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| | <section> | | <section> |
| | | | |
| | + | <h2>Biosensor</h2> |
| | + | <h3>Lactate detection system</h3> |
| | | | |
| − | <div id="Fluorescein"> | + | <p> |
| − | <h2>Fluorescein Fluorescence standard curve</h2> | + | One of our biosensor is to detect the level of lactate. In order to achieve this purpose, we constructed a lactate detection system (BBa_K2292005) in E. coli.<br/> |
| | + | In order to test our lactate detection system work or not, we add different concentrations of lactic acid into the liquid culture of E. coli DH5α which contain lactate detection system. |
| | | | |
| − | </div> | + | </p> |
| | | | |
| − | <div id="Fluorescein-Plate-reader"> | + | <img src="https://static.igem.org/mediawiki/2017/3/3f/DM_1.jpg" style="display:block; margin:auto;"> |
| − | <div class="aaa"></div> | + | <p style="text-align:center;">a. The fluorescence of each lactate concentration depends on time.</p> |
| − | <h3>Plate reader</h3>
| + | <br/> |
| − | | + | |
| − | <p>
| + | |
| − | microplate reader FLUOstar Omega</br>
| + | |
| − | emission filter: 520 nm</br>
| + | |
| − | excitation filter: 485 nm
| + | |
| − | </p>
| + | |
| | | | |
| − | </div> | + | <img src="https://static.igem.org/mediawiki/2017/5/5a/DM_2.jpg" style="display:block; margin:auto;"> |
| − |
| + | <p style="text-align:center;">b. The fluorescence of different time point depends on lactate level.</p> |
| − | <div id="Fluorescein-Material">
| + | |
| − | <div class="aaa"></div> | + | |
| − | <h3>Material</h3>
| + | |
| − | <p>
| + | |
| − | Fluorescein sodium salt</br>
| + | |
| − | 1xPBS</br>
| + | |
| − | Tissue culture testplate (black with flat bottom)
| + | |
| − | </p>
| + | |
| | | | |
| − | </div>
| + | <img src="https://static.igem.org/mediawiki/2017/2/29/DM_3.jpg" style="display:block; margin:auto;"> |
| | + | <p style="text-align:center;">c. Three-dimension diagram of fluorescence, time and lactate concentration</p> |
| | | | |
| − | <div id="Fluorescein-Method">
| |
| − | <div class="aaa"></div>
| |
| − | <h3>Method</h3>
| |
| − |
| |
| − | <ol><li>Prepare fluorescein stock solution</li></ol>
| |
| | <p> | | <p> |
| − | 1. Spin down fluorescein stock tube to make sure pellet is at the bottom of tube.</br>
| + | The fluorescence difference between each concentration of lactate is more obvious after 90 minutes. The experiment result demonstrates that the function of lactate detection is work. |
| − | 2. Prepare 2x fluorescein stock solution (100 μM) by resuspending fluorescein in 1 mL of 1xPBS. </br>
| + | |
| − | 3. Dilute the 2x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution and resulting concentration of fluorescein stock solution 50 μM </br></br>
| + | |
| | </p> | | </p> |
| − | <ol><li>Serial dilutions</li></ol> | + | |
| | + | <h3>CSP detection system</h3> |
| | + | <ol><li>PCR result：Prove that we successfully transformed our plasmid into E.coli, DH5α</ol></li> |
| | + | <p> i. pSBBS1C</p> |
| | + | |
| | + | <img src="https://static.igem.org/mediawiki/2017/b/b4/EP1.jpg" style="display:block; margin:auto;"> |
| | + | |
| | + | <p> ii. pSBBS4S</p> |
| | + | |
| | + | <img src="https://static.igem.org/mediawiki/2017/3/38/EP2.jpg" style="display:block; margin:auto;"> |
| | + | |
| | + | <ol><li>Functional test result</ol></li> |
| | + | |
| | <p> | | <p> |
| − | 1. Add 100 μl of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12</br>
| + | According to the measurement of absorbance of wavelength 595 nm, we can confirm that CSP isn’t toxin to Bacillus Subtilis. |
| − | 2. Add 200 μl of fluorescein 1x stock solution into A1, B1, C1, D1</br>
| + | |
| − | 3. Transfer 100 μl of fluorescein stock solution from A1 into A2. </br>
| + | |
| − | 4. Mix A2 by pipetting up and down 3x and transfer 100 μl into A3. </br>
| + | |
| − | 5. Mix A3 by pipetting up and down 3x and transfer 100 μl into A4. </br>
| + | |
| − | 6. Mix A4 by pipetting up and down 3x and transfer 100 μl into A5. </br>
| + | |
| − | 7. Mix A5 by pipetting up and down 3x and transfer 100 μl into A6. </br>
| + | |
| − | 8. Mix A6 by pipetting up and down 3x and transfer 100 μl into A7. </br>
| + | |
| − | 9. Mix A7 by pipetting up and down 3x and transfer 100 μl into A8. </br>
| + | |
| − | 10. Mix A8 by pipetting up and down 3x and transfer 100 μl into A9. </br>
| + | |
| − | 11. Mix A9 by pipetting up and down 3x and transfer 100 μl into A10.</br>
| + | |
| − | 12. Mix A10 by pipetting up and down 3x and transfer 100 μl into A11. </br>
| + | |
| − | 13. Mix A11 by pipetting up and down 3x and transfer 100 μl into liquid waste.
