Team:AUC TURKEY/Results/Cloning

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  <w:LsdException Locked="false" Priority="51"
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  <w:LsdException Locked="false" Priority="52"
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h2
h2
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mso-style-unhide:no;
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margin-bottom:0cm;
Line 78: Line 695:
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font-family:"Trebuchet MS","sans-serif";
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mso-fareast-theme-font:minor-fareast;
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color:black;}
h3
h3
-
{margin-top:8.0pt;
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{mso-style-priority:9;
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mso-style-unhide:no;
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mso-style-qformat:yes;
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mso-style-link:"Başlık 3 Char";
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margin-top:8.0pt;
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margin-right:0cm;
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margin-bottom:0cm;
Line 116: Line 715:
margin-bottom:.0001pt;
margin-bottom:.0001pt;
line-height:115%;
line-height:115%;
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mso-pagination:widow-orphan;
page-break-after:avoid;
page-break-after:avoid;
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font-size:12.0pt;
font-family:"Trebuchet MS","sans-serif";
font-family:"Trebuchet MS","sans-serif";
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color:#666666;}
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mso-fareast-font-family:"Times New Roman";
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h3.CxSpFirst
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mso-fareast-theme-font:minor-fareast;
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color:#666666;}
h4
h4
-
{margin-top:8.0pt;
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{mso-style-priority:9;
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mso-style-unhide:no;
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mso-style-qformat:yes;
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mso-style-link:"Başlık 4 Char";
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margin-top:8.0pt;
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margin-bottom:0cm;
Line 154: Line 735:
margin-bottom:.0001pt;
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line-height:115%;
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mso-pagination:widow-orphan;
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page-break-after:avoid;
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font-size:11.0pt;
font-family:"Trebuchet MS","sans-serif";
font-family:"Trebuchet MS","sans-serif";
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mso-fareast-font-family:"Times New Roman";
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mso-fareast-theme-font:minor-fareast;
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mso-bidi-font-family:"Times New Roman";
color:#666666;
color:#666666;
font-weight:normal;
font-weight:normal;
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h4.CxSpFirst
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{margin-top:8.0pt;
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h5
h5
-
{margin-top:8.0pt;
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{mso-style-priority:9;
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mso-style-unhide:no;
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mso-style-qformat:yes;
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mso-style-link:"Başlık 5 Char";
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margin-top:8.0pt;
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margin-right:0cm;
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margin-bottom:0cm;
Line 200: Line 758:
margin-bottom:.0001pt;
margin-bottom:.0001pt;
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line-height:115%;
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mso-pagination:widow-orphan;
page-break-after:avoid;
page-break-after:avoid;
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font-size:11.0pt;
font-family:"Trebuchet MS","sans-serif";
font-family:"Trebuchet MS","sans-serif";
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color:#666666;
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mso-fareast-theme-font:minor-fareast;
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h5.CxSpFirst
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mso-bidi-font-family:"Times New Roman";
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font-weight:normal;}
h6
h6
-
{margin-top:8.0pt;
+
{mso-style-priority:9;
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mso-style-unhide:no;
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mso-style-qformat:yes;
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mso-style-link:"Başlık 6 Char";
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margin-top:8.0pt;
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margin-right:0cm;
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margin-bottom:0cm;
Line 242: Line 779:
margin-bottom:.0001pt;
margin-bottom:.0001pt;
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line-height:115%;
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mso-pagination:widow-orphan;
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page-break-after:avoid;
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font-size:11.0pt;
font-family:"Trebuchet MS","sans-serif";
font-family:"Trebuchet MS","sans-serif";
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mso-fareast-font-family:"Times New Roman";
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mso-fareast-theme-font:minor-fareast;
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mso-bidi-font-family:"Times New Roman";
color:#666666;
color:#666666;
font-weight:normal;
font-weight:normal;
font-style:italic;}
font-style:italic;}
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h6.CxSpFirst
+
p.MsoTitle, li.MsoTitle, div.MsoTitle
-
{margin-top:8.0pt;
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{mso-style-priority:10;
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margin-right:0cm;
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mso-style-unhide:no;
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margin-bottom:0cm;
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mso-style-qformat:yes;
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margin-left:0cm;
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mso-style-link:"Konu Başlığı Char";
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margin:0cm;
margin-bottom:.0001pt;
margin-bottom:.0001pt;
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line-height:115%;
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color:black;}
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p.MsoSubtitle, li.MsoSubtitle, div.MsoSubtitle
-
margin-bottom:.0001pt;
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{mso-style-priority:11;
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mso-style-unhide:no;
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mso-style-qformat:yes;
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mso-style-link:"Altyazı Char";
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margin-top:0cm;
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margin-right:0cm;
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margin-bottom:10.0pt;
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line-height:115%;
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page-break-after:avoid;
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color:#666666;
color:#666666;
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font-style:italic;}
font-style:italic;}
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h6.CxSpLast
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span.Balk1Char
-
{margin:0cm;
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{mso-style-name:"Başlık 1 Char";
