100%; }
 +
          .row.gtr-0 {
 +
            margin-top: 0;
 +
            margin-left: 0rem; }
 +
            .row.gtr-0 > * {
 +
              padding: 0 0 0 0rem; }
 +
            .row.gtr-0.gtr-uniform {
 +
              margin-top: 0rem; }
 +
              .row.gtr-0.gtr-uniform > * {
 +
                padding-top: 0rem; }
 +
          .row.gtr-25 {
 +
            margin-top: 0;
 +
            margin-left: -0.3125rem; }
 +
            .row.gtr-25 > * {
 +
              padding: 0 0 0 0.3125rem; }
 +
            .row.gtr-25.gtr-uniform {
 +
              margin-top: -0.3125rem; }
 +
              .row.gtr-25.gtr-uniform > * {
 +
                padding-top: 0.3125rem; }
 +
          .row.gtr-50 {
 +
            margin-top: 0;
 +
            margin-left: -0.625rem; }
 +
            .row.gtr-50 > * {
 +
              padding: 0 0 0 0.625rem; }
 +
            .row.gtr-50.gtr-uniform {
 +
              margin-top: -0.625rem; }
 +
              .row.gtr-50.gtr-uniform > * {
 +
                padding-top: 0.625rem; }
 +
          .row {
 +
            margin-top: 0;
 +
            margin-left: -1.25rem; }
 +
            .row > * {
 +
              padding: 0 0 0 1.25rem; }
 +
            .row.gtr-uniform {
 +
              margin-top: -1.25rem; }
 +
              .row.gtr-uniform > * {
 +
                padding-top: 1.25rem; }
 +
          .row.gtr-150 {
 +
            margin-top: 0;
 +
            margin-left: -1.875rem; }
 +
            .row.gtr-150 > * {
 +
              padding: 0 0 0 1.875rem; }
 +
            .row.gtr-150.gtr-uniform {
 +
              margin-top: -1.875rem; }
 +
              .row.gtr-150.gtr-uniform > * {
 +
                padding-top: 1.875rem; }
 +
          .row.gtr-200 {
 +
            margin-top: 0;
 +
            margin-left: -2.5rem; }
 +
            .row.gtr-200 > * {
 +
              padding: 0 0 0 2.5rem; }
 +
            .row.gtr-200.gtr-uniform {
 +
              margin-top: -2.5rem; }
 +
              .row.gtr-200.gtr-uniform > * {
 +
                padding-top: 2.5rem; } }
 +
        @media screen and (max-width: 480px) {
 +
          .row {
 +
            display: flex;
 +
            flex-wrap: wrap;
 +
            box-sizing: border-box; }
 +
            .row > * {
 +
              box-sizing: border-box; }
 +
            .row.gtr-uniform > * > :last-child {
 +
              margin-bottom: 0; }
 +
       
 +
          .row > .imp-xsmall {
 +
            order: -1; }
 +
          .row > .col-1-xsmall {
 +
            width: 8.3333333333%; }
 +
          .row > .off-1-xsmall {
 +
            margin-left: 8.3333333333%; }
 +
          .row > .col-2-xsmall {
 +
            width: 16.6666666667%; }
 +
          .row > .off-2-xsmall {
 +
            margin-left: 16.6666666667%; }
 +
          .row > .col-3-xsmall {
 +
            width: 25%; }
 +
          .row > .off-3-xsmall {
 +
            margin-left: 25%; }
 +
          .row > .col-4-xsmall {
 +
            width: 33.3333333333%; }
 +
          .row > .off-4-xsmall {
 +
            margin-left: 33.3333333333%; }
 +
          .row > .col-5-xsmall {
 +
            width: 41.6666666667%; }
 +
          .row > .off-5-xsmall {
 +
            margin-left: 41.6666666667%; }
 +
          .row > .col-6-xsmall {
 +
            width: 50%; }
 +
          .row > .off-6-xsmall {
 +
            margin-left: 50%; }
 +
          .row > .col-7-xsmall {
 +
            width: 58.3333333333%; }
 +
          .row > .off-7-xsmall {
 +
            margin-left: 58.3333333333%; }
 +
          .row > .col-8-xsmall {
 +
            width: 66.6666666667%; }
 +
          .row > .off-8-xsmall {
 +
            margin-left: 66.6666666667%; }
 +
          .row > .col-9-xsmall {
 +
            width: 75%; }
 +
          .row > .off-9-xsmall {
 +
            margin-left: 75%; }
 +
          .row > .col-10-xsmall {
 +
            width: 83.3333333333%; }
 +
          .row > .off-10-xsmall {
 +
            margin-left: 83.3333333333%; }
 +
          .row > .col-11-xsmall {
 +
            width: 91.6666666667%; }
 +
          .row > .off-11-xsmall {
 +
            margin-left: 91.6666666667%; }
 +
          .row > .col-12-xsmall {
 +
            width: 100%; }
 +
          .row > .off-12-xsmall {
 +
            margin-left: 100%; }
 +
          .row.gtr-0 {
 +
            margin-top: 0;
 +
            margin-left: 0rem; }
 +
            .row.gtr-0 > * {
 +
              padding: 0 0 0 0rem; }
 +
            .row.gtr-0.gtr-uniform {
 +
              margin-top: 0rem; }
 +
              .row.gtr-0.gtr-uniform > * {
 +
                padding-top: 0rem; }
 +
          .row.gtr-25 {
 +
            margin-top: 0;
 +
            margin-left: -0.3125rem; }
 +
            .row.gtr-25 > * {
 +
              padding: 0 0 0 0.3125rem; }
 +
            .row.gtr-25.gtr-uniform {
 +
              margin-top: -0.3125rem; }
 +
              .row.gtr-25.gtr-uniform > * {
 +
                padding-top: 0.3125rem; }
 +
          .row.gtr-50 {
 +
            margin-top: 0;
 +
            margin-left: -0.625rem; }
 +
            .row.gtr-50 > * {
 +
              padding: 0 0 0 0.625rem; }
 +
            .row.gtr-50.gtr-uniform {
 +
              margin-top: -0.625rem; }
 +
              .row.gtr-50.gtr-uniform > * {
 +
                padding-top: 0.625rem; }
 +
          .row {
 +
            margin-top: 0;
 +
            margin-left: -1.25rem; }
 +
            .row > * {
 +
              padding: 0 0 0 1.25rem; }
 +
            .row.gtr-uniform {
 +
              margin-top: -1.25rem; }
 +
              .row.gtr-uniform > * {
 +
                padding-top: 1.25rem; }
 +
          .row.gtr-150 {
 +
            margin-top: 0;
 +
            margin-left: -1.875rem; }
 +
            .row.gtr-150 > * {
 +
              padding: 0 0 0 1.875rem; }
 +
            .row.gtr-150.gtr-uniform {
 +
              margin-top: -1.875rem; }
 +
              .row.gtr-150.gtr-uniform > * {
 +
                padding-top: 1.875rem; }
 +
          .row.gtr-200 {
 +
            margin-top: 0;
 +
            margin-left: -2.5rem; }
 +
            .row.gtr-200 > * {
 +
              padding: 0 0 0 2.5rem; }
 +
            .row.gtr-200.gtr-uniform {
 +
              margin-top: -2.5rem; }
 +
              .row.gtr-200.gtr-uniform > * {
 +
                padding-top: 2.5rem; } }
 +
        /* Icon */
 +
        .icon {
 +
          text-decoration: none;
 +
          border-bottom: none;
 +
          position: relative; }
 +
          .icon:before {
 +
            -moz-osx-font-smoothing: grayscale;
 +
            -webkit-font-smoothing: antialiased;
 +
            font-family: FontAwesome;
 +
            font-style: normal;
 +
            font-weight: normal;
 +
            text-transform: none !important; }
 +
          .icon > .label {
 +
            display: none; }
 +
       
 +
        /* Icons */
 +
        ul.icons {
 +
          cursor: default;
 +
          list-style: none;
 +
          padding-left: 0; }
 +
          ul.icons li {
 +
            display: inline-block;
 +
            padding: 0 1rem 0 0; }
 +
            ul.icons li:last-child {
 +
              padding-right: 0; }
 +
            ul.icons li .icon:before {
 +
              font-size: 2rem; }
 +
       
 +
        /* Image */
 +
        .image {
 +
          border-radius: 4px;
 +
          border: 0;
 +
          display: inline-block;
 +
          position: relative; }
 +
          .image img {
 +
            border-radius: 4px;
 +
            display: block; }
 +
          .image.left, .image.right {
 +
            max-width: 40%; }
 +
            .image.left img, .image.right img {
 +
              width: 100%; }
 +
          .image.left {
 +
            float: left;
 +
            margin: 0 2rem 2rem 0;
 +
            top: 0.25rem; }
 +
          .image.right {
 +
            float: right;
 +
            margin: 0 0 2rem 2rem;
 +
            top: 0.25rem; }
 +
          .image.fit {
 +
            display: block;
 +
            margin: 0 0 2rem 0;
 +
            width: 100%; }
 +
            .image.fit img {
 +
              width: 100%; }
 +
          .image.main {
 +
            display: block;
 +
            margin: 0 0 3rem 0;
 +
            width: 100%; }
 +
            .image.main img {
 +
              width: 100%; }
 +
       
 +
        /* Wrapper */
 +
        .wrapper {
 +
          padding: 8rem 0 6rem 0 ;
 +
          position: relative;
 +
          z-index: 1; }
 +
          @media screen and (max-width: 1280px) {
 +
            .wrapper {
 +
              padding: 4rem 0 2rem 0 ; } }
 +
          @media screen and (max-width: 736px) {
 +
            .wrapper {
 +
              padding: 3rem 0 1rem 0 ; } }
 +
          @media screen and (max-width: 480px) {
 +
            .wrapper {
 +
              padding: 2rem 0 0.1rem 0 ; } }
 +
       
