Difference between revisions of "Team:SHSBNU China/InterLab"

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<h1>InterLab</h1>
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<h1>Parts</h1>
 
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<div id="sec_content">
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<h2>I. Background</h2>
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<h2>I. Parts</h2>
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<p class="text">
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“All of 2018 iGEM teams are invited and encouraged to participate in the Fifth International InterLaboratory Measurement Study in synthetic biology.” Our team took part in this study which is aimed to figure out and correct the sources of systematic variability in synthetic biology measurements. The main task was to quantify expression of GFP in common, comparable or absolute units. In our case, we measured fluorescence using plate reader.
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</p>
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<h2>II. Design</h2>
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<p class="text">
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Fluorescence is widely used as a proxy for promoter activity by expressing fluorescent proteins such as green fluorescent protein (GFP). Despite this is an indirect measurement, it provides a useful insight into expression levels and has significant advantage that it could be a real-time monitor without disrupting cells.
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</p>
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<p class="text">
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Fluorescence/OD600 is routinely used to give an adjustment of the relative expression per cell.
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</p>
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<h2>III. Material and Methods</h2>
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<h3>a. Plasmids Used</h3>
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<td color="#FFFFF0">Name</td>
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<td>Negative control</td>
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<td color="#FFFFF0">Description</td>
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<td>BBa_R0040</td>
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<td color="#FFFFF0">Function</td>
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<td>(provided in kit plate)</td>
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<td>Positive control</td>
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<td>BBa_K52684000</td>
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<td>BBa_I20270</td>
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<td>CotA laccase of <em>B. subtilis</em></td>
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<td>(provided in kit plate)</td>
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<td>CotA of <em>B. subtilis</em> is a copper-dependet laccase, which can catalyze the oxidation of board range of synthetic dyes. </td>
 
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<td>Test Device 1</td>
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<td>BBa_K52684001</td>
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<td>BBa_J364000</td>
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<td>PelB-CotA</td>
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<td>(provided in kit plate)</td>
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<td>PelB signal peptide linking with CotA to transfer CotA out of the bacteria. </td>
 
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<td>Test Device 2</td>
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<td>BBa_K52684002</td>
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<td>BBa_J364001</td>
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<td>PhoA-CotA</td>
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<td>(provided in kit plate)</td>
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<td>PhoA signal peptide linking with CotA to transfer CotA out of the bacteria. </td>
 
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<td>Test Device 3</td>
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<td>BBa_K52684003</td>
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<td>BBa_J36400</td>
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<td>OmpA-CotA</td>
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<td>(provided in kit plate)</td>
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<td>OmpA signal peptide linking with CotA to transfer CotA out of the bacteria.</td>
 
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<td>Test Device 4</td>
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<td>BBa_K52684004</td>
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<td>BBa_J364007</td>
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<td>SpyCatcher-sfGFP</td>
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<td>(provided in kit plate)</td>
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<td>SfGFP fused to the SpyCatcher domain can be covalently attached onto the biofilm displaying SpyTag.This part is used to test our immobilization strategy of CotA.</td>
 
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<td>Test Device 5</td>
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<td>BBa_K52684005</td>
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<td>BBa_J364008</td>
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<td>SpyCatcher-CotA</td>
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<td>(provided in kit plate)</td>
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<td>CotA fused to the SpyCatcher domain can be covalently attached onto the biofilm displaying SpyTag.</td>
 
</tr>
 
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<td>Test Device 6</td>
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<td>BBa_K52684006</td>
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<td>BBa_J364009</td>
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<td>CsgA-SpyTag</td>
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<td>(provided in kit plate)</td>
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<td>CsgA is a major subunit of the biofilm of E. coli(1)(2). A SpyTag is fused to CsgA so that CotA laccase can be fixed on biofilm through SpyTag-SpyCatcher chemistry. </td>
 