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margin-bottom:.0001pt;
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mso-style-priority:9;
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mso-ascii-font-family:"Calibri Light";
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mso-fareast-font-family:"Times New Roman";
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mso-fareast-theme-font:major-fareast;
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mso-hansi-font-family:"Calibri Light";
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mso-hansi-theme-font:major-latin;
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mso-bidi-font-family:"Times New Roman";
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mso-bidi-theme-font:major-bidi;
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color:#2E74B5;
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mso-themecolor:accent1;
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mso-themeshade:191;}
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span.Balk2Char
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{mso-style-name:"Başlık 2 Char";
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mso-style-priority:9;
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mso-fareast-theme-font:major-fareast;
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mso-bidi-font-family:"Times New Roman";
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mso-bidi-theme-font:major-bidi;
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color:#2E74B5;
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mso-themecolor:accent1;
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mso-themeshade:191;}
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span.Balk3Char
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{mso-style-name:"Başlık 3 Char";
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mso-style-noshow:yes;
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mso-style-priority:9;
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mso-style-unhide:no;
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mso-style-locked:yes;
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mso-bidi-font-size:12.0pt;
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mso-fareast-theme-font:major-fareast;
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mso-hansi-font-family:"Calibri Light";
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mso-hansi-theme-font:major-latin;
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mso-bidi-font-family:"Times New Roman";
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mso-bidi-theme-font:major-bidi;
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color:#1F4D78;
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mso-themecolor:accent1;
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mso-themeshade:127;}
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span.Balk4Char
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{mso-style-name:"Başlık 4 Char";
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mso-style-noshow:yes;
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mso-style-priority:9;
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mso-style-unhide:no;
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mso-bidi-font-size:11.0pt;
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mso-bidi-theme-font:major-bidi;
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mso-themecolor:accent1;
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mso-themeshade:191;
font-style:italic;}
font-style:italic;}
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p.MsoTitle, li.MsoTitle, div.MsoTitle
+
span.Balk5Char
-
{margin:0cm;
+
{mso-style-name:"Başlık 5 Char";
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mso-style-noshow:yes;
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mso-style-priority:9;
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mso-bidi-font-size:11.0pt;
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mso-fareast-theme-font:major-fareast;
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mso-hansi-theme-font:major-latin;
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mso-bidi-font-family:"Times New Roman";
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mso-bidi-theme-font:major-bidi;
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color:#2E74B5;
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mso-themecolor:accent1;
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mso-themeshade:191;}
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span.Balk6Char
 +
{mso-style-name:"Başlık 6 Char";
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mso-style-noshow:yes;
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mso-style-priority:9;
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mso-style-unhide:no;
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mso-style-locked:yes;
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mso-style-link:"Başlık 6";
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mso-bidi-font-size:11.0pt;
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mso-ascii-font-family:"Calibri Light";
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mso-fareast-theme-font:major-fareast;
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mso-hansi-font-family:"Calibri Light";
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mso-hansi-theme-font:major-latin;
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mso-bidi-font-family:"Times New Roman";
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mso-bidi-theme-font:major-bidi;
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color:#1F4D78;
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mso-themecolor:accent1;
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mso-themeshade:127;}
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span.KonuBalChar
 +
{mso-style-name:"Konu Başlığı Char";
 +
mso-style-priority:10;
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mso-style-unhide:no;
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mso-style-locked:yes;
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mso-style-link:"Konu Başlığı";
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mso-bidi-font-size:28.0pt;
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mso-ascii-theme-font:major-latin;
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mso-fareast-font-family:"Times New Roman";
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mso-fareast-theme-font:major-fareast;
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mso-hansi-font-family:"Calibri Light";
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mso-hansi-theme-font:major-latin;
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mso-bidi-theme-font:major-bidi;
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mso-font-kerning:14.0pt;}
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p.msotitlecxspfirst, li.msotitlecxspfirst, div.msotitlecxspfirst
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{mso-style-name:msotitlecxspfirst;
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mso-style-unhide:no;
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margin-bottom:.0001pt;
margin-bottom:.0001pt;
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line-height:115%;
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page-break-after:avoid;
page-break-after:avoid;
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font-size:21.0pt;
font-family:"Trebuchet MS","sans-serif";
font-family:"Trebuchet MS","sans-serif";
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mso-fareast-font-family:"Times New Roman";
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mso-fareast-theme-font:minor-fareast;
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mso-bidi-font-family:"Times New Roman";
color:black;}
color:black;}
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p.MsoTitleCxSpFirst, li.MsoTitleCxSpFirst, div.MsoTitleCxSpFirst
+
p.msotitlecxspmiddle, li.msotitlecxspmiddle, div.msotitlecxspmiddle
-
{margin:0cm;
+
{mso-style-name:msotitlecxspmiddle;