 +
        /* Banner */
 +
        #banner {
 +
          -ms-flex-align: center;
 +
          -ms-flex-pack: center;
 +
          background-color: #111111;
 +
          color: rgba(255, 255, 255, 0.5);
 +
          -moz-align-items: center;
 +
          -webkit-align-items: center;
 +
          -ms-align-items: center;
 +
          align-items: center;
 +
          display: -moz-flex;
 +
          display: -webkit-flex;
 +
          display: -ms-flex;
 +
          display: flex;
 +
          -moz-justify-content: center;
 +
          -webkit-justify-content: center;
 +
          -ms-justify-content: center;
 +
          justify-content: center;
 +
          background-image: url(https://static.igem.org/mediawiki/2019/3/36/T--Waterloo--banner.jpeg);
 +
          background-position: center;
 +
          background-repeat: no-repeat;
 +
          background-size: cover;
 +
          border-top: 0;
 +
          display: -ms-flexbox;
 +
          height: 35rem !important;
 +
          min-height: 35rem;
 +
          overflow: hidden;
 +
          position: relative;
 +
          text-align: center;
 +
          width: 100%; }
 +
          #banner input, #banner select, #banner textarea {
 +
            color: #ffffff; }
 +
          #banner a {
 +
            color: #B3DC77; }
 +
          #banner strong, #banner b {
 +
            color: #ffffff; }
 +
          #banner h1, #banner h2, #banner h3, #banner h4, #banner h5, #banner h6 {
 +
            color: #ffffff; }
 +
          #banner blockquote {
 +
            border-left-color: rgba(255, 255, 255, 0.25); }
 +
          #banner code {
 +
            background: rgba(255, 255, 255, 0.075);
 +
            border-color: rgba(255, 255, 255, 0.25); }
 +
          #banner hr {
 +
            border-bottom-color: rgba(255, 255, 255, 0.25); }
 +
          #banner input[type="submit"],
 +
          #banner input[type="reset"],
 +
          #banner input[type="button"],
 +
          #banner button,
 +
          #banner .button {
 +
            background-color: transparent;
 +
            box-shadow: inset 0 0 0 1px #ffffff;
 +
            color: #ffffff !important; }
 +
            #banner input[type="submit"]:hover,
 +
            #banner input[type="reset"]:hover,
 +
            #banner input[type="button"]:hover,
 +
            #banner button:hover,
 +
            #banner .button:hover {
 +
              box-shadow: inset 0 0 0 1px #B3DC77;
 +
              color: #B3DC77 !important; }
 +
              #banner input[type="submit"]:hover:active,
 +
              #banner input[type="reset"]:hover:active,
 +
              #banner input[type="button"]:hover:active,
 +
              #banner button:hover:active,
 +
              #banner .button:hover:active {
 +
                background-color: rgba(206, 27, 40, 0.25); }
 +
            #banner input[type="submit"].primary,
 +
            #banner input[type="reset"].primary,
 +
            #banner input[type="button"].primary,
 +
            #banner button.primary,
 +
            #banner .button.primary {
 +
              box-shadow: none;
 +
              background-color: #B3DC77;
 +
              color: #ffffff !important; }
 +
              #banner input[type="submit"].primary:hover,
 +
              #banner input[type="reset"].primary:hover,
 +
              #banner input[type="button"].primary:hover,
 +
              #banner button.primary:hover,
 +
              #banner .button.primary:hover {
 +
                background-color: #B3DC77;
 +
                box-shadow: none; }
 +
                #banner input[type="submit"].primary:hover:active,
 +
                #banner input[type="reset"].primary:hover:active,
 +
                #banner input[type="button"].primary:hover:active,
 +
                #banner button.primary:hover:active,
 +
                #banner .button.primary:hover:active {
 +
                  background-color: #02b31f; }
 +
          #banner label {
 +
            color: #ffffff; }
 +
          #banner input[type="text"],
 +
          #banner input[type="password"],
 +
          #banner input[type="email"],
 +
          #banner input[type="tel"],
 +
          #banner input[type="search"],
 +
          #banner input[type="url"],
 +
          #banner select,
 +
          #banner textarea {
 +
            background-color: rgba(255, 255, 255, 0.075);
 +
            border-color: rgba(255, 255, 255, 0.25); }
 +
            #banner input[type="text"]:focus,
 +
            #banner input[type="password"]:focus,
 +
            #banner input[type="email"]:focus,
 +
            #banner input[type="tel"]:focus,
 +
            #banner input[type="search"]:focus,
 +
            #banner input[type="url"]:focus,
 +
            #banner select:focus,
 +
            #banner textarea:focus {
 +
              border-color: #B3DC77;
 +
              box-shadow: 0 0 0 1px #B3DC77; }
 +
          #banner select {
 +
            background-image: url("data:image/svg+xml;charset=utf8,%3Csvg xmlns='http://www.w3.org/2000/svg' width='40' height='40' preserveAspectRatio='none' viewBox='0 0 40 40'%3E%3Cpath d='M9.4,12.3l10.4,10.4l10.4-10.4c0.2-0.2,0.5-0.4,0.9-0.4c0.3,0,0.6,0.1,0.9,0.4l3.3,3.3c0.2,0.2,0.4,0.5,0.4,0.9 c0,0.4-0.1,0.6-0.4,0.9L20.7,31.9c-0.2,0.2-0.5,0.4-0.9,0.4c-0.3,0-0.6-0.1-0.9-0.4L4.3,17.3c-0.2-0.2-0.4-0.5-0.4-0.9 c0-0.4,0.1-0.6,0.4-0.9l3.3-3.3c0.2-0.2,0.5-0.4,0.9-0.4S9.1,12.1,9.4,12.3z' fill='rgba(255, 255, 255, 0.25)' /%3E%3C/svg%3E"); }
 +
            #banner select option {
 +
              color: rgba(255, 255, 255, 0.5);
 +
              background-color: #111111; }
 +
          #banner input[type="checkbox"] + label,
 +
          #banner input[type="radio"] + label {
 +
            color: rgba(255, 255, 255, 0.5); }
 +
            #banner input[type="checkbox"] + label:before,
 +
            #banner input[type="radio"] + label:before {
 +
              background: rgba(255, 255, 255, 0.075);
 +
              border-color: rgba(255, 255, 255, 0.25); }
 +
          #banner input[type="checkbox"]:checked + label:before,
 +
          #banner input[type="radio"]:checked + label:before {
 +
            background-color: #B3DC77;
 +
            border-color: #B3DC77;
 +
            color: #ffffff; }
 +
          #banner input[type="checkbox"]:focus + label:before,
 +
          #banner input[type="radio"]:focus + label:before {
 +
            border-color: #B3DC77;
 +
            box-shadow: 0 0 0 1px #B3DC77; }
 +
          #banner ::-webkit-input-placeholder {
 +
            color: rgba(255, 255, 255, 0.4) !important; }
 +
          #banner :-moz-placeholder {
 +
            color: rgba(255, 255, 255, 0.4) !important; }
 +
          #banner ::-moz-placeholder {
 +
            color: rgba(255, 255, 255, 0.4) !important; }
 +
          #banner :-ms-input-placeholder {
 +
            color: rgba(255, 255, 255, 0.4) !important; }
 +
          #banner ul.alt li {
 +
            border-top-color: rgba(255, 255, 255, 0.25); }
 +
          #banner table tbody tr {
 +
            border-color: rgba(255, 255, 255, 0.25); }
 +
            #banner table tbody tr:nth-child(2n + 1) {
 +
              background-color: rgba(255, 255, 255, 0.075); }
 +
          #banner table th {
 +
            color: #ffffff; }
 +
          #banner table thead {
 +
            border-bottom-color: rgba(255, 255, 255, 0.25); }
 +
          #banner table tfoot {
 +
            border-top-color: rgba(255, 255, 255, 0.25); }
 +
          #banner table.alt tbody tr td {
 +
            border-color: rgba(255, 255, 255, 0.25); }
 +
          #banner .highlights .content {
 +
            background: #111111;
 +
            box-shadow: 0px 0px 4px 1px rgba(255, 255, 255, 0.025); }
 +
          #banner .testimonials .content {
 +
            background: #111111;
 +
            box-shadow: 0px 0px 4px 1px rgba(255, 255, 255, 0.025); }
 +
            #banner .testimonials .content .credit strong {
 +
              color: #B3DC77; }
 +
          #banner > .inner {
 +
            -moz-transform: scale(1.0);
 +
            -webkit-transform: scale(1.0);
 +
            -ms-transform: scale(1.0);
 +
            transform: scale(1.0);
 +
            -moz-transition: opacity 1s ease, -moz-transform 1s ease;
 +
            -webkit-transition: opacity 1s ease, -webkit-transform 1s ease;
 +
            -ms-transition: opacity 1s ease, -ms-transform 1s ease;
 +
            transition: opacity 1s ease, transform 1s ease;
 +
            opacity: 1;
 +
            position: relative;
 +
            z-index: 3; }
 +
            #banner > .inner > :last-child {
 +
              margin-bottom: 0; }
 +
          #banner h1 {
 +
            font-size: 4rem;
 +
            margin-bottom: 1rem; }
 +
          #banner p {
 +
            font-size: 1.5rem; }
 +
          #banner a {
 +
            color: rgba(255, 255, 255, 0.5);
 +
            text-decoration: none; }
 +
            #banner a:hover {
 +
              color: #ffffff; }
 +
          #banner video {
 +
            -moz-transform: translateX(50%) translateY(50%);
 +
            -webkit-transform: translateX(50%) translateY(50%);
 +
            -ms-transform: translateX(50%) translateY(50%);
 +
            transform: translateX(50%) translateY(50%);
 +
            bottom: 50%;
 +
            height: auto;
 +
            min-height: 100%;
 +
            min-width: 100%;
 +
            overflow: hidden;
 +
            position: absolute;
 +
            right: 50%;
 +
            width: auto; }
 +
          #banner:before {
 +
            -moz-transition: opacity 3s ease;
 +
            -webkit-transition: opacity 3s ease;
 +
            -ms-transition: opacity 3s ease;
 +
            transition: opacity 3s ease;
 +
            -moz-transition-delay: 1.25s;
 +
            -webkit-transition-delay: 1.25s;
 +
            -ms-transition-delay: 1.25s;
 +
            transition-delay: 1.25s;
 +
            background: #111111;
 +
            content: '';
 +
            display: block;
 +
            height: 100%;
 +
            left: 0;
 +
            opacity: 0.45;
 +
            position: absolute;
 +
            top: 0;
 +
            width: 100%;
 +
            z-index: 1; }
 +
          #banner:after {
 +
            background: linear-gradient(135deg, #B3DC77 0%, #111111 74%);
 +
            content: ' ';
 +
            display: block;
 +
            height: 100%;
 +
            left: 0;
 +
            opacity: 0.6;
 +
            position: absolute;
 +
            top: 0;
 +
            webkit-linear-gradientidth: 100%;
 +
            width: 100%;
 +
            z-index: 1; }
 +
          #banner.small {
 +
            height: 30vh !important;
 +
            min-height: 30vh; }
 +
          @media screen and (max-width: 1280px) {
 +
            #banner video {
 +
              display: none; } }
 +
          @media screen and (max-width: 736px) {
 +
            #banner {
 +
              height: auto !important;
 +
              min-height: 0;
 +
              padding: 4rem 2rem 4rem 2rem; }
 +
              #banner .inner {
 +
                width: 100%; }
 +
              #banner h1 {
 +
                font-size: 1.75rem;
 +
                margin-bottom: 0.5rem;
 +
                padding-bottom: 0; }
 +
              #banner p {
 +
                font-size: 1.25rem; }
 +
              #banner br {
 +
                display: none; }
 +
              #banner .button {
 +
                width: 100%; } }
 +
          @media screen and (max-width: 480px) {
 +
            #banner p {
 +
              font-size: 1rem; } }
 +
          body.is-preload #banner .inner {
 +
            -moz-transform: scale(0.99);
 +
            -webkit-transform: scale(0.99);
 +
            -ms-transform: scale(0.99);
 +
            transform: scale(0.99);
 +
            opacity: 0; }
 +
          body.is-preload #banner:before {
 +
            opacity: 1; }
 +
       
