Difference between revisions of "Team:SCUT/Measurement"

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<h2>Discription</h2>
 
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<p class=MsoNormal><span lang=EN-US style='font-size:16.0pt;font-family:"Arial Unicode MS",sans-serif'>Overview<o:p></o:p></span></p>
 
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<p class=MsoNormal><span lang=EN-US style='font-size:12.0pt;font-family:"Arial Unicode MS",sans-serif;
 
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color:black;background:white'>In the iGEM competition, teams specify, design,
 
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build, and test simple biological systems made from standard, interchangeable
 
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biological parts.<span class=apple-converted-space>&nbsp;</span>Most BioBrick
 
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parts have never been characterized. And it was important to make a
 
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characterization for parts, which people could use the parameter as the experimental
 
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basis. Protein expression was a key parameter for a promoter. So in this part,
 
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we aimed at measuring the fluorescence of GFP expression which was activated by
 
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our promoter, using a plate reader.<o:p></o:p></span></p>
 
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<p class=MsoNormal><span lang=EN-US style='font-size:16.0pt;font-family:"Arial Unicode MS",sans-serif;
 
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color:black;background:white'>Introduction<o:p></o:p></span></p>
 
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<p class=MsoNormal><span lang=EN-US style='font-size:12.0pt;font-family:"Arial Unicode MS",sans-serif;
 
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color:black;background:white'>We chose a promoter that had never been
 
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characterized in the register M36247 as our improvement work. M36247 was a constitutive
 
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promoter </span><span lang=EN-US style='font-size:12.0pt;font-family:"Arial Unicode MS",sans-serif;
 
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background:white'>at medium strength in E.coli</span><span lang=EN-US
 
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style='font-size:12.0pt;font-family:"Arial Unicode MS",sans-serif'>. The
 
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construction were inserted I13504 as a back insert into the promoter. <o:p></o:p></span></p>
 
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<p class=MsoNormal align=left style='margin-bottom:3.6pt;text-align:left;
 
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line-height:14.3pt;mso-pagination:widow-orphan;mso-outline-level:3;background:
 
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white'><span lang=EN-US style='font-size:14.0pt;font-family:"Arial Unicode MS",sans-serif;
 
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color:black;mso-font-kerning:0pt;mso-bidi-font-weight:bold'>Strains:<o:p></o:p></span></p>
 
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<p class=MsoNormal align=left style='margin-top:4.8pt;margin-right:0cm;
 
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margin-bottom:6.0pt;margin-left:0cm;text-align:left;line-height:14.3pt;
 
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mso-pagination:widow-orphan;background:white'><span lang=EN-US
 
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style='font-size:12.0pt;font-family:"Arial Unicode MS",sans-serif;color:black;
 
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mso-font-kerning:0pt'>The system should be measured in the strain of E.coli
 
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BL21.<o:p></o:p></span></p>
 
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<p class=MsoNormal align=left style='margin-bottom:3.6pt;text-align:left;
 
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line-height:14.3pt;mso-pagination:widow-orphan;mso-outline-level:3;background:
 
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white'><span lang=EN-US style='font-size:14.0pt;font-family:"Arial Unicode MS",sans-serif;
 
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color:black;mso-font-kerning:0pt;mso-bidi-font-weight:bold'>Plasmid:<o:p></o:p></span></p>
 
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<p class=MsoNormal align=left style='margin-top:4.8pt;margin-right:0cm;
 
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mso-pagination:widow-orphan;background:white'><span lang=EN-US
 
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style='font-size:12.0pt;font-family:"Arial Unicode MS",sans-serif;color:black;
 
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mso-font-kerning:0pt'>The Biobrick parts measured must be supplied in the
 
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plasmid pSB1C3.<o:p></o:p></span></p>
 
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<p class=MsoNormal align=left style='margin-bottom:3.6pt;text-align:left;
 
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line-height:14.3pt;mso-pagination:widow-orphan;mso-outline-level:3;background:
 
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white'><span lang=EN-US style='font-size:14.0pt;font-family:"Arial Unicode MS",sans-serif;
 