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mso-style-unhide:no;
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margin-bottom:.0001pt;
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line-height:115%;
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font-size:21.0pt;
font-family:"Trebuchet MS","sans-serif";
font-family:"Trebuchet MS","sans-serif";
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mso-fareast-font-family:"Times New Roman";
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Line 383: Line 1,179:
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>&nbsp;</span></p>
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>&nbsp;</span></p>
-
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>Plasmids
+
<p class=MsoNormal style='text-align:justify'><span class=SpellE><span
-
containing the BBa_K1291071 (Horseradish peroxidase composite part) and
+
style='font-family:"Calibri","sans-serif"'>Plasmids</span></span><span
-
BBa_K1291299 (Lignin Peroxidase composite part) parts were transferred into our
+
style='font-family:"Calibri","sans-serif"'> <span class=SpellE>containing</span>
-
T10 strains, which were prepared through the competent cell preparation
+
<span class=SpellE>the</span> BBa_K1291071 (<span class=SpellE>Horseradish</span>
-
protocol, using transformation protocols. Colonies were seen in agar plates
+
<span class=SpellE>peroxidase</span> <span class=SpellE>composite</span> <span
-
that had the appropriate antibiotics after a 16 hour long incubation process. </span></p>
+
class=SpellE>part</span>) <span class=SpellE>and</span> BBa_K1291299 (Lignin <span
 +
class=SpellE>Peroxidase</span> <span class=SpellE>composite</span> <span
 +
class=SpellE>part</span>) <span class=SpellE>parts</span> <span class=SpellE>were</span>
 +
<span class=SpellE>transferred</span> <span class=SpellE>into</span> <span
 +
class=SpellE>our</span> T10 <span class=SpellE>strains</span>, <span
 +
class=SpellE>which</span> <span class=SpellE>were</span> <span class=SpellE>prepared</span>
 +
<span class=SpellE>through</span> <span class=SpellE>the</span> <span
 +
class=SpellE>competent</span> <span class=SpellE>cell</span> <span
 +
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 +
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 +
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 +
class=SpellE>were</span> <span class=SpellE>seen</span> in <span class=SpellE>agar</span>
 +
<span class=SpellE>plates</span> <span class=SpellE>that</span> had <span
 +
class=SpellE>the</span> <span class=SpellE>appropriate</span> <span
 +
class=SpellE>antibiotics</span> <span class=SpellE>after</span> a 16 <span
 +
class=SpellE>hour</span> <span class=SpellE>long</span> <span class=SpellE>incubation</span>
 +
<span class=SpellE>process</span>. </span></p>
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>&nbsp;</span></p>
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>&nbsp;</span></p>
Line 397: Line 1,209:
line-height:115%;font-family:"Calibri","sans-serif"'>PLASMID ISOLATION</span></p>
line-height:115%;font-family:"Calibri","sans-serif"'>PLASMID ISOLATION</span></p>
-
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'> The
+
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>&nbsp;<span
-
colonies taken from plates were incubated in LB Broth containing AMP for 16
+
class=SpellE>The</span> <span class=SpellE>colonies</span> <span class=SpellE>taken</span>
-
hours. DNA isolation was done on the bacteria culture to check the efficiency.
+
<span class=SpellE>from</span> <span class=SpellE>plates</span> <span
-
The adequacy of the amount of the nucleic acid concentration for digestion was
+
class=SpellE>were</span> <span class=SpellE>incubated</span> in LB <span
-
met in the acquired samples and it was seen that the samples had peaks at OD
+
class=SpellE>Broth</span> <span class=SpellE>containing</span> AMP <span
-
260, which was captured by the nanodrop.</span><span style='position:relative;
+
class=SpellE>for</span> 16 <span class=SpellE>hours</span>. DNA <span
-
z-index:251658240;left:-20px;top:0px;width:586px;height:295px'>
+
class=SpellE>isolation</span> <span class=SpellE>was</span> <span class=GramE>done</span>
 +
on <span class=SpellE>the</span> <span class=SpellE>bacteria</span> <span
 +
class=SpellE>culture</span> <span class=SpellE>to</span> <span class=SpellE>check</span>
 +
<span class=SpellE>the</span> <span class=SpellE>efficiency</span>. <span
 +
class=SpellE>The</span> <span class=SpellE>adequacy</span> of <span
 +
class=SpellE>the</span> <span class=SpellE>amount</span> of <span class=SpellE>the</span>
 +
<span class=SpellE>nucleic</span> <span class=SpellE>acid</span> <span
 +
class=SpellE>concentration</span> <span class=SpellE>for</span> <span
 +
class=SpellE>digestion</span> <span class=SpellE>was</span> met in <span
 +
class=SpellE>the</span> <span class=SpellE>acquired</span> <span class=SpellE>samples</span>
 +
<span class=SpellE>and</span> it <span class=SpellE>was</span> <span
 +
class=SpellE>seen</span> <span class=SpellE>that</span> <span class=SpellE>the</span>
 +
<span class=SpellE>samples</span> had <span class=SpellE>peaks</span> at OD
 +
260, <span class=SpellE>which</span> <span class=SpellE>was</span> <span
 +
class=SpellE>captured</span> <span class=SpellE>by</span> <span class=SpellE>the</span>
 +
<span class=SpellE>nanodrop</span>.</span><span style='left:-20px;position:
 +
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height=486 src="https://static.igem.org/mediawiki/2014hs/f/f4/Cimage003.jpg"></span><br
+
type="#_x0000_t75" alt="https://static.igem.org/mediawiki/2014hs/f/f4/Cimage003.jpg"
-
clear=ALL>
+
style='position:absolute;left:0;text-align:left;margin-left:0;margin-top:0;
 +
width:570.75pt;height:364.5pt;z-index:-251656192;visibility:visible;
 +
mso-wrap-style:square;mso-wrap-distance-left:9pt;mso-wrap-distance-top:0;
 +
mso-wrap-distance-right:9pt;mso-wrap-distance-bottom:0;
 +
mso-position-horizontal:absolute;mso-position-horizontal-relative:text;
 +
mso-position-vertical:absolute;mso-position-vertical-relative:text'>
 +
<v:imagedata src="https://static.igem.org/mediawiki/2014hs/f/f4/Cimage003.jpg"/>
 +
<w:wrap type="tight"/>
 +
</v:shape><![endif]--><![if !vml]><img width=761 height=486
 +
src="https://static.igem.org/mediawiki/2014hs/f/f4/Cimage003.jpg" align=left
 +
hspace=12 alt="https://static.igem.org/mediawiki/2014hs/f/f4/Cimage003.jpg" v:shapes="_x0000_s1031"><![endif]><br
 +
clear=all style='mso-special-character:line-break'>
</p>
</p>
-
<p class=MsoNormal style='text-align:justify'><img width=300 height=372
+
<p class=MsoNormal style='text-align:justify'><!--[if gte vml 1]><v:shape id="Resim_x0020_2"
-
src="https://static.igem.org/mediawiki/2014hs/9/98/Cimage004.jpg" align=left hspace=12
+
o:spid="_x0000_s1030" type="#_x0000_t75" alt="https://static.igem.org/mediawiki/2014hs/9/98/Cimage004.jpg"
-
vspace=12></p>
+
style='position:absolute;left:0;text-align:left;margin-left:0;margin-top:0;
 +
width:225pt;height:279pt;z-index:-251658240;visibility:visible;
 +
mso-wrap-style:square;mso-width-percent:0;mso-height-percent:0;
 +
mso-wrap-distance-left:9pt;mso-wrap-distance-top:9pt;
 +
mso-wrap-distance-right:9pt;mso-wrap-distance-bottom:9pt;
 +
mso-position-horizontal:left;mso-position-horizontal-relative:text;
 +
mso-position-vertical:absolute;mso-position-vertical-relative:line;
 +
mso-width-percent:0;mso-height-percent:0;mso-width-relative:page;
 +
mso-height-relative:page' o:allowoverlap="f">
 +
<v:imagedata src="https://static.igem.org/mediawiki/2014hs/9/98/Cimage004.jpg"/>
 +
<w:wrap type="tight" anchory="line"/>
 +
</v:shape><![endif]--><![if !vml]><img width=300 height=372
 +
src="https://static.igem.org/mediawiki/2014hs/9/98/Cimage004.jpg" align=left
 +
hspace=12 vspace=12 alt="https://static.igem.org/mediawiki/2014hs/9/98/Cimage004.jpg"
 +
v:shapes="Resim_x0020_2"><![endif]><span style='font-family:"Calibri","sans-serif"'>&nbsp;</span></p>
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>&nbsp;</span></p>
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>&nbsp;</span></p>
-
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>&nbsp;</span></p>
+
<p class=MsoNormal style='text-align:justify'><span class=SpellE><span
-
 