 +
        /* CTA */
 +
        #cta {
 +
          background-color: #B3DC77;
 +
          color: rgba(255, 255, 255, 0.75);
 +
          background-attachment: fixed;
 +
          background-image: linear-gradient(rgba(206, 27, 40, 0.25), rgba(206, 27, 40, 0.25)), url(../../images/cta01.jpg);
 +
          background-position: bottom;
 +
          background-repeat: no-repeat;
 +
          background-size: cover;
 +
          position: relative;
 +
          text-align: center;
 +
          z-index: 1; }
 +
          #cta input, #cta select, #cta textarea {
 +
            color: #ffffff; }
 +
          #cta a {
 +
            color: #ffffff; }
 +
          #cta strong, #cta b {
 +
            color: #ffffff; }
 +
          #cta h1, #cta h2, #cta h3, #cta h4, #cta h5, #cta h6 {
 +
            color: #ffffff; }
 +
          #cta blockquote {
 +
            border-left-color: rgba(255, 255, 255, 0.25); }
 +
          #cta code {
 +
            background: rgba(255, 255, 255, 0.075);
 +
            border-color: rgba(255, 255, 255, 0.25); }
 +
          #cta hr {
 +
            border-bottom-color: rgba(255, 255, 255, 0.25); }
 +
          #cta input[type="submit"],
 +
          #cta input[type="reset"],
 +
          #cta input[type="button"],
 +
          #cta button,
 +
          #cta .button {
 +
            background-color: transparent;
 +
            box-shadow: inset 0 0 0 1px #ffffff;
 +
            color: #ffffff !important; }
 +
            #cta input[type="submit"]:hover,
 +
            #cta input[type="reset"]:hover,
 +
            #cta input[type="button"]:hover,
 +
            #cta button:hover,
 +
            #cta .button:hover {
 +
              background-color: rgba(255, 255, 255, 0.1); }
 +
              #cta input[type="submit"]:hover:active,
 +
              #cta input[type="reset"]:hover:active,
 +
              #cta input[type="button"]:hover:active,
 +
              #cta button:hover:active,
 +
              #cta .button:hover:active {
 +
                background-color: rgba(255, 255, 255, 0.25); }
 +
            #cta input[type="submit"].primary,
 +
            #cta input[type="reset"].primary,
 +
            #cta input[type="button"].primary,
 +
            #cta button.primary,
 +
            #cta .button.primary {
 +
              box-shadow: none;
 +
              background-color: #ffffff;
 +
              color: #B3DC77 !important; }
 +
              #cta input[type="submit"].primary:hover,
 +
              #cta input[type="reset"].primary:hover,
 +
              #cta input[type="button"].primary:hover,
 +
              #cta button.primary:hover,
 +
              #cta .button.primary:hover {
 +
                background-color: rgba(255, 255, 255, 0.875);
 +
                box-shadow: none; }
 +
                #cta input[type="submit"].primary:hover:active,
 +
                #cta input[type="reset"].primary:hover:active,
 +
                #cta input[type="button"].primary:hover:active,
 +
                #cta button.primary:hover:active,
 +
                #cta .button.primary:hover:active {
 +
                  background-color: rgba(255, 255, 255, 0.5); }
 +
          #cta label {
 +
            color: #ffffff; }
 +
          #cta input[type="text"],
 +
          #cta input[type="password"],
 +
          #cta input[type="email"],
 +
          #cta input[type="tel"],
 +
          #cta input[type="search"],
 +
          #cta input[type="url"],
 +
          #cta select,
 +
          #cta textarea {
 +
            background-color: rgba(255, 255, 255, 0.075);
 +
            border-color: rgba(255, 255, 255, 0.25); }
 +
            #cta input[type="text"]:focus,
 +
            #cta input[type="password"]:focus,
 +
            #cta input[type="email"]:focus,
 +
            #cta input[type="tel"]:focus,
 +
            #cta input[type="search"]:focus,
 +
            #cta input[type="url"]:focus,
 +
            #cta select:focus,
 +
            #cta textarea:focus {
 +
              border-color: #ffffff;
 +
              box-shadow: 0 0 0 1px #ffffff; }
 +
          #cta select {
 +
            background-image: url("data:image/svg+xml;charset=utf8,%3Csvg xmlns='http://www.w3.org/2000/svg' width='40' height='40' preserveAspectRatio='none' viewBox='0 0 40 40'%3E%3Cpath d='M9.4,12.3l10.4,10.4l10.4-10.4c0.2-0.2,0.5-0.4,0.9-0.4c0.3,0,0.6,0.1,0.9,0.4l3.3,3.3c0.2,0.2,0.4,0.5,0.4,0.9 c0,0.4-0.1,0.6-0.4,0.9L20.7,31.9c-0.2,0.2-0.5,0.4-0.9,0.4c-0.3,0-0.6-0.1-0.9-0.4L4.3,17.3c-0.2-0.2-0.4-0.5-0.4-0.9 c0-0.4,0.1-0.6,0.4-0.9l3.3-3.3c0.2-0.2,0.5-0.4,0.9-0.4S9.1,12.1,9.4,12.3z' fill='rgba(255, 255, 255, 0.25)' /%3E%3C/svg%3E"); }
 +
            #cta select option {
 +
              color: rgba(255, 255, 255, 0.75);
 +
              background-color: #B3DC77; }
 +
          #cta input[type="checkbox"] + label,
 +
          #cta input[type="radio"] + label {
 +
            color: rgba(255, 255, 255, 0.75); }
 +
            #cta input[type="checkbox"] + label:before,
 +
            #cta input[type="radio"] + label:before {
 +
              background: rgba(255, 255, 255, 0.075);
 +
              border-color: rgba(255, 255, 255, 0.25); }
 +
          #cta input[type="checkbox"]:checked + label:before,
 +
          #cta input[type="radio"]:checked + label:before {
 +
            background-color: #ffffff;
 +
            border-color: #ffffff;
 +
            color: #B3DC77; }
 +
          #cta input[type="checkbox"]:focus + label:before,
 +
          #cta input[type="radio"]:focus + label:before {
 +
            border-color: #ffffff;
 +
            box-shadow: 0 0 0 1px #ffffff; }
 +
          #cta ::-webkit-input-placeholder {
 +
            color: rgba(255, 255, 255, 0.4) !important; }
 +
          #cta :-moz-placeholder {
 +
            color: rgba(255, 255, 255, 0.4) !important; }
 +
          #cta ::-moz-placeholder {
 +
            color: rgba(255, 255, 255, 0.4) !important; }
 +
          #cta :-ms-input-placeholder {
 +
            color: rgba(255, 255, 255, 0.4) !important; }
 +
          #cta ul.alt li {
 +
            border-top-color: rgba(255, 255, 255, 0.25); }
 +
          #cta table tbody tr {
 +
            border-color: rgba(255, 255, 255, 0.25); }
 +
            #cta table tbody tr:nth-child(2n + 1) {
 +
              background-color: rgba(255, 255, 255, 0.075); }
 +
          #cta table th {
 +
            color: #ffffff; }
 +
          #cta table thead {
 +
            border-bottom-color: rgba(255, 255, 255, 0.25); }
 +
          #cta table tfoot {
 +
            border-top-color: rgba(255, 255, 255, 0.25); }
 +
          #cta table.alt tbody tr td {
 +
            border-color: rgba(255, 255, 255, 0.25); }
 +
          #cta .highlights .content {
 +
            background: #B3DC77;
 +
            box-shadow: 0px 0px 4px 1px rgba(255, 255, 255, 0.025); }
 +
          #cta .testimonials .content {
 +
            background: #B3DC77;
 +
            box-shadow: 0px 0px 4px 1px rgba(255, 255, 255, 0.025); }
 +
            #cta .testimonials .content .credit strong {
 +
              color: #B3DC77; }
 +
          #cta .inner {
 +
            position: relative;
 +
            z-index: 3; }
 +
          @media screen and (max-width: 980px) {
 +
            #cta {
 +
              background-attachment: scroll; } }
 +
       