| + | |
| − | (Caution: Do not to continue serial dilution into column 12.)</br>
| + | |
| | </p> | | </p> |
| − | <ol><li>repeat serial dilute for Row B、D、E</strong></li></ol>
| |
| − | <ol><li>Measure fluorescence of all samples in all standard measurement modes in instrument Record the data in your notebook</strong></li></ol>
| |
| − | <ol><li>Import data into Excel (fluorescein standard curve tab ) Sheet_1 provided</li></ol>
| |
| − |
| |
| − | <br/><br/>
| |
| | | | |
| − | </div> | + | <img src="https://static.igem.org/mediawiki/2017/f/f7/DM_7.jpg" style="display:block; margin:auto;"> |
| | + | <img src="https://static.igem.org/mediawiki/2017/4/4a/DM_8.png" style="display:block; margin:auto;"> |
| | | | |
| − | <div id="Fluorescein-Data-result"> | + | <p> |
| − | <div class="aaa"></div>
| + | And for the measurement of the fluorescent plate reader (excitation: 485 nm, emission: 520 nm), we found out that the best amount of bacteria for measuring, the CSP conc. is at the value of OD600 = 0.2 after a series of experiment. |
| − | <h3>Data result</h3>
| + | </p> |
| − | <br/>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2017/8/85/FFs_1.png" style="display:block; margin:auto;"><br/><br/>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2017/a/a4/FFs_2.png" style="display:block; margin:auto;"><br/><br/>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2017/5/5c/FFs_3.png" style="display:block; margin:auto;"><br/>
| + | |
| − | </div>
| + | |
| − | </section>
| + | |
| | | | |
| − | <section>
| + | <img src="https://static.igem.org/mediawiki/2017/2/24/DM_10.tif" style="display:block; margin:auto;"> |
| | + | |
| | + | <p> |
| | + | The intensity of fluorescence slowly drops after a sharp rise in a small amount of time, hence we can tell that the reaction starts right away as soon as CSP is added. |
| | + | </p> |
| | | | |
| | | | |
| − | <div id="OD600">
| |
| − | <h2>OD600 Reference point</h2>
| |
| | | | |
| − | </div>
| |
| | | | |
| | | | |
| − | <div id="OD600-Plate-reader">
| |
| − | <div class="aaa"></div>
| |
| − | <h3>Plate reader</h3>
| |
| | | | |
| − | <p>
| |
| − | Thermo Scientific™ Multiskan™ FC Filter-based Microplate Photometer</br>
| |
| − | Filter: 595 nm</br>
| |
| − | </p>
| |
| | | | |
| − | </div>
| |
| | | | |
| − | <div id="OD600-Material">
| |
| − | <div class="aaa"></div>
| |
| − | <h3>Material</h3>
| |
| − | <p>
| |
| − | 1 ml LUDOX</br>
| |
| − | mQH<sub>2</sub>O</br>
| |
| − | 96 well cell culture plate (clear with flat-bottom)
| |
| − | </p>
| |
| | | | |
| − | </div>
| |
| | | | |
| − | <div id="OD600-Method">
| |
| − | <div class="aaa"></div>
| |
| − | <h3>Method</h3>
| |
| | | | |
| − | <p>
| |
| − | 1. Add 100 μl LUDOX into wells A1, B1, C1, D1 (or 1 mL LUDOX into cuvette)</br>
| |
| − | 2. Add 100 μl of H<sub>2</sub>O into wells A2, B2, C2, D2 (or 1 mL H<sub>2</sub>O into cuvette)</br>
| |
| − | 3. Measure absorbance 600 nm of all samples in all standard measurement modes in instrument</br>
| |
| − | 4. Record the data in excel and Import data into Excel ( OD600 reference point tab ) Sheet_1 provided</br>
| |
| − | </p>
| |
| − |
| |
| − | </div>
| |
| | | | |