+
style='font-size:16.0pt;line-height:115%;font-family:"Calibri","sans-serif"'>Digestion</span></span><span
-
<p class=MsoNormal style='text-align:justify'><span style='font-size:16.0pt;
+
style='font-size:16.0pt;line-height:115%;font-family:"Calibri","sans-serif"'> <span
-
line-height:115%;font-family:"Calibri","sans-serif"'>Digestion and DNA
+
class=SpellE>and</span> DNA <span class=SpellE>Electrophoresis</span></span></p>
-
Electrophoresis</span></p>
+
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>&nbsp;</span></p>
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>&nbsp;</span></p>
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>Gel
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>Gel
-
electrophoresis was conducted on the samples so that the enzymes which were
+
<span class=SpellE>electrophoresis</span> <span class=SpellE>was</span> <span
-
digested with the EcoRI and PstI could be verified in terms of their
+
class=SpellE>conducted</span> on <span class=SpellE>the</span> <span
-
wavelength. The acquired results showed that the plasmid transfer process was
+
class=SpellE>samples</span> <span class=SpellE>so</span> <span class=SpellE>that</span>
-
successful for the Top10 strains while the NEB10 strains, which were taken from
+
<span class=SpellE>the</span> <span class=SpellE>enzymes</span> <span
-
the stock, were disfunctional thus were unusable.</span></p>
+
class=SpellE>which</span> <span class=SpellE>were</span> <span class=SpellE>digested</span>
 +
<span class=SpellE>with</span> <span class=SpellE>the</span> <span
 +
class=SpellE>EcoRI</span> <span class=SpellE>and</span> <span class=SpellE>PstI</span>
 +
<span class=SpellE>could</span> be <span class=SpellE>verified</span> in <span
 +
class=SpellE>terms</span> of <span class=SpellE>their</span> <span
 +
class=SpellE>wavelength</span>. <span class=SpellE>The</span> <span
 +
class=SpellE>acquired</span> <span class=SpellE>results</span> <span
 +
class=SpellE>showed</span> <span class=SpellE>that</span> <span class=SpellE>the</span>
 +
<span class=SpellE>plasmid</span> transfer <span class=SpellE>process</span> <span
 +
class=SpellE>was</span> <span class=SpellE>successful</span> <span
 +
class=SpellE>for</span> <span class=SpellE>the</span> Top10 <span class=SpellE>strains</span>
 +
<span class=SpellE>while</span> <span class=SpellE>the</span> NEB10 <span
 +
class=SpellE>strains</span>, <span class=SpellE>which</span> <span
 +
class=SpellE>were</span> <span class=SpellE>taken</span> <span class=SpellE>from</span>
 +
<span class=SpellE>the</span> <span class=SpellE>stock</span>, <span
 +
class=SpellE>were</span> <span class=SpellE>disfunctional</span> <span
 +
class=SpellE>thus</span> <span class=SpellE>were</span> <span class=SpellE>unusable</span>.</span></p>
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>&nbsp;</span></p>
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>&nbsp;</span></p>
Line 467: Line 1,387:
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>Since
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>Since
-
we require a high concentration of proteins in our project, we decided to use the
+
<span class=SpellE>we</span> <span class=SpellE>require</span> a <span
-
BL-21 strain of E.coli instead of Top10 strains due to its enhanced protein
+
class=SpellE>high</span> <span class=SpellE>concentration</span> of <span
-
synthesis capacity. Plasmids containing the BBa_K1291071 (Horseradish
+
class=SpellE>proteins</span> in <span class=SpellE>our</span> <span
-
peroxidase composite part) and BBa_K1291299 (Lignin Peroxidase composite part)
+
class=SpellE>project</span>, <span class=SpellE>we</span> <span class=SpellE>decided</span>
-
parts were transferred into our BL-21 strains, which were prepared through the
+
<span class=SpellE>to</span> <span class=SpellE>use</span> <span class=SpellE>the</span>
-
competent cell preparation protocol, using the transformation protocol. The
+
BL-21 <span class=SpellE>strain</span> of <span class=SpellE>E.coli</span> <span
-
colonies successfully acquired after a 16-hour long incubation process.</span></p>
+
class=SpellE>instead</span> of Top10 <span class=SpellE>strains</span> <span
 +
class=SpellE>due</span> <span class=SpellE>to</span> <span class=SpellE>its</span>
 +
<span class=SpellE>enhanced</span> protein <span class=SpellE>synthesis</span> <span
 +
class=SpellE>capacity</span>. <span class=SpellE>Plasmids</span> <span
 +
class=SpellE>containing</span> <span class=SpellE>the</span> BBa_K1291071 (<span
 +
class=SpellE>Horseradish</span> <span class=SpellE>peroxidase</span> <span
 +
class=SpellE>composite</span> <span class=SpellE>part</span>) <span
 +
class=SpellE>and</span> BBa_K1291299 (Lignin <span class=SpellE>Peroxidase</span>
 +
<span class=SpellE>composite</span> <span class=SpellE>part</span>) <span
 +