 +
        /* Footer */
 +
        #footer {
 +
          background-color: #111111;
 +
          color: rgba(255, 255, 255, 0.5);
 +
          padding: 8rem 0 6rem 0 ; }
 +
          #footer input, #footer select, #footer textarea {
 +
            color: #ffffff; }
 +
          #footer a {
 +
            color: #B3DC77; }
 +
          #footer strong, #footer b {
 +
            color: #ffffff; }
 +
          #footer h1, #footer h2, #footer h3, #footer h4, #footer h5, #footer h6 {
 +
            color: #ffffff; }
 +
          #footer blockquote {
 +
            border-left-color: rgba(255, 255, 255, 0.25); }
 +
          #footer code {
 +
            background: rgba(255, 255, 255, 0.075);
 +
            border-color: rgba(255, 255, 255, 0.25); }
 +
          #footer hr {
 +
            border-bottom-color: rgba(255, 255, 255, 0.25); }
 +
          #footer input[type="submit"],
 +
          #footer input[type="reset"],
 +
          #footer input[type="button"],
 +
          #footer button,
 +
          #footer .button {
 +
            background-color: transparent;
 +
            box-shadow: inset 0 0 0 1px #ffffff;
 +
            color: #ffffff !important; }
 +
            #footer input[type="submit"]:hover,
 +
            #footer input[type="reset"]:hover,
 +
            #footer input[type="button"]:hover,
 +
            #footer button:hover,
 +
            #footer .button:hover {
 +
              box-shadow: inset 0 0 0 1px #B3DC77;
 +
              color: #B3DC77 !important; }
 +
              #footer input[type="submit"]:hover:active,
 +
              #footer input[type="reset"]:hover:active,
 +
              #footer input[type="button"]:hover:active,
 +
              #footer button:hover:active,
 +
              #footer .button:hover:active {
 +
                background-color: rgba(206, 27, 40, 0.25); }
 +
            #footer input[type="submit"].primary,
 +
            #footer input[type="reset"].primary,
 +
            #footer input[type="button"].primary,
 +
            #footer button.primary,
 +
            #footer .button.primary {
 +
              box-shadow: none;
 +
              background-color: #B3DC77;
 +
              color: #ffffff !important; }
 +
              #footer input[type="submit"].primary:hover,
 +
              #footer input[type="reset"].primary:hover,
 +
              #footer input[type="button"].primary:hover,
 +
              #footer button.primary:hover,
 +
              #footer .button.primary:hover {
 +
                background-color: #B3DC77;
 +
                box-shadow: none; }
 +
                #footer input[type="submit"].primary:hover:active,
 +
                #footer input[type="reset"].primary:hover:active,
 +
                #footer input[type="button"].primary:hover:active,
 +
                #footer button.primary:hover:active,
 +
                #footer .button.primary:hover:active {
 +
                  background-color: #02b31f; }
 +
          #footer label {
 +
            color: #ffffff; }
 +
          #footer input[type="text"],
 +
          #footer input[type="password"],
 +
          #footer input[type="email"],
 +
          #footer input[type="tel"],
 +
          #footer input[type="search"],
 +
          #footer input[type="url"],
 +
          #footer select,
 +
          #footer textarea {
 +
            background-color: rgba(255, 255, 255, 0.075);
 +
            border-color: rgba(255, 255, 255, 0.25); }
 +
            #footer input[type="text"]:focus,
 +
            #footer input[type="password"]:focus,
 +
            #footer input[type="email"]:focus,
 +
            #footer input[type="tel"]:focus,
 +
            #footer input[type="search"]:focus,
 +
            #footer input[type="url"]:focus,
 +
            #footer select:focus,
 +
            #footer textarea:focus {
 +
              border-color: #B3DC77;
 +
              box-shadow: 0 0 0 1px #B3DC77; }
 +
          #footer select {
 +
            background-image: url("data:image/svg+xml;charset=utf8,%3Csvg xmlns='http://www.w3.org/2000/svg' width='40' height='40' preserveAspectRatio='none' viewBox='0 0 40 40'%3E%3Cpath d='M9.4,12.3l10.4,10.4l10.4-10.4c0.2-0.2,0.5-0.4,0.9-0.4c0.3,0,0.6,0.1,0.9,0.4l3.3,3.3c0.2,0.2,0.4,0.5,0.4,0.9 c0,0.4-0.1,0.6-0.4,0.9L20.7,31.9c-0.2,0.2-0.5,0.4-0.9,0.4c-0.3,0-0.6-0.1-0.9-0.4L4.3,17.3c-0.2-0.2-0.4-0.5-0.4-0.9 c0-0.4,0.1-0.6,0.4-0.9l3.3-3.3c0.2-0.2,0.5-0.4,0.9-0.4S9.1,12.1,9.4,12.3z' fill='rgba(255, 255, 255, 0.25)' /%3E%3C/svg%3E"); }
 +
            #footer select option {
 +
              color: rgba(255, 255, 255, 0.5);
 +
              background-color: #111111; }
 +
          #footer input[type="checkbox"] + label,
 +
          #footer input[type="radio"] + label {
 +
            color: rgba(255, 255, 255, 0.5); }
 +
            #footer input[type="checkbox"] + label:before,
 +
            #footer input[type="radio"] + label:before {
 +
              background: rgba(255, 255, 255, 0.075);
 +
              border-color: rgba(255, 255, 255, 0.25); }
 +
          #footer input[type="checkbox"]:checked + label:before,
 +
          #footer input[type="radio"]:checked + label:before {
 +
            background-color: #B3DC77;
 +
            border-color: #B3DC77;
 +
            color: #ffffff; }
 +
          #footer input[type="checkbox"]:focus + label:before,
 +
          #footer input[type="radio"]:focus + label:before {
 +
            border-color: #B3DC77;
 +
            box-shadow: 0 0 0 1px #B3DC77; }
 +
          #footer ::-webkit-input-placeholder {
 +
            color: rgba(255, 255, 255, 0.4) !important; }
 +
          #footer :-moz-placeholder {
 +
            color: rgba(255, 255, 255, 0.4) !important; }
 +
          #footer ::-moz-placeholder {
 +
            color: rgba(255, 255, 255, 0.4) !important; }
 +
          #footer :-ms-input-placeholder {
 +
            color: rgba(255, 255, 255, 0.4) !important; }
 +
          #footer ul.alt li {
 +
            border-top-color: rgba(255, 255, 255, 0.25); }
 +
          #footer table tbody tr {
 +
            border-color: rgba(255, 255, 255, 0.25); }
 +
            #footer table tbody tr:nth-child(2n + 1) {
 +
              background-color: rgba(255, 255, 255, 0.075); }
 +
          #footer table th {
 +
            color: #ffffff; }
 +
          #footer table thead {
 +
            border-bottom-color: rgba(255, 255, 255, 0.25); }
 +
          #footer table tfoot {
 +
            border-top-color: rgba(255, 255, 255, 0.25); }
 +
          #footer table.alt tbody tr td {
 +
            border-color: rgba(255, 255, 255, 0.25); }
 +
          #footer .highlights .content {
 +
            background: #111111;
 +
            box-shadow: 0px 0px 4px 1px rgba(255, 255, 255, 0.025); }
 +
          #footer .testimonials .content {
 +
            background: #111111;
 +
            box-shadow: 0px 0px 4px 1px rgba(255, 255, 255, 0.025); }
 +
            #footer .testimonials .content .credit strong {
 +
              color: #B3DC77; }
 +
          #footer a {
 +
            color: rgba(255, 255, 255, 0.5);
 +
            text-decoration: none; }
 +
            #footer a:hover {
 +
              color: #B3DC77; }
 +
          #footer .content {
 +
            display: -moz-flex;
 +
            display: -webkit-flex;
 +
            display: -ms-flex;
 +
            display: flex; }
 +
            #footer .content section {
 +
              width: 25%; }
 +
              #footer .content section:first-child {
 +
                width: 50%;
 +
                padding-right: 4rem; }
 +
              #footer .content section:last-child {
 +
                padding-left: 4rem; }
 +
          #footer .copyright {
 +
            border-top: 1px solid;
 +
            font-size: 0.8rem;
 +
            opacity: 0.5;
 +
            padding: 2rem 0;
 +
            text-align: center; }
 +
          @media screen and (max-width: 1280px) {
 +
            #footer {
 +
              padding: 4rem 0 2rem 0 ; } }
 +
          @media screen and (max-width: 980px) {
 +
            #footer .content {
 +
              -moz-flex-wrap: wrap;
 +
              -webkit-flex-wrap: wrap;
 +
              -ms-flex-wrap: wrap;
 +
              flex-wrap: wrap; }
 +
              #footer .content section {
 +
                width: 50%; }
 +
                #footer .content section:first-child {
 +
                  width: 100%;
 +
                  padding-right: 0; } }
 +
          @media screen and (max-width: 736px) {
 +
            #footer {
 +
              padding: 3rem 0 1rem 0 ; }
 +
              #footer .content section {
 +
                width: 100%; }
 +
                #footer .content section:last-child {
 +
                  padding-left: 0; } }
 +
          @media screen and (max-width: 480px) {
 +
            #footer {
 +
              padding: 2rem 0 0.1rem 0 ; } }
 +
       
 +
        /* Header */
 +
        body {
 +
          padding-top: 3.25rem; }
 +
          body:before {
 +
            content: ' ';
 +
            background-image: url(https://static.igem.org/mediawiki/2019/3/37/T--Waterloo--bg_image.jpeg);
 +
            background-size: cover;
 +
            background-repeat: no-repeat;
 +
            background-attachment: fixed;
 +
            display: block;
 +
            position: fixed;
 +
            left: 0;
 +
            top: 0;
 +
            width: 100%;
 +
            height: 100%;
 +
            z-index: -1;
 +
            opacity: 0.05; }
 +
       