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color:black;mso-font-kerning:0pt;mso-bidi-font-weight:bold'>Reporter:<o:p></o:p></span></p>
 
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<p class=MsoNormal align=left style='margin-top:4.8pt;margin-right:0cm;
 
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margin-bottom:6.0pt;margin-left:0cm;text-align:left;line-height:14.3pt;
 
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mso-pagination:widow-orphan;background:white'><span lang=EN-US
 
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style='font-size:12.0pt;font-family:"Arial Unicode MS",sans-serif;color:black;
 
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mso-font-kerning:0pt'>The Part BBa_I13504 is chosen as the reporter of our reporter.<o:p></o:p></span></p>
 
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<p class=MsoNormal align=left style='margin-top:4.8pt;margin-right:0cm;
 
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margin-bottom:6.0pt;margin-left:0cm;text-align:left;line-height:14.3pt;
 
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mso-pagination:widow-orphan;background:white'><span lang=EN-US
 
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style='font-size:14.0pt;font-family:"Arial Unicode MS",sans-serif;background:
 
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white;mso-highlight:white;mso-font-kerning:0pt'>Equipment</span><span
 
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lang=EN-US style='font-size:14.0pt;font-family:"Arial Unicode MS",sans-serif;
 
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mso-font-kerning:0pt'>:<o:p></o:p></span></p>
 
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<p class=MsoNormal align=left style='margin-top:4.8pt;margin-right:0cm;
 
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margin-bottom:6.0pt;margin-left:0cm;text-align:left;line-height:14.3pt;
 
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mso-pagination:widow-orphan;background:white'><span lang=EN-US
 
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style='font-size:12.0pt;font-family:"Arial Unicode MS",sans-serif;color:black;
 
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background:white;mso-highlight:white;mso-font-kerning:0pt'>Infinite M200 with
 
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the software Magellan 6.5</span><b style='mso-bidi-font-weight:normal'><span
 
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lang=EN-US style='font-size:12.0pt;font-family:"Arial Unicode MS",sans-serif;
 
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mso-font-kerning:0pt'><o:p></o:p></span></b></p>
 
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<p class=MsoNormal><span lang=EN-US style='font-size:16.0pt;font-family:"Arial Unicode MS",sans-serif'>Protocol<o:p></o:p></span></p>
 
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<p class=MsoNormal style='margin-left:21.0pt;text-indent:-21.0pt;mso-list:l0 level1 lfo1'><![if !supportLists]><span
 
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lang=EN-US style='font-size:14.0pt;font-family:Wingdings;mso-fareast-font-family:
 
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Wingdings;mso-bidi-font-family:Wingdings;color:black'><span style='mso-list:
 
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Ignore'>l<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span></span><![endif]><span
 
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lang=EN-US style='font-size:14.0pt;font-family:"Arial Unicode MS",sans-serif;
 
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color:black;background:white'>Construction <o:p></o:p></span></p>
 
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<p class=MsoNormal><span lang=EN-US style='font-size:12.0pt;font-family:"Arial Unicode MS",sans-serif;
 
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color:black;background:white'>We got the promoter by the way of overlap PCR. After
 
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sequencing, </span><span lang=EN-US style='font-size:12.0pt;font-family:"Arial Unicode MS",sans-serif'>the
 
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construction were inserted I13504 as a back insert into the promoter.<span
 
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style='color:black;background:white'> <o:p></o:p></span></span></p>
 
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<p class=MsoNormal style='margin-left:21.0pt;text-indent:-21.0pt;mso-list:l0 level1 lfo1'><![if !supportLists]><span
 
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lang=EN-US style='font-size:14.0pt;font-family:Wingdings;mso-fareast-font-family:
 
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Wingdings;mso-bidi-font-family:Wingdings;color:black'><span style='mso-list:
 
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Ignore'>l<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span></span><![endif]><span
 
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lang=EN-US style='font-size:14.0pt;font-family:"Arial Unicode MS",sans-serif;
 
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color:black;background:white'>Growing and measuring<o:p></o:p></span></p>
 
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<p style='margin-top:4.8pt;margin-right:0cm;margin-bottom:6.0pt;margin-left:
 
