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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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| − |
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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> </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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| − |
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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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| − |
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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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| − |
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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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| − |
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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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| − |
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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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| − |
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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 Biobrick parts measured must be supplied in the
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| − | plasmid pSB1C3.<o:p></o:p></span></p>
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| − |
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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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| − |
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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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| − |
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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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| − |
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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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| − |
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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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| − |
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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"'> </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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| − |
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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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| − |
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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"'> </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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| − |
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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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| − |
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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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| − |
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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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| − |
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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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| − |
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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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| − |
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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"'>
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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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| − |
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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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| − |
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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"'>
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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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| − |
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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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| − |
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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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| − |
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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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| − |
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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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| − |
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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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| − |
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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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| − |
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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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