| − | <div id="OD600-Data-result">
| |
| − | <div class="aaa"></div>
| |
| − | <h3>Data result</h3>
| |
| − | <br/>
| |
| − | <img src="https://static.igem.org/mediawiki/2017/c/cd/Ee.jpeg" style="display:block; margin:auto;"><br/>
| |
| − | </div>
| |
| − | </section>
| |
| | | | |
| − | <section>
| |
| | | | |
| | | | |
| − | <div id="Cell">
| |
| − | <h2>Cell measure</h2>
| |
| | | | |
| − | </div>
| |
| | | | |
| | | | |
| − | <div id="Cell-Material">
| |
| − | <div class="aaa"></div>
| |
| − | <h3>Material</h3>
| |
| − | <p>
| |
| − | Competent cells ( Escherichia coli strain DH5α)</br>
| |
| − | LB (Luria Bertani) media</br>
| |
| − | Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH - working stock 25 μg /mL)</br>
| |
| − | 50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block light)</br>
| |
| − | Incubator at 37°C</br>
| |
| − | 1.5 ml eppendorf tubes for sample storage</br>
| |
| − | Ice bucket with ice</br>
| |
| − | Pipettes</br>
| |
| − | 96 well plate(cell culture 96 well plate、tissue culture testplate)</br>
| |
| − | Devices (from InterLab Measurement Kit):</br>
| |
| − | 1. Negative control(BBa_R0040)</br>
| |
| − | 2. Positive control(J23151+B0032+E0040+B0010+B0012)</br>
| |
| − | 3. Test Device 1: J23101+I13504</br>
| |
| − | 4. Test Device 2: J23106+I13504</br>
| |
| − | 5. Test Device 3: J23117+I13504</br>
| |
| − | 6. Test Device 4: J23101+BCD2+E0040+B0015</br>
| |
| − | 7. Test Device 5: J23106+BCD2+E0040+B0015</br>
| |
| − | 8. Test Device 6: J23117+BCD2+E0040+B0015</br>
| |
| − | </p>
| |
| | | | |
| − | </div>
| |
| | | | |
| − | <div id="Cell-Method">
| |
| − | <div class="aaa"></div>
| |
| − | <h3>Method</h3>
| |
| | | | |
| − | <p>
| |
| − | 1. Day 1 : Resuspended each plasmid in plate 7 and transform into Escherichia coli DH5α.</br>
| |
| − | (Transformation protocol is from iGEM)</br>
| |
| − | 2. Day 2 : Pick 2 colonies from each of plate and inoculate it on 5-10 mL LB medium +Chloramphenicol.Grow the cells overnight (16-18 hours) at 37°C and 170 rpm.</br>
| |
| − | 3. Day 3 : Set instrument to read OD600 (as OD calibration setting)and measure OD600 of the overnight cultures</br>
| |
| − | 4. Dilute the cultures to a target OD 600 of 0.02 in 12 ml LB medium + Chloramphenicol in 50 mL falcon tube (covered with foil to block light).</br>
| |
| − | 5. Incubate the cultures at 37°C and 170 rpm.</br>
| |
| − | 6. Take 1000 μL samples of the cultures at 0, 2, 4, and 6 hours of incubation and place samples on ice</br>
| |
| − | 7. 4 replicates of 100 uL samples were taken from each culture at 0, 2, 4, and 6 hours of incubation and placed in a 96 well plate for OD and fluorescence measurements using the setup described above</br>
| |
| − | </p>
| |
| | | | |
| − | </div>
| |
| | | | |
| − | <div id="Cell-Data-result">
| |
| − | <div class="aaa"></div>
| |
| − | <h3>Data result</h3>
| |
| − | <br/>
| |
| − | <img src="https://static.igem.org/mediawiki/2017/f/f7/IL_cell1.jpg" style="display:block; margin:auto;"><br/><br/>
| |
| − | <img src="https://static.igem.org/mediawiki/2017/4/41/IL_cell2.jpg" style="display:block; margin:auto;"><br/>
| |
| − | </div>
| |
| | </section> | | </section> |
| | | | |