class=SpellE>parts</span> <span class=SpellE>were</span> <span class=SpellE>transferred</span>
 +
<span class=SpellE>into</span> <span class=SpellE>our</span> BL-21 <span
 +
class=SpellE>strains</span>, <span class=SpellE>which</span> <span
 +
class=SpellE>were</span> <span class=SpellE>prepared</span> <span class=SpellE>through</span>
 +
<span class=SpellE>the</span> <span class=SpellE>competent</span> <span
 +
class=SpellE>cell</span> <span class=SpellE>preparation</span> <span
 +
class=SpellE>protocol</span>, <span class=SpellE>using</span> <span
 +
class=SpellE>the</span> <span class=SpellE>transformation</span> <span
 +
class=SpellE>protocol</span>. <span class=SpellE>The</span> <span class=SpellE>colonies</span>
 +
<span class=SpellE>successfully</span> <span class=SpellE>acquired</span> <span
 +
class=SpellE>after</span> a 16-hour <span class=SpellE>long</span> <span
 +
class=SpellE>incubation</span> <span class=SpellE>process</span>.</span></p>
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>&nbsp;</span></p>
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>&nbsp;</span></p>
Line 481: Line 1,421:
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>&nbsp;</span></p>
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>&nbsp;</span></p>
-
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>PLASMID
+
<p class=MsoNormal style='text-align:justify'><span style='font-size:16.0pt;
-
ISOLATION</span><span style='position:relative;z-index:251662336;left:-20px;
+
mso-bidi-font-size:11.0pt;line-height:115%;font-family:"Calibri","sans-serif"'>PLASMID
-
top:0px;width:624px;height:309px'>
+
ISOLATION</span><span style='left:-20px;position:relative;top:0px;z-index:251662336'><span
 +
style='font-size:16.0pt;mso-bidi-font-size:11.0pt;line-height:115%'> <o:p></o:p></span></p>
-
<table cellpadding=0 cellspacing=0>
+
<table class=MsoNormalTable border=0 cellspacing=0 cellpadding=0
-
  <tr>
+
style='mso-cellspacing:0cm;mso-yfti-tbllook:1184;mso-padding-alt:0cm 0cm 0cm 0cm'>
-
   <td width=0 height=0></td>
+
  <tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'>
-
   <td width=309></td>
+
   <td width=0 style='width:.3pt;padding:0cm 0cm 0cm 0cm'></td>
-
   <td width=34></td>
+
   <td width=309 style='width:231.75pt;padding:0cm 0cm 0cm 0cm'></td>
-
   <td width=281></td>
+
   <td width=34 style='width:25.5pt;padding:0cm 0cm 0cm 0cm'></td>
 +
   <td width=281 style='width:210.75pt;padding:0cm 0cm 0cm 0cm'></td>
  </tr>
  </tr>
-
  <tr>
+
  <tr style='mso-yfti-irow:1;height:230.25pt'>
-
   <td height=307></td>
+
   <td style='padding:0cm 0cm 0cm 0cm;height:230.25pt'></td>
-
   <td rowspan=2 align=left valign=top><img width=309 height=309
+
   <td rowspan=2 valign=top style='padding:0cm 0cm 0cm 0cm;height:230.25pt'>
-
   src="https://static.igem.org/mediawiki/2014hs/1/1b/Cimage005.jpg"></td>
+
  <p class=MsoNormal><!--[if gte vml 1]><v:shape id="Resim_x0020_4" o:spid="_x0000_s1029"
-
   <td></td>
+
  type="#_x0000_t75" alt="https://static.igem.org/mediawiki/2014hs/1/1b/Cimage005.jpg"
-
   <td align=left valign=top><img width=281 height=307
+
  style='position:absolute;margin-left:0;margin-top:0;width:231.75pt;height:231.75pt;
-
   src="https://static.igem.org/mediawiki/2014hs/1/1f/Cimage006.jpg"></td>
+
  z-index:-251653120;visibility:visible;mso-wrap-style:square;
 +
  mso-wrap-distance-left:9pt;mso-wrap-distance-top:0;
 +
  mso-wrap-distance-right:9pt;mso-wrap-distance-bottom:0;
 +
  mso-position-horizontal:absolute;mso-position-horizontal-relative:text;
 +
  mso-position-vertical:absolute;mso-position-vertical-relative:text'>
 +
  <v:imagedata src="https://static.igem.org/mediawiki/2014hs/1/1b/Cimage005.jpg"/>
 +
  <w:wrap type="tight"/>
 +
  </v:shape><![endif]--><![if !vml]><img width=309 height=309
 +
   src="https://static.igem.org/mediawiki/2014hs/1/1b/Cimage005.jpg" align=left
 +
  hspace=12 alt="https://static.igem.org/mediawiki/2014hs/1/1b/Cimage005.jpg" v:shapes="Resim_x0020_4"><![endif]><span
 +
  style='font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif";
 +
  mso-fareast-font-family:"Times New Roman";color:windowtext'><o:p></o:p></span></p>
 +
  </td>
 +
   <td style='padding:0cm 0cm 0cm 0cm;height:230.25pt'></td>
 +
   <td valign=top style='padding:0cm 0cm 0cm 0cm;height:230.25pt'>
 +
  <p class=MsoNormal><!--[if gte vml 1]><v:shape id="Resim_x0020_5" o:spid="_x0000_s1028"
 +
  type="#_x0000_t75" alt="https://static.igem.org/mediawiki/2014hs/1/1f/Cimage006.jpg"
 +
  style='position:absolute;margin-left:0;margin-top:0;width:210.75pt;height:230.25pt;
 +
  z-index:-251652096;visibility:visible;mso-wrap-style:square;
 +
  mso-wrap-distance-left:9pt;mso-wrap-distance-top:0;
 +
  mso-wrap-distance-right:9pt;mso-wrap-distance-bottom:0;
 +
  mso-position-horizontal:absolute;mso-position-horizontal-relative:text;
 +
  mso-position-vertical:absolute;mso-position-vertical-relative:text'>
 +
