Difference between revisions of "Team:SCAU-China/Parts"

 
(2 intermediate revisions by one other user not shown)
Line 22: Line 22:
 
}
 
}
 
img {
 
img {
−
width: 90%;
 
 
height: auto;
 
height: auto;
 
max-width: 90%;
 
max-width: 90%;
Line 226: Line 225:
 
line-height: 60px;
 
line-height: 60px;
 
text-align: center;
 
text-align: center;
 +
 
display: none;
 
display: none;
 
}
 
}
Line 501: Line 501:
 
<td style="text-align: center;"><a href="http://parts.igem.org/Part:BBa_K2559013">BBa_K2559013</a></td>
 
<td style="text-align: center;"><a href="http://parts.igem.org/Part:BBa_K2559013">BBa_K2559013</a></td>
 
<td style="text-align: center;">Basic</td>
 
<td style="text-align: center;">Basic</td>
−
<td style="text-align: center;">Terminator of RuBisCO(Prbc)</td>
+
<td style="text-align: center;">Terminator of RuBisCO(Trbc)</td>
 
<td style="text-align: center;">Unsubmit </td>
 
<td style="text-align: center;">Unsubmit </td>
 
</tr>
 
</tr>
Line 519: Line 519:
 
</tbody>
 
</tbody>
 
</table>
 
</table>
−
<p>Prepare competent cells (Escherichia coli strain DH5α) and perform transformation according to the cell measurement protocol. Meanwhile, we followed the calibration protocol supplied by the iGEM HQ to generate OD600 reference point, particle standard curve and  fluorescence standard curve to calibrate the plate reader (96 well plate) and then measured both the fluorescence and absorbance of our samples in 0-hour and 6-hour.  </p>
 
−
<p>We used the setted protocol that we obtained so as to ensure everyone was doing correctly and repeatable. These can be found at <a href="https://2018.igem.org/Team:SCAU-China/Protocol">this page.</a></p>
 
−
<h1>Results and discussion</h1>
 
−
<p>We submitted the iGEM’s completed spreadsheet to the measurement committee before the deadline, and received confirmation that the results were shown here: 2018/7/30</p>
 
−
<p>OD600 calibration</p>
 
−
<table style="">
 
−
<tr>
 
−
<th class="no-border" style="border: 0;text-align: left; background-color: #ffffff;"></th>
 
−
<th colspan="2" class="no-border" style="border: 0;text-align: left; background-color: #ffffff;">LUDOX CL-X  H2O</th>
 
−
</tr>
 
−
<tr>
 
−
<td class="no-border" style="border: 0;text-align: left;">Replicate 1</td>
 
−
<td class="blue" style=" background-color: #00ccff;text-align: left; border: 1px solid #000000;">0.059</td>
 
−
<td class="blue" style=" background-color: #00ccff;text-align: left; border: 1px solid #000000;">0.039</td>
 
−
</tr>
 
−
<tr>
 
−
<td class="no-border" style="border: 0;text-align: left;">Replicate 2</td>
 
−
<td class="blue" style=" background-color: #00ccff;text-align: left; border: 1px solid #000000;">0.060</td>
 
−
<td class="blue" style=" background-color: #00ccff;text-align: left; border: 1px solid #000000;">0.035</td>
 
−
</tr>
 
−
<tr>
 
−
<td class="no-border" style="border: 0;text-align: left;">Replicate 3</td>
 
−
<td class="blue" style=" background-color: #00ccff;text-align: left; border: 1px solid #000000;">0.060</td>
 
−
<td class="blue" style=" background-color: #00ccff;text-align: left; border: 1px solid #000000;">0.035</td>
 
−
</tr>
 
−
<tr>
 
−
<td class="no-border" style="border: 0;text-align: left;">Replicate 4</td>
 
−
<td class="blue" style=" background-color: #00ccff;text-align: left; border: 1px solid #000000;">0.061</td>
 
−
<td class="blue" style=" background-color: #00ccff;text-align: left; border: 1px solid #000000;">0.040</td>
 