 +
        #header {
 +
          -moz-align-items: center;
 +
          -webkit-align-items: center;
 +
          -ms-align-items: center;
 +
          align-items: center;
 +
          display: -moz-flex;
 +
          display: -webkit-flex;
 +
          display: -ms-flex;
 +
          display: flex;
 +
          -moz-justify-content: space-between;
 +
          -webkit-justify-content: space-between;
 +
          -ms-justify-content: space-between;
 +
          justify-content: space-between;
 +
          background: #111111;
 +
          color: rgba(255, 255, 255, 0.5);
 +
          cursor: default;
 +
          height: 3.25rem;
 +
          left: 0;
 +
          line-height: 4.25rem;
 +
          position: fixed;
 +
          top: 0;
 +
          width: 100%;
 +
          z-index: 10001; }
 +
          #header > .logo {
 +
            color: #ffffff;
 +
            font-size: 1rem;
 +
            font-weight: 600;
 +
            height: inherit;
 +
            line-height: inherit;
 +
            padding: 0 1.25rem;
 +
            text-decoration: none; }
 +
          #header > nav > a {
 +
            color: inherit;
 +
            display: inline-block;
 +
            padding: 0 0.75rem;
 +
            text-decoration: none; }
 +
            #header > nav > a:hover {
 +
              color: #ffffff; }
 +
            #header > nav > a[href="#menu"] {
 +
              text-decoration: none;
 +
              -webkit-tap-highlight-color: rgba(0, 0, 0, 0); }
 +
              #header > nav > a[href="#menu"]:before {
 +
                -moz-osx-font-smoothing: grayscale;
 +
                -webkit-font-smoothing: antialiased;
 +
                font-family: FontAwesome;
 +
                font-style: normal;
 +
                font-weight: normal;
 +
                text-transform: none !important; }
 +
              #header > nav > a[href="#menu"]:before {
 +
                content: '\f0c9';
 +
                margin: 0 0.5rem 0 0; }
 +
            #header > nav > a + a[href="#menu"]:last-child {
 +
              border-left: solid 1px rgba(255, 255, 255, 0.25);
 +
              margin-left: 0.5rem;
 +
              padding-left: 1.25rem; }
 +
            #header > nav > a:last-child {
 +
              padding-right: 1.25rem; }
 +
            @media screen and (max-width: 736px) {
 +
              #header > nav > a {
 +
                padding: 0 0.5rem; }
 +
                #header > nav > a + a[href="#menu"]:last-child {
 +
                  margin-left: 0.25rem;
 +
                  padding-left: 1rem; }
 +
                #header > nav > a:last-child {
 +
                  padding-right: 1rem; } }
 +
       
 +
        @media screen and (max-width: 980px) {
 +
          body {
 +
            padding-top: 44px; }
 +
       
 +
          #header {
 +
            height: 44px;
 +
            line-height: 44px; } }
 +
        @media screen and (max-width: 480px) {
 +
          #header {
 +
            min-width: 320px; } }
 +
        /* Heading */
 +
        #heading {
 +
          -ms-flex-align: center;
 +
          -ms-flex-pack: center;
 +
          background-color: #111111;
 +
          color: rgba(255, 255, 255, 0.5);
 +
          -moz-align-items: center;
 +
          -webkit-align-items: center;
 +
          -ms-align-items: center;
 +
          align-items: center;
 +
          display: -moz-flex;
 +
          display: -webkit-flex;
 +
          display: -ms-flex;
 +
          display: flex;
 +
          -moz-justify-content: center;
 +
          -webkit-justify-content: center;
 +
          -ms-justify-content: center;
 +
          justify-content: center;
 +
          background-image: linear-gradient(rgba(17, 17, 17, 0.25), rgba(17, 17, 17, 0.25)), url(https://static.igem.org/mediawiki/2019/3/36/T--Waterloo--banner.jpeg);
 +
          background-position: center;
 +
          background-repeat: no-repeat;
 +
          background-size: cover;
 +
          border-top: 0;
 +
          display: -ms-flexbox;
 +
          height: 15rem !important;
 +
          min-height: 15rem;
 +
          overflow: hidden;
 +
          position: relative;
 +
          text-align: center;
 +
          width: 100%; }
 +
          #heading input, #heading select, #heading textarea {
 +
            color: #ffffff; }
 +
          #heading a {
 +
            color: #B3DC77; }
 +
          #heading strong, #heading b {
 +
            color: #ffffff; }
 +
          #heading h1, #heading h2, #heading h3, #heading h4, #heading h5, #heading h6 {
 +
            color: #ffffff; }
 +
          #heading blockquote {
 +
            border-left-color: rgba(255, 255, 255, 0.25); }
 +
          #heading code {
 +
            background: rgba(255, 255, 255, 0.075);
 +
            border-color: rgba(255, 255, 255, 0.25); }
 +
          #heading hr {
 +
            border-bottom-color: rgba(255, 255, 255, 0.25); }
 +
          #heading input[type="submit"],
 +
          #heading input[type="reset"],
 +
          #heading input[type="button"],
 +
          #heading button,
 +
          #heading .button {
 +
            background-color: transparent;
 +
            box-shadow: inset 0 0 0 1px #ffffff;
 +
            color: #ffffff !important; }
 +
            #heading input[type="submit"]:hover,
 +
            #heading input[type="reset"]:hover,
 +
            #heading input[type="button"]:hover,
 +
            #heading button:hover,
 +
            #heading .button:hover {
 +
              box-shadow: inset 0 0 0 1px #B3DC77;
 +
              color: #B3DC77 !important; }
 +
              #heading input[type="submit"]:hover:active,
 +
              #heading input[type="reset"]:hover:active,
 +
              #heading input[type="button"]:hover:active,
 +
              #heading button:hover:active,
 +
              #heading .button:hover:active {
 +
                background-color: rgba(206, 27, 40, 0.25); }
 +
            #heading input[type="submit"].primary,
 +
            #heading input[type="reset"].primary,
 +
            #heading input[type="button"].primary,
 +
            #heading button.primary,
 +
            #heading .button.primary {
 +
              box-shadow: none;
 +
              background-color: #B3DC77;
 +
              color: #ffffff !important; }
 +
              #heading input[type="submit"].primary:hover,
 +
              #heading input[type="reset"].primary:hover,
 +
              #heading input[type="button"].primary:hover,
 +
              #heading button.primary:hover,
 +
              #heading .button.primary:hover {
 +
                background-color: #B3DC77;
 +
                box-shadow: none; }
 +
                #heading input[type="submit"].primary:hover:active,
 +
                #heading input[type="reset"].primary:hover:active,
 +
                #heading input[type="button"].primary:hover:active,
 +
                #heading button.primary:hover:active,
 +
                #heading .button.primary:hover:active {
 +
                  background-color: #02b31f; }
 +
          #heading label {
 +
            color: #ffffff; }
 +
          #heading input[type="text"],
 +
          #heading input[type="password"],
 +
          #heading input[type="email"],
 +
          #heading input[type="tel"],
 +
          #heading input[type="search"],
 +
          #heading input[type="url"],
 +
          #heading select,
 +
          #heading textarea {
 +
            background-color: rgba(255, 255, 255, 0.075);
 +
            border-color: rgba(255, 255, 255, 0.25); }
 +
            #heading input[type="text"]:focus,
 +
            #heading input[type="password"]:focus,
 +
            #heading input[type="email"]:focus,
 +
            #heading input[type="tel"]:focus,
 +
            #heading input[type="search"]:focus,
 +
            #heading input[type="url"]:focus,
 +
            #heading select:focus,
 +
            #heading textarea:focus {
 +
              border-color: #B3DC77;
 +
              box-shadow: 0 0 0 1px #B3DC77; }
 +
          #heading select {
 +
            background-image: url("data:image/svg+xml;charset=utf8,%3Csvg xmlns='http://www.w3.org/2000/svg' width='40' height='40' preserveAspectRatio='none' viewBox='0 0 40 40'%3E%3Cpath d='M9.4,12.3l10.4,10.4l10.4-10.4c0.2-0.2,0.5-0.4,0.9-0.4c0.3,0,0.6,0.1,0.9,0.4l3.3,3.3c0.2,0.2,0.4,0.5,0.4,0.9 c0,0.4-0.1,0.6-0.4,0.9L20.7,31.9c-0.2,0.2-0.5,0.4-0.9,0.4c-0.3,0-0.6-0.1-0.9-0.4L4.3,17.3c-0.2-0.2-0.4-0.5-0.4-0.9 c0-0.4,0.1-0.6,0.4-0.9l3.3-3.3c0.2-0.2,0.5-0.4,0.9-0.4S9.1,12.1,9.4,12.3z' fill='rgba(255, 255, 255, 0.25)' /%3E%3C/svg%3E"); }
 +
            #heading select option {
 +
              color: rgba(255, 255, 255, 0.5);
 +
              background-color: #111111; }
 +
          #heading input[type="checkbox"] + label,
 +
          #heading input[type="radio"] + label {
 +
            color: rgba(255, 255, 255, 0.5); }
 +
            #heading input[type="checkbox"] + label:before,
 +
            #heading input[type="radio"] + label:before {
 +
              background: rgba(255, 255, 255, 0.075);
 +
              border-color: rgba(255, 255, 255, 0.25); }
 +
          #heading input[type="checkbox"]:checked + label:before,
 +
          #heading input[type="radio"]:checked + label:before {
 +
            background-color: #B3DC77;
 +
            border-color: #B3DC77;
 +
            color: #ffffff; }
 +
          #heading input[type="checkbox"]:focus + label:before,
 +
          #heading input[type="radio"]:focus + label:before {
 +
            border-color: #B3DC77;
 +
            box-shadow: 0 0 0 1px #B3DC77; }
 +
          #heading ::-webkit-input-placeholder {
 +
            color: rgba(255, 255, 255, 0.4) !important; }
 +
          #heading :-moz-placeholder {
 +
            color: rgba(255, 255, 255, 0.4) !important; }
 +
          #heading ::-moz-placeholder {
 +
            color: rgba(255, 255, 255, 0.4) !important; }
 +
          #heading :-ms-input-placeholder {
 +
            color: rgba(255, 255, 255, 0.4) !important; }
 +
          #heading ul.alt li {
 +
            border-top-color: rgba(255, 255, 255, 0.25); }
 +
          #heading table tbody tr {
 +
            border-color: rgba(255, 255, 255, 0.25); }
 +
            #heading table tbody tr:nth-child(2n + 1) {
 +
              background-color: rgba(255, 255, 255, 0.075); }
 +
          #heading table th {
 +
            color: #ffffff; }
 +
          #heading table thead {
 +
            border-bottom-color: rgba(255, 255, 255, 0.25); }
 +
          #heading table tfoot {
 +
            border-top-color: rgba(255, 255, 255, 0.25); }
 +
          #heading table.alt tbody tr td {
 +
            border-color: rgba(255, 255, 255, 0.25); }
 +
          #heading .highlights .content {
 +
            background: #111111;
 +
            box-shadow: 0px 0px 4px 1px rgba(255, 255, 255, 0.025); }
 +
          #heading .testimonials .content {
 +
            background: #111111;
 +
            box-shadow: 0px 0px 4px 1px rgba(255, 255, 255, 0.025); }
 +
            #heading .testimonials .content .credit strong {
 +
              color: #B3DC77; }
 +
          #heading:before {
 +
            background: linear-gradient(135deg, #B3DC77 0%, #111111 74%);
 +
            content: ' ';
 +
            display: block;
 +
            height: 100%;
 +
            left: 0;
 +
            opacity: 0.6;
 +
            position: absolute;
 +
            top: 0;
 +
            width: 100%;
 +
            z-index: 1; }
 +
          #heading h1 {
 +
            margin-bottom: 0;
 +
            position: relative;
 +
            z-index: 2; }
 +
          @media screen and (max-width: 980px) {
 +
            #heading {
 +
              padding: 2rem; } }
 +
       