  <v:imagedata src="https://static.igem.org/mediawiki/2014hs/1/1f/Cimage006.jpg"/>
 +
  <w:wrap type="tight"/>
 +
  </v:shape><![endif]--><![if !vml]><img width=281 height=307
 +
   src="https://static.igem.org/mediawiki/2014hs/1/1f/Cimage006.jpg" align=left
 +
  hspace=12 alt="https://static.igem.org/mediawiki/2014hs/1/1f/Cimage006.jpg" v:shapes="Resim_x0020_5"><![endif]><span
 +
  style='font-size:12.0pt;line-height:115%;font-family:"Times New Roman","serif";
 +
  mso-fareast-font-family:"Times New Roman";color:windowtext'><o:p></o:p></span></p>
 +
  </td>
  </tr>
  </tr>
-
  <tr>
+
  <tr style='mso-yfti-irow:2;mso-yfti-lastrow:yes;height:1.5pt'>
-
   <td height=2></td>
+
   <td style='padding:0cm 0cm 0cm 0cm;height:1.5pt'></td>
 +
  <td style='padding:0cm 0cm 0cm 0cm;height:1.5pt'></td>
 +
  <td style='padding:0cm 0cm 0cm 0cm;height:1.5pt'></td>
  </tr>
  </tr>
</table>
</table>
-
</span><br clear=ALL>
+
<p class=MsoNormal style='text-align:justify'><br clear=all style='mso-special-character:
-
</p>
+
line-break'>
 +
</span></p>
-
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>The
+
<p class=MsoNormal style='text-align:justify'><!--[if gte vml 1]><v:shape id="Resim_x0020_6"
-
chosen colonies were incubated for 16-hours in AMP containing LB Broth. DNA
+
o:spid="_x0000_s1027" type="#_x0000_t75" alt="https://static.igem.org/mediawiki/2014hs/5/55/Cimage007.gif"
-
Isolation was done to test the efficiency of the transformation. A peek at OD
+
style='position:absolute;left:0;text-align:left;margin-left:0;margin-top:1442.45pt;
-
260 was observed and sufficient levels of DNA concentration was acquired to
+
width:567.75pt;height:362.25pt;z-index:-251651072;visibility:visible;
-
continue to digestion.</span><span style='position:relative;z-index:251664384;
+
mso-wrap-style:square;mso-wrap-distance-left:9pt;mso-wrap-distance-top:0;
-
left:-78px;top:0px;width:757px;height:483px'><img width=757 height=483
+
mso-wrap-distance-right:9pt;mso-wrap-distance-bottom:0;
-
src="https://static.igem.org/mediawiki/2014hs/5/55/Cimage007.gif"></span><br clear=ALL>
+
mso-position-horizontal:left;mso-position-horizontal-relative:text;
-
</p>
+
mso-position-vertical:absolute;mso-position-vertical-relative:text'>
 +
<v:imagedata src="https://static.igem.org/mediawiki/2014hs/5/55/Cimage007.gif"/>
 +
<w:wrap type="tight"/>
 +
</v:shape><![endif]--><![if !vml]><img width=757 height=483
 +
src="https://static.igem.org/mediawiki/2014hs/5/55/Cimage007.gif" align=left
 +
hspace=12 alt="https://static.igem.org/mediawiki/2014hs/5/55/Cimage007.gif" v:shapes="Resim_x0020_6"><![endif]><span
 +
style='font-family:"Calibri","sans-serif"'><span
 +
style='mso-spacerun:yes'>                                                                                                                                                                                                          
 +
</span><span style='mso-spacerun:yes'>                     </span><span
 +
style='mso-spacerun:yes'>          </span><span class=SpellE>The</span> <span
 +
class=SpellE>chosen</span> <span class=SpellE>colonies</span> <span
 +
class=SpellE>were</span> <span class=SpellE>incubated</span> <span
 +
class=SpellE>for</span> 16-hours in AMP <span class=SpellE>containing</span> LB
 +
<span class=SpellE>Broth</span>. DNA <span class=SpellE>Isolation</span> <span
 +
class=SpellE>was</span> <span class=GramE>done</span> <span class=SpellE>to</span>
 +
test <span class=SpellE>the</span> <span class=SpellE>efficiency</span> of <span
 +
class=SpellE>the</span> <span class=SpellE>transformation</span>. A <span
 +
class=SpellE>peek</span> at OD 260 <span class=SpellE>was</span> <span
 +
class=SpellE>observed</span> <span class=SpellE>and</span> <span class=SpellE>sufficient</span>
 +
<span class=SpellE>levels</span> of DNA <span class=SpellE>concentration</span>
 +
<span class=SpellE>was</span> <span class=SpellE>acquired</span> <span
 +
class=SpellE>to</span> <span class=SpellE>continue</span> <span class=SpellE>to</span>
 +
<span class=SpellE>digestion</span>.</span><span style='left:-78px;position:
 +
relative;top:0px;z-index:251664384'> <br clear=all style='mso-special-character:
 +
line-break'>
 +
<span style='font-family:"Calibri","sans-serif"'><o:p></o:p></span></p>
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>&nbsp;</span></p>
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>&nbsp;</span></p>
-
<p class=MsoNormal style='text-align:justify'><img width=340 height=357
+
<p class=MsoNormal style='text-align:justify'><!--[if gte vml 1]><v:shape id="Resim_x0020_3"
-
src="https://static.igem.org/mediawiki/2014hs/c/cf/Cimage008.jpg" align=left hspace=12
+
o:spid="_x0000_s1026" type="#_x0000_t75" alt="https://static.igem.org/mediawiki/2014hs/c/cf/Cimage008.jpg"
-
vspace=12></p>
+
style='position:absolute;left:0;text-align:left;margin-left:0;margin-top:0;
-
 