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0cm;line-height:14.65pt;background:white'><span lang=EN-US style='font-family:
 
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"Arial Unicode MS",sans-serif'>1. Streaked a plate of the strain which contained
 
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M36247 listed in pSB1C3 .<o:p></o:p></span></p>
 
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<p style='margin-top:4.8pt;margin-right:0cm;margin-bottom:6.0pt;margin-left:
 
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0cm;line-height:14.65pt;background:white'><span lang=EN-US style='font-family:
 
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"Arial Unicode MS",sans-serif'>2. Inoculated three 10ml cultures of
 
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supplemented M9 Medium and antibiotic (chloramphenicol <span style='background:
 
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white;mso-highlight:white'>25<span style='letter-spacing:-.55pt'>μ</span></span>g/ml)
 
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with single colony from the plate.<o:p></o:p></span></p>
 
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<p style='margin-top:4.8pt;margin-right:0cm;margin-bottom:6.0pt;margin-left:
 
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0cm;line-height:14.65pt;background:white'><span lang=EN-US style='font-family:
 
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"Arial Unicode MS",sans-serif'>3. Cultures were grown in<span style='color:
 
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black;background:white;mso-highlight:white'> 50ml conical tube</span> for 16hours
 
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at 37℃ with shaking at 250rpm.<o:p></o:p></span></p>
 
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<p style='margin-top:4.8pt;margin-right:0cm;margin-bottom:6.0pt;margin-left:
 
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0cm;line-height:14.65pt;background:white'><span lang=EN-US style='font-family:
 
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"Arial Unicode MS",sans-serif'>4. Cultures were diluted 1:100 into 3ml fresh
 
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medium and grown for 3hrs.<o:p></o:p></span></p>
 
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<p style='margin-top:4.8pt;margin-right:0cm;margin-bottom:6.0pt;margin-left:
 
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0cm;line-height:14.65pt;background:white'><span lang=EN-US style='font-family:
 
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"Arial Unicode MS",sans-serif'>5. Measure the fluorescence (<span
 
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style='color:black;background:white;mso-highlight:white'>Infinite M200 with the
 
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software Magellan 6.5</span><span style='color:black'>,</span> 485 nm
 
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excitation, 528 nm emission) and absorbance (600nm) every 30 minutes in the
 
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next 4 hours.<o:p></o:p></span></p>
 
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<p style='margin-top:4.8pt;margin-right:0cm;margin-bottom:6.0pt;margin-left:
 
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21.0pt;text-indent:-21.0pt;line-height:14.65pt;mso-list:l0 level1 lfo1;
 
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background:white'><![if !supportLists]><span lang=EN-US style='font-size:14.0pt;
 
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font-family:Wingdings;mso-fareast-font-family:Wingdings;mso-bidi-font-family:
 
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Wingdings'><span style='mso-list:Ignore'>l<span style='font:7.0pt "Times New Roman"'>&nbsp;
 
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</span></span></span><![endif]><span lang=EN-US style='font-size:14.0pt;
 
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font-family:"Arial Unicode MS",sans-serif'>Processing the data<o:p></o:p></span></p>
 
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<p style='margin-top:4.8pt;margin-right:0cm;margin-bottom:6.0pt;margin-left:
 
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0cm;line-height:14.65pt;background:white'><span lang=EN-US style='font-family:
 
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"Arial Unicode MS",sans-serif;color:#282828'>Every device was measured thrice.
 
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The data was the arithmetic average of the three row data. Then they were subtracted
 
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the background controlling LB.<o:p></o:p></span></p>
 
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<p style='margin-top:4.8pt;margin-right:0cm;margin-bottom:6.0pt;margin-left:
 
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21.0pt;text-indent:-21.0pt;line-height:14.65pt;mso-list:l0 level1 lfo1;
 
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background:white'><![if !supportLists]><span lang=EN-US style='font-size:14.0pt;
 
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font-family:Wingdings;mso-fareast-font-family:Wingdings;mso-bidi-font-family:
 
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Wingdings;color:#282828'><span style='mso-list:Ignore'>l<span style='font:7.0pt "Times New Roman"'>&nbsp;
 