 +
        /* Main */
 +
        #main .content {
 +
          background: #ffffff;
 +
          border-radius: 4px;
 +
          box-shadow: 0px 0px 4px 1px rgba(0, 0, 0, 0.025);
 +
          margin-bottom: 2rem;
 +
          padding: 3rem; }
 +
          @media screen and (max-width: 980px) {
 +
            #main .content {
 +
              padding: 2rem; } }
 +
          @media screen and (max-width: 480px) {
 +
            #main .content {
 +
              padding: 1.5rem; } }
 +
       
 +
        /* Menu */
 +
        #menu {
 +
          background-color: #111111;
 +
          color: rgba(255, 255, 255, 0.5);
 +
          -moz-transform: translateX(20rem);
 +
          -webkit-transform: translateX(20rem);
 +
          -ms-transform: translateX(20rem);
 +
          transform: translateX(20rem);
 +
          -moz-transition: -moz-transform 0.5s ease, box-shadow 0.5s ease, visibility 0.5s;
 +
          -webkit-transition: -webkit-transform 0.5s ease, box-shadow 0.5s ease, visibility 0.5s;
 +
          -ms-transition: -ms-transform 0.5s ease, box-shadow 0.5s ease, visibility 0.5s;
 +
          transition: transform 0.5s ease, box-shadow 0.5s ease, visibility 0.5s;
 +
          -webkit-overflow-scrolling: touch;
 +
          box-shadow: none;
 +
          height: 100%;
 +
          max-width: 80%;
 +
          overflow-y: auto;
 +
          padding: 3rem 2rem;
 +
          position: fixed;
 +
          right: 0;
 +
          top: 0;
 +
          visibility: hidden;
 +
          width: 20rem;
 +
          z-index: 10002; }
 +
          #menu input, #menu select, #menu textarea {
 +
            color: #ffffff; }
 +
          #menu a {
 +
            color: #B3DC77; }
 +
          #menu strong, #menu b {
 +
            color: #ffffff; }
 +
          #menu h1, #menu h2, #menu h3, #menu h4, #menu h5, #menu h6 {
 +
            color: #ffffff; }
 +
          #menu blockquote {
 +
            border-left-color: rgba(255, 255, 255, 0.25); }
 +
          #menu code {
 +
            background: rgba(255, 255, 255, 0.075);
 +
            border-color: rgba(255, 255, 255, 0.25); }
 +
          #menu hr {
 +
            border-bottom-color: rgba(255, 255, 255, 0.25); }
 +
          #menu input[type="submit"],
 +
          #menu input[type="reset"],
 +
          #menu input[type="button"],
 +
          #menu button,
 +
          #menu .button {
 +
            background-color: transparent;
 +
            box-shadow: inset 0 0 0 1px #ffffff;
 +
            color: #ffffff !important; }
 +
            #menu input[type="submit"]:hover,
 +
            #menu input[type="reset"]:hover,
 +
            #menu input[type="button"]:hover,
 +
            #menu button:hover,
 +
            #menu .button:hover {
 +
              box-shadow: inset 0 0 0 1px #B3DC77;
 +
              color: #B3DC77 !important; }
 +
              #menu input[type="submit"]:hover:active,
 +
              #menu input[type="reset"]:hover:active,
 +
              #menu input[type="button"]:hover:active,
 +
              #menu button:hover:active,
 +
              #menu .button:hover:active {
 +
                background-color: rgba(206, 27, 40, 0.25); }
 +
            #menu input[type="submit"].primary,
 +
            #menu input[type="reset"].primary,
 +
            #menu input[type="button"].primary,
 +
            #menu button.primary,
 +
            #menu .button.primary {
 +
              box-shadow: none;
 +
              background-color: #B3DC77;
 +
              color: #ffffff !important; }
 +
              #menu input[type="submit"].primary:hover,
 +
              #menu input[type="reset"].primary:hover,
 +
              #menu input[type="button"].primary:hover,
 +
              #menu button.primary:hover,
 +
              #menu .button.primary:hover {
 +
                background-color: #B3DC77;
 +
                box-shadow: none; }
 +
                #menu input[type="submit"].primary:hover:active,
 +
                #menu input[type="reset"].primary:hover:active,
 +
                #menu input[type="button"].primary:hover:active,
 +
                #menu button.primary:hover:active,
 +
                #menu .button.primary:hover:active {
 +
                  background-color: #02b31f; }
 +
          #menu > ul {
 +
            margin: 0 0 1rem 0; }
 +
            #menu > ul.links {
 +
              list-style: none;
 +
              padding: 0; }
 +
              #menu > ul.links > li {
 +
                padding: 0; }
 +
                #menu > ul.links > li > a {
 +
                  border: 0;
 +
                  border-top: solid 1px rgba(255, 255, 255, 0.25);
 +
                  color: inherit;
 +
                  display: block;
 +
                  line-height: 3.5rem;
 +
                  text-decoration: none; }
 +
                  #menu > ul.links > li > a:hover {
 +
                    color: #ffffff; }
 +
                #menu > ul.links > li:first-child > a {
 +
                  border-top: 0; }
 +
          #menu .close {
 +
            text-decoration: none;
 +
            -moz-transition: color 0.2s ease-in-out;
 +
            -webkit-transition: color 0.2s ease-in-out;
 +
            -ms-transition: color 0.2s ease-in-out;
 +
            transition: color 0.2s ease-in-out;
 +
            -webkit-tap-highlight-color: rgba(0, 0, 0, 0);
 +
            border: 0;
 +
            color: rgba(255, 255, 255, 0.4);
 +
            cursor: pointer;
 +
            display: block;
 +
            height: 3.25rem;
 +
            line-height: 3.25rem;
 +
            padding-right: 1.25rem;
 +
            position: absolute;
 +
            right: 0;
 +
            text-align: right;
 +
            top: 0;
 +
            vertical-align: middle;
 +
            width: 7rem; }
 +
            #menu .close:before {
 +
              -moz-osx-font-smoothing: grayscale;
 +
              -webkit-font-smoothing: antialiased;
 +
              font-family: FontAwesome;
 +
              font-style: normal;
 +
              font-weight: normal;
 +
              text-transform: none !important; }
 +
            #menu .close:before {
 +
              content: '\f00d';
 +
              font-size: 1.25rem; }
 +
            #menu .close:hover {
 +
              color: #ffffff; }
 +
            @media screen and (max-width: 736px) {
 +
              #menu .close {
 +
                height: 4rem;
 +
                line-height: 4rem; } }
 +
          @media screen and (max-width: 736px) {
 +
            #menu {
 +
              padding: 2.5rem 1.75rem; } }
 +
       
 +
        body.is-menu-visible #menu {
 +
          -moz-transform: translateX(0);
 +
          -webkit-transform: translateX(0);
 +
          -ms-transform: translateX(0);
 +
          transform: translateX(0);
 +
          box-shadow: 0 0 1.5rem 0 rgba(0, 0, 0, 0.2);
 +
          visibility: visible; }
 +
       
 +
        </style>
 +
<!--
 +
Template Information:
 +
Industrious by TEMPLATED
 +
templated.co @templatedco
 +
Released for free under the Creative Commons Attribution 3.0 license (templated.co/license)
 +
-->
  