+
width:255pt;height:267.75pt;z-index:-251657216;visibility:visible;
-
<p class=MsoNormal style='text-align:justify'><span style='font-size:16.0pt;
+
mso-wrap-style:square;mso-width-percent:0;mso-height-percent:0;
-
line-height:115%;font-family:"Calibri","sans-serif"'>Digestion and DNA
+
mso-wrap-distance-left:9pt;mso-wrap-distance-top:9pt;
-
Electrophoresis</span></p>
+
mso-wrap-distance-right:9pt;mso-wrap-distance-bottom:9pt;
 +
mso-position-horizontal:left;mso-position-horizontal-relative:text;
 +
mso-position-vertical:absolute;mso-position-vertical-relative:line;
 +
mso-width-percent:0;mso-height-percent:0;mso-width-relative:page;
 +
mso-height-relative:page' o:allowoverlap="f">
 +
<v:imagedata src="https://static.igem.org/mediawiki/2014hs/c/cf/Cimage008.jpg"/>
 +
<w:wrap type="tight" anchory="line"/>
 +
</v:shape><![endif]--><![if !vml]><img width=340 height=357
 +
src="https://static.igem.org/mediawiki/2014hs/c/cf/Cimage008.jpg" align=left
 +
hspace=12 vspace=12 alt="https://static.igem.org/mediawiki/2014hs/c/cf/Cimage008.jpg"
 +
v:shapes="Resim_x0020_3"><![endif]><span class=SpellE><span style='font-size:
 +
16.0pt;line-height:115%;font-family:"Calibri","sans-serif"'>Digestion</span></span><span
 +
style='font-size:16.0pt;line-height:115%;font-family:"Calibri","sans-serif"'> <span
 +
class=SpellE>and</span> DNA <span class=SpellE>Electrophoresis</span></span></p>
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>&nbsp;</span></p>
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>&nbsp;</span></p>
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>Gel
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>Gel
-
electrophoresis was conducted on the samples so that the enzymes which were
+
<span class=SpellE>electrophoresis</span> <span class=SpellE>was</span> <span
-
digested with the EcoRI and PstI could be verified in terms of their
+
class=SpellE>conducted</span> on <span class=SpellE>the</span> <span
-
wavelength. The acquired results showed that the plasmid transfer process was
+
class=SpellE>samples</span> <span class=SpellE>so</span> <span class=SpellE>that</span>
-
successful for BL-21 Strains and it was possible to continue to the next step.</span></p>
+
<span class=SpellE>the</span> <span class=SpellE>enzymes</span> <span
 +
class=SpellE>which</span> <span class=SpellE>were</span> <span class=SpellE>digested</span>
 +
<span class=SpellE>with</span> <span class=SpellE>the</span> <span
 +
class=SpellE>EcoRI</span> <span class=SpellE>and</span> <span class=SpellE>PstI</span>
 +
<span class=SpellE>could</span> be <span class=SpellE>verified</span> in <span
 +
class=SpellE>terms</span> of <span class=SpellE>their</span> <span
 +
class=SpellE>wavelength</span>. <span class=SpellE>The</span> <span
 +
class=SpellE>acquired</span> <span class=SpellE>results</span> <span
 +
class=SpellE>showed</span> <span class=SpellE>that</span> <span class=SpellE>the</span>
 +
<span class=SpellE>plasmid</span> transfer <span class=SpellE>process</span> <span
 +
class=SpellE>was</span> <span class=SpellE>successful</span> <span
 +
class=SpellE>for</span> BL-21 <span class=SpellE>Strains</span> <span
 +
class=SpellE>and</span> it <span class=SpellE>was</span> <span class=SpellE>possible</span>
 +
<span class=SpellE>to</span> <span class=SpellE>continue</span> <span
 +
class=SpellE>to</span> <span class=SpellE>the</span> <span class=SpellE>next</span>
 +
step.</span></p>
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Revision as of 03:11, 21 June 2014