−
</tr>
 
−
<tr>
 
−
<td class="no-border" style="border: 0;text-align: left;">Arith. Mean</td>
 
−
<td class="yellow" style="background-color: #ffcc00;text-align: left; border: 1px solid #000000;">0.059</td>
 
−
<td class="yellow" style="background-color: #ffcc00;text-align: left; border: 1px solid #000000;">0.039</td>
 
−
</tr>
 
−
<tr>
 
−
<td class="no-border" style="border: 0;text-align: left;">Corrected Abs600</td>
 
−
<td class="yellow" style="background-color: #ffcc00;text-align: left; border: 1px solid #000000;">0.023</td>
 
−
<td style="border: 0; background-color: transparent;"></td>
 
−
</tr>
 
−
<tr>
 
−
<td class="no-border" style="border: 0;text-align: left;">Reference OD600</td>
 
−
<td class="yellow" style="background-color: #ffcc00;text-align: left; border: 1px solid #000000;">0.063</td>
 
−
<td style="border: 0; background-color: transparent;"></td>
 
−
</tr>
 
−
<tr>
 
−
<td class="no-border" style="border: 0;text-align: left;">OD600/Abs600</td>
 
−
<td class="yellow" style="background-color: #ffcc00;text-align: left; border: 1px solid #000000;">2.769</td>
 
−
<td style="border: 0; background-color: transparent;"></td>
 
−
</tr>
 
−
</table>
 
−
<p>Fluorescein standard curves</p>
 
−
<img src="https://static.igem.org/mediawiki/2018/4/40/T--SCAU-China--Fluoresceinstandardcurves.jpg">
 
−
<p>Particle standard curve</p>
 
−
<img src="https://static.igem.org/mediawiki/2018/8/8d/T--SCAU-China--Particlestandardcurve.jpg">
 
−
<p>Our goal is to reduce lab-to-lab variations in fluorescent intensity measurements, in order to test the variation in our sample, we caculated coefficient of variation for both colony, and compared the CV value by using different normalization method (fluorescence per OD or  fluorescence per particle)</p>
 
−
<p>0th</p>
 
−
<img src="https://static.igem.org/mediawiki/2018/8/8a/T--SCAU-China--0th1.jpg">
 
−
<img src="https://static.igem.org/mediawiki/2018/3/37/T--SCAU-China--0th2.jpg">
 
−
<p>6th</p>
 
−
<img src="https://static.igem.org/mediawiki/2018/9/94/T--SCAU-China--6th1.jpg">
 
−
<img src="https://static.igem.org/mediawiki/2018/a/af/T--SCAU-China--6th2.jpg">
 
−
<p>After 6 hr growth, we found that the variation of each type of cells (devices) is decreased, but the CV value of devices 4 and device5 are much higher than the others. The variation of fluorescence per OD is much smaller than fluorescence per particle. Furthermore, we also found that the data from colony 2’s is usually more steady compared with colony 1.
 
−
To conveniently compare the normalized value with original value, we removed the data of devices 4 and device 5, and only compare to the 6-hour’s data of colony 2 .
 
−
</p>
 
−
<img src="https://static.igem.org/mediawiki/2018/6/65/T--SCAU-China--interlabLast.jpg">
 
−
<p>According to the graph, we found that fluorescence per particle could decrease the variation but it  depending on the devices. It seemed that usage ofe OD value to normalize is still better than particle. But one thing need to be pointed out that too steady normalized value will lose sensitivity when test the influences of some factors.  You can see this <a href="https://static.igem.org/mediawiki/2017/e/ef/T--oxford--interlab-data.xlsx">excel file</a> to check our raw and manipulated data.
 
−
</p>
 
 
</div>
 
</div>
 
<div class="DBoard" id="title">
 
<div class="DBoard" id="title">
Line 600: Line 532:
 
<img src="https://static.igem.org/mediawiki/2018/2/2f/T--SCAU-China--parts-2.jpg" style="width: 100%" />
 
<img src="https://static.igem.org/mediawiki/2018/2/2f/T--SCAU-China--parts-2.jpg" style="width: 100%" />
 
<p>
 
<p>
−
These three parts were obtained from the Acetobacter xylinum bacterial cellulose synthase, as they were suspected to function together to regulate the expression and secretion of bacterial cellulose.  
+
This three parts were obtained from the Acetobacter Xylinum bacterial cellulose synthase, as they were suspected to function together to regulate the expression and secretion of bacterial cellulose.  
 