 +
<head>
 +
<title>iGEM UWaterloo 2019 - Results</title>
 +
<meta charset="utf-8" />
 +
<meta name="viewport" content="width=device-width, initial-scale=1, user-scalable=no" />
 +
<meta name="description" content="" />
 +
<meta name="keywords" content="" />
 +
<link rel="stylesheet" href="assets/css/uwaterloo-main.css" />
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<h1 id="cloning">Cloning</h1>
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<h2 id="linuron-construct">Linuron construct</h2>
 +
<p><img src="https://static.igem.org/mediawiki/2019/d/d2/T--Waterloo--Linuron.png" alt="Linuron"></p>
 +
<p>In this construct, the insert is was cloned into the BglII site between the Paph promoter and bjGFP sequence on the pRJPaph-bjGFP to give a transcriptional fusion with the linuron degradation genes.  The insert itself was made by inserting the BBa_B0034 ribosome binding site ahead of each the <em>libA</em> and <em>nat1</em> genes through PCR.  These were then inserted downstream of the Paph promoter in the order of <em>libA</em> then <em>nat1</em> by using Gibson Assembly/NEBuild.  The coding sequences for the <em>libA</em> and <em>nat1</em> genes were codon optimised for <em>B. diazoefficiens</em> USDA 110.5.</p>
 +
<p>We successfully assembled this construct! It was validated by colony PCR (amplifying inserted genes) and sequencing. We also confirmed operon expression by measuring GFP fluorescence.</p>
 +
<h2 id="glyphosate-construct">Glyphosate construct</h2>
 +
<p><img src="https://static.igem.org/mediawiki/2019/a/a4/T--Waterloo--Glyphosate.png" alt="glyphosate"></p>
 +
<p>Similar to the construction of our plasmid for resistance to linuron, we planned to insert the BBa_B0034 ribosome binding site and coding sequence of the CP4 EPSP synthase gene into the BglII site present between the Paph promoter and bjGFP coding sequence on the pRJPaph-bjGFP plasmid to give a transcriptional fusion of our EPSP gene with the bjGFP. The coding sequence of the CP4 EPSP synthase gene used was derived from the amino acid sequence claimed in the Monsanto patent, reverse translated to be codon optimised for <em>B. diazoefficiens</em> USDA 110.5 Attempts to assemble this construct through NEBuilder were not successful as the no conditions could be found to amplify the coding sequence with required overhangs and ribosome binding site by PCR. This likely was due to the high GC content of the coding sequence (71.2%), however the lengths of the primers could have also contributed to the difficulty. Unfortunately, we decided to drop the glyphosate part of the project and only focus on Linuron due to time constraints.</p>
 +
              <h1 id="mic-assay-results">MIC assay results</h1>
 +
              <h2 id="growth-curve-experiment-with-wild-type-b-diazoefficiens">Growth curve experiment with wild-type <em>B. diazoefficiens</em></h2>
 +
              <p>Although we were unable to evaluate the tolerance of engineered <em>B. diazoefficiens</em> strains due to time constraints, we nonetheless investigated the tolerance of wild-type <em>B. diazoefficiens</em> to linuron and 3,4-Dichloroaniline (3,4-DCA). This was evaluated using growth curve Minimum Inhibitory Concentration (MIC) experiments performed with hourly absorbance readings from 20 to 25 hours of incubation at 30 °C. The absorbances at the five timepoints were plotted versus time and fit with exponential curves of the form y = ae^(kx) + b, where k is growth rate. Sextuplicates were used for each toxin and concentration. </p>
 +
              <p>The results of these experiments are presented in figures 5, 6, and 7.</p>
 +
              <p><img src="https://static.igem.org/mediawiki/2019/6/6d/T--Waterloo--LinBd2.png" alt="LinBd2"></p>
 +
              <p><em>Figure 5</em>. Growth rate versus linuron concentration from 20 to 25 hours. Error bars represent standard deviations of sextuplicates. The growth rate with no toxin was 0.08639 +/- 0.01232 1/h. The sigmoidal fit parameters A, B, C, and D are 3.23E-03, 3.46E-02, 2.96, and 2.30E+01 respectively.</p>
 +
              <p><img src="https://static.igem.org/mediawiki/2019/0/06/T--Waterloo--DCABd2.png" alt="DCABd2"></p>
 +
              <p><em>Figure 6</em>. Growth rate versus linuron concentration from 20 to 25 hours. Error bars represent standard deviations of sextuplicates. The growth rate with no toxin was 0.08639 +/- 0.01232 1/h. The sigmoidal fit parameters A, B, C, and D are 7.43E-03, 3.62E-02, 8.32E-01, and 2.08E+01 respectively.</p>
 +
              <p><em>Figure 7</em>. Summary of key inhibitory concentrations for linuron and 3,4-DCA with <em>B. diazoefficiens</em> based on growth curves from 20 to 25 hours. </p>
 +
              <p><img style="width:30%;" src="https://static.igem.org/mediawiki/2019/8/83/T--Waterloo--T1.png" alt="T1"></p>
 +
              <p>These differences in toxicity profiles are demonstrated by the key inhibitory concentrations in figure 7. Linuron’s IC<sub>50</sub> value is higher than that of 3,4-DCA, demonstrating that linuron is generally less toxic than 3,4-DCA. This agrees with reported toxicity values (Tixier, 2002) (Santos, 2014). Comparing IC<sub>10</sub> values shows that 3,4-DCA starts showing toxic effects at a much lower concentration than linuron, due to steeper slope of the plot for linuron. The steeper slope of linuron’s plot also means that linuron’s IC<sub>90</sub> is much lower than that of 3,4-DCA. This means that full growth inhibition actually requires a lower concentration of linuron than of 3,4-DCA, despite linuron’s lower IC<sub>50</sub>. These differences highlight the importance of examining the entire toxicity profile and various inhibitory concentrations, since these convey much more information that the IC<sub>50</sub> value alone. </p>
 +
              <p>We have therefore experimentally deteremined the toxicity profiles of both linuron and 3,4-DCA toward wild type <em>B. diazoefficiens</em>, validating our experimental approach. This information provides also a reference point against which to compare engineered strains, other toxins, or bacterial species.</p>
 +
              <h2 id="single-timepoint-experiment-with-wild-type-b-diazoefficiens">Single-timepoint experiment with wild-type <em>B. diazoefficiens</em></h2>
 +
              <p>In addition to the growth curve experiment peformed with <em>B. diazoefficiens</em> both in linuron and 3,4-DCA, we also completed this experiment in single-time point format. This was done using one Abs<sub>600</sub> after 22 hours incubation at 30 C. Figures 8 and 9 show plots of Abs<sub>600</sub> versus toxin concentration. These results are shown in figures 8, 9, and 10. Fitting was performed to the four parameter in equation 1.</p>
 +
              <p><img src="https://static.igem.org/mediawiki/2019/5/5b/T--Waterloo--LinBd1.png" alt="LinBd1"></p>
 +
              <p><em>Figure 8</em>. Abs<sub>600</sub> versus linuron concentration at 22 hours. Error bars represent standard deviations of sextuplicates. The sigmoidal fit parameters A, B, C, and D are 1.28E-02, 5.52E-02, 1.62, and 1.28E+01 respectively.</p>
 +
              <p><img src="https://static.igem.org/mediawiki/2019/e/ed/T--Waterloo--DCABd1.png" alt="DCABd1"></p>
 +
              <p><em>Figure 9</em>. Abs<sub>600</sub> versus linuron concentration at 22 hours. Error bars represent standard deviations of sextuplicates. The sigmoidal fit parameters A, B, C, and D are 1.50E-02, 5.47E-02, 8.97E-01, and 3.08E+01 respectively.  </p>
 +
              <p><em>Figure 10</em>. Summary of key inhibitory concentrations for linuron and 3,4-DCA with <em>B. diazoefficiens</em> after 22 hours. </p>
 +
              <p><img style="width:30%;"  src="https://static.igem.org/mediawiki/2019/2/2d/T--Waterloo--T2.png" alt="T2"></p>
 +
              <p>Figures 8 and 9 both show the characteristic sigmoidal shapes expected for MIC experiments. All datapoints are very close to the sigmoidal fit, indicating that this model is a good representation of the data. </p>
 +
              <p>Figure 10 summarizes some key inhibitory concentrations as calculated based on these growth curves. Although the two curves have the same general shape as those determined using the growth curve MICs, the calculated inbitory concentrations are somewhat different. The single timepoint data’s IC<sub>50</sub> values suggest that linuron is more toxic that 3,4-DCA, which disagrees with the results obtained using growth curves and literature toxicity data (Tixier, 2002; Santos, 2014). This highlights the shortfalls of determining toxicity from Abs<sub>600</sub> at a single timepoint and the importance of using growth curves to evaluate a toxin’s overall effect on an organism’s growth rate. </p>
 +
              <h2 id="single-timepoint-experiment-with-e-coli-empty-vector-and-constructs">Single-timepoint experiment with <em>E. coli</em> empty vector and constructs</h2>
 +
              <p>We also chose to evaluate the linuron and 3,4-DCA tolerance of our engineered DH5α strains. We performed single-timepoint MICs after 7 hours incubation at 30 C using empty vector pRJPaph-GFP and two constructs, assigned the numerical values of 14 and 21. These constructs had been vertified by colony PCR, sequencing, and GFP fluorescence reading. Each single-timepoint MIC was completed in triplicate, using LB supplemented with tetracycline to promote vector retention. Figures 3 and 4 show plots of Abs<sub>600</sub> versus log toxin concentration for each.</p>
 +
              <p><img src="https://static.igem.org/mediawiki/2019/c/c9/T--Waterloo--LinEc.png" alt="LinEc"></p>
 +
              <p><em>Figure 3</em>. Abs<sub>600</sub> versus linuron concentration for 3 engineered DH5α strains at 7 hours. “EV” is pRJPaph-GFP and “14” and “21” each contain the construct with LibA and NAT1. Datapoints are averages of triplicates.</p>
 +
              <p><img src="https://static.igem.org/mediawiki/2019/a/a4/T--Waterloo--DCAEc.png" alt="DCAEc"></p>
 +
              <p>Figure 4. Abs<sub>600</sub> versus 3,4-DCA concentration for 3 engineered DH5α strains at 7 hours. “EV” is pRJPaph-GFP and “14” and “21” each contain the construct with LibA and NAT1. Datapoints are averages of triplicates.</p>
 +
              <p>Linuron (figure 3) does not show any significant inhibition, even at the highest linuron concentrations. This means that it is not possible to compute parameters such as IC<sub>50</sub>, so sigmoidal fitting was not applied. Since higher linuron concentrations would not be possible due to solubility constraints, this type of assay is a poor method for evaluating LibA and NAT1 activity in <em>E. coli</em>. Additionally, there are no significant differences between the Abs<sub>600</sub> values of the different strains, suggesting that the construct and empty vector do not cause measurable differences in growth rate. </p>
 +
              <p>3,4-DCA (figure 4) shows nearly equal inhibition of all three strains beginning at 226 µM 3,4-DCA. However, since the lower “minimal growth rate” region is absent, sigmoidal fitting was not applied, so it is not possible to calculate paramters such as IC<sub>50</sub>. As with linuron, obtained data at higher toxin concentrations would be challenging due to solubility constraints. Additionally, there are no significant differences between the Abs<sub>600</sub> values of the different strains at any toxin concentration, suggesting that the construct and empty vector do not cause differences in growth.</p>
 +
              <p>These experiments therefore demonstrate that the construct does not lend linuron or 3,4-DCA tolerance to <em>E. coli</em> DH5α. However, the lack of difference in growth rate provides some evidence that LibA and NAT1 do not impose a significant metabolic burden on DH5α when used in this expression system.</p>
 +
              <h1 id="references">References</h1>
 +
              <p>Santos, S., Romeu, V., Maria, F., Sharon, C., António, M., Joaqium, V., &amp; Amália, J. (2014). Toxicity of the herbicide linuron as assessed by bacterial and mitochondrial model systems. Toxicology in Vitro, 28 doi:10.1016/j.tiv.2014.04.004</p>
 +
              <p>Tixier, C., Sancelme, M., Aït-Aïssa, W., P., Bonneymoy, F., Cuer, A., Fruffaut, N., &amp; Veschambre, H. (2002). Biotransformation of phenylurea herbicides by a soil bacterial strain, arthrobacter sp. N2: Structure, ecotoxicity and fate of diuron metabolite with soil fungi. Chemosphere, 46, 519-526. doi:10.1016/S0045-6535(01)00193-X</p>
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Latest revision as of 02:30, 22 October 2019