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CLONING THE TOP10 STRAINS

 

TRANSFORMATION

 

Plasmids containing the BBa_K1291071 (Horseradish peroxidase composite part) and BBa_K1291299 (Lignin Peroxidase composite part) parts were transferred into our T10 strains, which were prepared through the competent cell preparation protocol, using transformation protocols. Colonies were seen in agar plates that had the appropriate antibiotics after a 16 hour long incubation process.

 

 

PLASMID ISOLATION

 The colonies taken from plates were incubated in LB Broth containing AMP for 16 hours. DNA isolation was done on the bacteria culture to check the efficiency. The adequacy of the amount of the nucleic acid concentration for digestion was met in the acquired samples and it was seen that the samples had peaks at OD 260, which was captured by the nanodrop.

https://static.igem.org/mediawiki/2014hs/a/a8/Cimage001.jpg

https://static.igem.org/mediawiki/2014hs/e/e0/Cimage002.jpg


https://static.igem.org/mediawiki/2014hs/f/f4/Cimage003.jpg

https://static.igem.org/mediawiki/2014hs/9/98/Cimage004.jpg 

 

Digestion and DNA Electrophoresis

 

Gel electrophoresis was conducted on the samples so that the enzymes which were digested with the EcoRI and PstI could be verified in terms of their wavelength. The acquired results showed that the plasmid transfer process was successful for the Top10 strains while the NEB10 strains, which were taken from the stock, were disfunctional thus were unusable.

 

 

 

 

CLONING THE BL-21 STRAINS

 

Since we require a high concentration of proteins in our project, we decided to use the BL-21 strain of E.coli instead of Top10 strains due to its enhanced protein synthesis capacity. Plasmids containing the BBa_K1291071 (Horseradish peroxidase composite part) and BBa_K1291299 (Lignin Peroxidase composite part) parts were transferred into our BL-21 strains, which were prepared through the competent cell preparation protocol, using the transformation protocol. The colonies successfully acquired after a 16-hour long incubation process.

 

 

 

PLASMID ISOLATION

https://static.igem.org/mediawiki/2014hs/1/1b/Cimage005.jpg

https://static.igem.org/mediawiki/2014hs/1/1f/Cimage006.jpg


https://static.igem.org/mediawiki/2014hs/5/55/Cimage007.gif                                                                                                                                                                                                                                          The chosen colonies were incubated for 16-hours in AMP containing LB Broth. DNA Isolation was done to test the efficiency of the transformation. A peek at OD 260 was observed and sufficient levels of DNA concentration was acquired to continue to digestion.

 

https://static.igem.org/mediawiki/2014hs/c/cf/Cimage008.jpgDigestion and DNA Electrophoresis

 

Gel electrophoresis was conducted on the samples so that the enzymes which were digested with the EcoRI and PstI could be verified in terms of their wavelength. The acquired results showed that the plasmid transfer process was successful for BL-21 Strains and it was possible to continue to the next step.

Retrieved from "http://2014hs.igem.org/Team:AUC_TURKEY/Results/Cloning"