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<h3>b. Strain used</h3>
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<div class="content">
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<h2>II. References</h2>
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<li class="content_list">Competent cells (Escherichia coli strain DH5α)</li>
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<li class="content_list">1x PBS</li>
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<li class="content_list">ddH2O</li>
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<li class="content_list">LB media</li>
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<li class="content_list">Chloramphenicol</li>
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<li class="content_list">50 ml Falcon tube</li>
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<li class="content_list">1.5 ml Eppendorf tubes</li>
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<li class="content_list">Ice bucket with ice</li>
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<li class="content_list">Micropipettes</li>
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<li class="content_list">Micropipette tips</li>
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<li class="content_list">96 well plates, black with clear flat bottom preferred</li>
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<li class="content_list">LUDOX CL-X (provided in kit)</li>
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<li class="content_list">Fluorescein sodium salt (provided in kit)</li>
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<li class="content_list">Silica Beads (Provided in kit)</li>
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<h3>c. Machines</h3>
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<p class="text">
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Spectrophotometer: Thermo scientific Varioskan Flash
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</p>
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<h3>d. Software</h3>
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<p class="text">
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Microsoft Excel 2016
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</p>
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</div>
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<h3>f. Results</h3>
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<h5 class="text">
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OD600 Reference Point
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<img class="pictures" id = "29400" src=""/>
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<p class="pic_text">Table 1. OD600 Reference Point.</p>
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<h5 class="text">
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Fluorescein Standard Curve
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<div style="float:" class="content_pic_left">
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<img class="pictures" id = "29401" src=""/>
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<p class="pic_text">Table2. Original data of Fluorescein standard curve</p>
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</div>
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<div style="float:" class="content_pic_left">
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<img class="pictures" id = "29402" src=""/>
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<p class="pic_text">Figure1. Fluorescein Standard Curve</p>
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<h5 class="text">
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Particle Standard Curve
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<div style="float:" class="content_pic_left">
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<img class="pictures" id = "29403" src=""/>
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<p class="pic_text">Table 3. Original data of Particle Standard Curve</p>
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<div style="float:" class="content_pic_left">
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<img class="pictures" id = "29404" src=""/>
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<p class="pic_text">Figure 2. Particle Standard Curve.</p>
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<img class="pictures" id = "29405" src=""/>
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<p class="pic_text">Figure 3. Particle Standard Curve (log scale).</p>
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</div>
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<h5 class="text">
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Cell measurement
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</h5>
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<div style="float:" class="content_pic_left">
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<img class="pictures" id = "29406" src=""/>
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<p class="pic_text">Table 4. Fluorescence Intensity of E.coli transformed with Device 1 to 6 and two control groups. Intensities was measured under OD600 using Plate Reader for 6 hours.</p>
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</div>
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<p class="text">
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As shown in Table.4, the combination of RBS and promoter in Device exhibited the strongest fluorescent intensity in 6h. Among these six devices, Device 3 almost had no fluorescent intensity.
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</p>
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<h5 class="text">
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Original data link:
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<img class="pictures" id = "29407" src=""/>
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<p class="pic_text">Table 5. Bacterial concentration of E.coli transformed with Device1-6, Negative Control & Positive Control. Intensities were measured under OD600 using plate for 6 hours. </p>
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<p class="text">
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As shown in Table.5, except Device 1, the concentrations of E.coli transformed with another five devices were almost the same.
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</p>
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</div>
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<h5 class="text">
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Normalizing
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</h5>
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<p class="text">
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Counting colony-forming units
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</p>
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<div style="float:" class="content_pic_left">
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<img class="pictures" id = "29408" src=""/>
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<p class="pic_text">Figure 5. Counted by Promega Colony Counter, bacterial concentration of E.coli transformed with Positive Control & Negative Control. </p>
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</div>
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<p class="text">
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As shown in Figure.5, bacterial colonies was counted in order to determine whether absolute cell count or colony-forming units (CFUs) can reduce lab-to-lab variability in fluorescence measurements.
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</p>
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<p class="text">
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It is evident that the promoter of the Device 1 is strongest followed by the promoter of the Device 2 and Device 3. Additionally, CFUs cannot replace the lab measurement because bacterial colonies is so crowded that it is impossible to count precisely by Promega Colony Counter.
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This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. 
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(1). Wang, X, et al. “Programming Cells for Dynamic Assembly of Inorganic Nano-Objects with Spatiotemporal Control.” <em>Advanced Materials (Deerfield Beach, Fla.).,</em> U.S. National Library of Medicine, Apr. 2018, www.ncbi.nlm.nih.gov/pubmed/29516606.  
 
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<p class="pic_text">This is a Picture. This is a Picture. This is a Picture. This is a Picture.</p>
 
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This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. 
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(2). Chen, Allen Y., et al. “Synthesis and Patterning of Tunable Multiscale Materials with Engineered Cells .” <em>Nature Materials,</em> U.S. National Library of Medicine, May 2014, www.ncbi.nlm.nih.gov/pmc/articles/PMC4063449/.
 
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Revision as of 15:46, 16 October 2018

Section Sample

Parts

I. Parts

Name Description Function
BBa_K52684000 CotA laccase of B. subtilis CotA of B. subtilis is a copper-dependet laccase, which can catalyze the oxidation of board range of synthetic dyes.
BBa_K52684001 PelB-CotA PelB signal peptide linking with CotA to transfer CotA out of the bacteria.
BBa_K52684002 PhoA-CotA PhoA signal peptide linking with CotA to transfer CotA out of the bacteria.
BBa_K52684003 OmpA-CotA OmpA signal peptide linking with CotA to transfer CotA out of the bacteria.
BBa_K52684004 SpyCatcher-sfGFP SfGFP fused to the SpyCatcher domain can be covalently attached onto the biofilm displaying SpyTag.This part is used to test our immobilization strategy of CotA.
BBa_K52684005 SpyCatcher-CotA CotA fused to the SpyCatcher domain can be covalently attached onto the biofilm displaying SpyTag.
BBa_K52684006 CsgA-SpyTag CsgA is a major subunit of the biofilm of E. coli(1)(2). A SpyTag is fused to CsgA so that CotA laccase can be fixed on biofilm through SpyTag-SpyCatcher chemistry.

II. References

(1). Wang, X, et al. “Programming Cells for Dynamic Assembly of Inorganic Nano-Objects with Spatiotemporal Control.” Advanced Materials (Deerfield Beach, Fla.)., U.S. National Library of Medicine, Apr. 2018, www.ncbi.nlm.nih.gov/pubmed/29516606.

(2). Chen, Allen Y., et al. “Synthesis and Patterning of Tunable Multiscale Materials with Engineered Cells .” Nature Materials, U.S. National Library of Medicine, May 2014, www.ncbi.nlm.nih.gov/pmc/articles/PMC4063449/.