iGEM UWaterloo 2019 - Results

Results

Cloning

Linuron construct

Linuron

In this construct, the insert is was cloned into the BglII site between the Paph promoter and bjGFP sequence on the pRJPaph-bjGFP to give a transcriptional fusion with the linuron degradation genes. The insert itself was made by inserting the BBa_B0034 ribosome binding site ahead of each the libA and nat1 genes through PCR. These were then inserted downstream of the Paph promoter in the order of libA then nat1 by using Gibson Assembly/NEBuild. The coding sequences for the libA and nat1 genes were codon optimised for B. diazoefficiens USDA 110.5.

We successfully assembled this construct! It was validated by colony PCR (amplifying inserted genes) and sequencing. We also confirmed operon expression by measuring GFP fluorescence.

Glyphosate construct

glyphosate

Similar to the construction of our plasmid for resistance to linuron, we planned to insert the BBa_B0034 ribosome binding site and coding sequence of the CP4 EPSP synthase gene into the BglII site present between the Paph promoter and bjGFP coding sequence on the pRJPaph-bjGFP plasmid to give a transcriptional fusion of our EPSP gene with the bjGFP. The coding sequence of the CP4 EPSP synthase gene used was derived from the amino acid sequence claimed in the Monsanto patent, reverse translated to be codon optimised for B. diazoefficiens USDA 110.5 Attempts to assemble this construct through NEBuilder were not successful as the no conditions could be found to amplify the coding sequence with required overhangs and ribosome binding site by PCR. This likely was due to the high GC content of the coding sequence (71.2%), however the lengths of the primers could have also contributed to the difficulty. Unfortunately, we decided to drop the glyphosate part of the project and only focus on Linuron due to time constraints.

MIC assay results

Growth curve experiment with wild-type B. diazoefficiens

Although we were unable to evaluate the tolerance of engineered B. diazoefficiens strains due to time constraints, we nonetheless investigated the tolerance of wild-type B. diazoefficiens to linuron and 3,4-Dichloroaniline (3,4-DCA). This was evaluated using growth curve Minimum Inhibitory Concentration (MIC) experiments performed with hourly absorbance readings from 20 to 25 hours of incubation at 30 °C. The absorbances at the five timepoints were plotted versus time and fit with exponential curves of the form y = ae^(kx) + b, where k is growth rate. Sextuplicates were used for each toxin and concentration.

The results of these experiments are presented in figures 5, 6, and 7.

LinBd2

Figure 5. Growth rate versus linuron concentration from 20 to 25 hours. Error bars represent standard deviations of sextuplicates. The growth rate with no toxin was 0.08639 +/- 0.01232 1/h. The sigmoidal fit parameters A, B, C, and D are 3.23E-03, 3.46E-02, 2.96, and 2.30E+01 respectively.

DCABd2

Figure 6. Growth rate versus linuron concentration from 20 to 25 hours. Error bars represent standard deviations of sextuplicates. The growth rate with no toxin was 0.08639 +/- 0.01232 1/h. The sigmoidal fit parameters A, B, C, and D are 7.43E-03, 3.62E-02, 8.32E-01, and 2.08E+01 respectively.

Figure 7. Summary of key inhibitory concentrations for linuron and 3,4-DCA with B. diazoefficiens based on growth curves from 20 to 25 hours.

T1

These differences in toxicity profiles are demonstrated by the key inhibitory concentrations in figure 7. Linuron’s IC50 value is higher than that of 3,4-DCA, demonstrating that linuron is generally less toxic than 3,4-DCA. This agrees with reported toxicity values (Tixier, 2002) (Santos, 2014). Comparing IC10 values shows that 3,4-DCA starts showing toxic effects at a much lower concentration than linuron, due to steeper slope of the plot for linuron. The steeper slope of linuron’s plot also means that linuron’s IC90 is much lower than that of 3,4-DCA. This means that full growth inhibition actually requires a lower concentration of linuron than of 3,4-DCA, despite linuron’s lower IC50. These differences highlight the importance of examining the entire toxicity profile and various inhibitory concentrations, since these convey much more information that the IC50 value alone.

We have therefore experimentally deteremined the toxicity profiles of both linuron and 3,4-DCA toward wild type B. diazoefficiens, validating our experimental approach. This information provides also a reference point against which to compare engineered strains, other toxins, or bacterial species.

Single-timepoint experiment with wild-type B. diazoefficiens

In addition to the growth curve experiment peformed with B. diazoefficiens both in linuron and 3,4-DCA, we also completed this experiment in single-time point format. This was done using one Abs600 after 22 hours incubation at 30 C. Figures 8 and 9 show plots of Abs600 versus toxin concentration. These results are shown in figures 8, 9, and 10. Fitting was performed to the four parameter in equation 1.

LinBd1

Figure 8. Abs600 versus linuron concentration at 22 hours. Error bars represent standard deviations of sextuplicates. The sigmoidal fit parameters A, B, C, and D are 1.28E-02, 5.52E-02, 1.62, and 1.28E+01 respectively.

DCABd1

Figure 9. Abs600 versus linuron concentration at 22 hours. Error bars represent standard deviations of sextuplicates. The sigmoidal fit parameters A, B, C, and D are 1.50E-02, 5.47E-02, 8.97E-01, and 3.08E+01 respectively.

Figure 10. Summary of key inhibitory concentrations for linuron and 3,4-DCA with B. diazoefficiens after 22 hours.

T2

Figures 8 and 9 both show the characteristic sigmoidal shapes expected for MIC experiments. All datapoints are very close to the sigmoidal fit, indicating that this model is a good representation of the data.

Figure 10 summarizes some key inhibitory concentrations as calculated based on these growth curves. Although the two curves have the same general shape as those determined using the growth curve MICs, the calculated inbitory concentrations are somewhat different. The single timepoint data’s IC50 values suggest that linuron is more toxic that 3,4-DCA, which disagrees with the results obtained using growth curves and literature toxicity data (Tixier, 2002; Santos, 2014). This highlights the shortfalls of determining toxicity from Abs600 at a single timepoint and the importance of using growth curves to evaluate a toxin’s overall effect on an organism’s growth rate.

Single-timepoint experiment with E. coli empty vector and constructs

We also chose to evaluate the linuron and 3,4-DCA tolerance of our engineered DH5α strains. We performed single-timepoint MICs after 7 hours incubation at 30 C using empty vector pRJPaph-GFP and two constructs, assigned the numerical values of 14 and 21. These constructs had been vertified by colony PCR, sequencing, and GFP fluorescence reading. Each single-timepoint MIC was completed in triplicate, using LB supplemented with tetracycline to promote vector retention. Figures 3 and 4 show plots of Abs600 versus log toxin concentration for each.

LinEc

Figure 3. Abs600 versus linuron concentration for 3 engineered DH5α strains at 7 hours. “EV” is pRJPaph-GFP and “14” and “21” each contain the construct with LibA and NAT1. Datapoints are averages of triplicates.

DCAEc

Figure 4. Abs600 versus 3,4-DCA concentration for 3 engineered DH5α strains at 7 hours. “EV” is pRJPaph-GFP and “14” and “21” each contain the construct with LibA and NAT1. Datapoints are averages of triplicates.

Linuron (figure 3) does not show any significant inhibition, even at the highest linuron concentrations. This means that it is not possible to compute parameters such as IC50, so sigmoidal fitting was not applied. Since higher linuron concentrations would not be possible due to solubility constraints, this type of assay is a poor method for evaluating LibA and NAT1 activity in E. coli. Additionally, there are no significant differences between the Abs600 values of the different strains, suggesting that the construct and empty vector do not cause measurable differences in growth rate.

3,4-DCA (figure 4) shows nearly equal inhibition of all three strains beginning at 226 µM 3,4-DCA. However, since the lower “minimal growth rate” region is absent, sigmoidal fitting was not applied, so it is not possible to calculate paramters such as IC50. As with linuron, obtained data at higher toxin concentrations would be challenging due to solubility constraints. Additionally, there are no significant differences between the Abs600 values of the different strains at any toxin concentration, suggesting that the construct and empty vector do not cause differences in growth.

These experiments therefore demonstrate that the construct does not lend linuron or 3,4-DCA tolerance to E. coli DH5α. However, the lack of difference in growth rate provides some evidence that LibA and NAT1 do not impose a significant metabolic burden on DH5α when used in this expression system.

References

Santos, S., Romeu, V., Maria, F., Sharon, C., António, M., Joaqium, V., & Amália, J. (2014). Toxicity of the herbicide linuron as assessed by bacterial and mitochondrial model systems. Toxicology in Vitro, 28 doi:10.1016/j.tiv.2014.04.004

Tixier, C., Sancelme, M., Aït-Aïssa, W., P., Bonneymoy, F., Cuer, A., Fruffaut, N., & Veschambre, H. (2002). Biotransformation of phenylurea herbicides by a soil bacterial strain, arthrobacter sp. N2: Structure, ecotoxicity and fate of diuron metabolite with soil fungi. Chemosphere, 46, 519-526. doi:10.1016/S0045-6535(01)00193-X