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</span></span></span><![endif]><span lang=EN-US style='font-size:14.0pt;
 
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font-family:"Arial Unicode MS",sans-serif;color:#282828'>Positive and negative
 
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control<o:p></o:p></span></p>
 
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<p style='margin-top:4.8pt;margin-right:0cm;margin-bottom:6.0pt;margin-left:
 
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0cm;line-height:14.65pt;background:white'><span lang=EN-US style='font-family:
 
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"Arial Unicode MS",sans-serif;color:#282828'>As our positive control, J23101
 
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was medium strength promoter in constitutive family with close strength to our
 
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promoter, to exclude false negative results caused by the operation or low
 
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content. R0040 and BL21 without any plasmid were our negative control. R0040
 
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was the part of ptet, which could regard as an empty plasmid to exclude false
 
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negative results caused by the operation or low content. Differed from the
 
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positive control, there was no back insert I13504. <o:p></o:p></span></p>
 
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<p class=MsoNormal><span lang=EN-US style='font-size:16.0pt;font-family:"Arial Unicode MS",sans-serif;
 
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color:black;background:white'>Result<o:p></o:p></span></p>
 
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<p class=MsoNormal><span lang=EN-US style='font-size:12.0pt;font-family:"Arial Unicode MS",sans-serif;
 
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color:black;background:white'>Figure 1: The expression of fluorescence was
 
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growing with the increasing of OD.</span><span lang=EN-US style='font-size:
 
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22.0pt;font-family:"Arial Unicode MS",sans-serif;color:black;background:white'><o:p></o:p></span></p>
 
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<p class=MsoNormal><span lang=EN-US style='font-size:12.0pt;font-family:"Arial Unicode MS",sans-serif;
 
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color:black;background:white;mso-no-proof:yes'>Figure 2</span><span
 
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style='font-size:12.0pt;font-family:"Arial Unicode MS",sans-serif;color:black;
 
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background:white;mso-no-proof:yes'>： <span lang=EN-US>OD of the three
 
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biological replicates were growing in the four hours. <o:p></o:p></span></span></p>
 
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<p class=MsoNormal><span lang=EN-US style='font-size:12.0pt;font-family:"Arial Unicode MS",sans-serif;
 
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color:black;background:white;mso-no-proof:yes'>Figure 3: The fluorescence of
 
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three biological replicate of M36247+I13504 were growing in four hours.</span><span
 
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lang=EN-US style='font-size:12.0pt;font-family:"Arial Unicode MS",sans-serif;
 
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color:black;background:white'><o:p></o:p></span></p>
 
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<p class=MsoNormal><span lang=EN-US style='font-size:16.0pt;font-family:"Arial Unicode MS",sans-serif;
 
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color:black;background:white'>Discussion <o:p></o:p></span></p>
 
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<p class=MsoNormal><span lang=EN-US style='font-size:12.0pt;font-family:"Arial Unicode MS",sans-serif;
 
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color:black;background:white'>It could be seen that M36247 was a constitutive
 
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promoter </span><span lang=EN-US style='font-size:12.0pt;font-family:"Arial Unicode MS",sans-serif;
 
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background:white'>at medium strength, which could activated the expression of
 
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GFP without any inducer added. And compared with our positive control J23101,
 
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M36247 were slightly stronger. <span style='color:black'><o:p></o:p></span></span></p>
 
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<h6>PROJECT PHOTOS</h6>
 
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<h3>About Us</h3>
 
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<p class="percent-two-third">In 2015, we SCUT teams won top ten innovative and entrepreneurial team set up by SCUT.Because of the strong support of the college, our team is being on the right track, and increasing understanding of the subject and experience.  </p>
 
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<h3 style="margin-left:20px;">Thanks</h3>
 
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<ul>
 
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<li>Zhang Zhenwu,Prof. Guo Shouqian,Dr. Li, Dr. Li Cheng,Dr. Wang Meng,Chen Kejie</li>
 
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<li>Guangzhou Municipal Environmental Protection Bureau<br/>
 
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Revision as of 15:18, 18 September 2015

Team:SCUT