</p>
 
</p>
 
<p>
 
<p>
−
The bcs Z(BBa_K2559000) is coding an endoglucanase in the process of cellulose hydrolysis, bcs H (BBa_K2559001) can express cellulose complement protein A which affects the expression levels of bcsB and bcsC, interacts with bcsD.
+
This bcs Z(BBa_K2559000) is coding an endoglucanase in the process of cellulose hydrolysis, bcs H (BBa_K2559001) can express cellulose complement protein A which affects the expression levels of bcsB and bcsC, interacts with bcsD.
 
bglX (BBa_K2559003) is a β-glucosidase.  
 
bglX (BBa_K2559003) is a β-glucosidase.  
 +
  
 
</p>
 
</p>
Line 611: Line 544:
 
</p>
 
</p>
 
<img src="https://static.igem.org/mediawiki/2018/e/ec/T--SCAU-China--parts-3.jpg" style="width: 100%" />
 
<img src="https://static.igem.org/mediawiki/2018/e/ec/T--SCAU-China--parts-3.jpg" style="width: 100%" />
−
<p>
+
<p>This three parts BBa_K2559000, BBa_K2559001 and BBa_K2559002 were obtained from E. coli, and but the promoters (PrplJ, PdapA, PcaiF) are totally different in expressional intensity.  
−
The three parts BBa_K2559003, BBa_K2559004 and BBa_K2559011 were obtained from E. coli, and but all the promoter pRplj , pDapa and pCaif are totally different in expressional intensity(Figure1).  
+
 
</p>
 
</p>
 
<p></p>
 
<p></p>
Line 620: Line 552:
 
</p>
 
</p>
 
<img src="https://static.igem.org/mediawiki/2018/5/52/T--SCAU-China--parts-4.jpg" style="width: 100%" />
 
<img src="https://static.igem.org/mediawiki/2018/5/52/T--SCAU-China--parts-4.jpg" style="width: 100%" />
−
<p>The four parts BBa_K2559007, BBa_K2559008 ,BBa_K2559012 BBa_K2559013 were used for the construction of our synergistic recombination and CRISPR/cas9 Kit</p>
+
<p>This four parts BBa_K2559007, BBa_K2559008, BBa_K2559012 and BBa_K2559013 were used for the construction of our synergistic recombination and CRISPR/cas9 kit</p>
 
</div>
 
</div>
 
<p>School's name:SCAU</p>
 
<p>School's name:SCAU</p>

Latest revision as of 16:56, 17 October 2018

SCAU-2018
1. Parts collection

The parts we used are listed at the table below.

Part number Type Function description Medal
BBa_K515107 Composite Dendra2 cording Bronze
BBa_K2559000 Basic endoglucanase Silver
BBa_K2559001 Basic cellulose complement protein A Silver
BBa_K2559002 Basic β-glucosidase Silver
BBa_K2559003 Basic E. coli. Promoter PrplJ Unsubmit
BBa_K2559004 Basic E. coli. Promoter Pdapa Unsubmit
BBa_K2559011 Basic E. coli. Promoter PcaiF Unsubmit
BBa_K2559007 Basic Promoter of Heat Shock Protein Pgroel Unsubmit
BBa_K2559008 Basic slr0168 Unsubmit
BBa_K2559012 Basic Promoter of RuBisCO(Prbc) Unsubmit
BBa_K2559013 Basic Terminator of RuBisCO(Trbc) Unsubmit
BBa_K2559005 Basic enhance green fluorescent protein Gold
BBa_K2559009 Composite expression of eGFP Gold
2. Basic parts and Composite Part

(1) BBa_K515107

We characterized the BBa_K515107 part comprising the coding sequence BBa_K515007 which coded for Dendra2, a photoconvertible reporter protein under the control of the repressible promoter TetR (BBa_R0040) and the RBS (BBa_B0034).

(2) BBa_K2559000/ BBa_K2559001/ BBa_K2559002

This three parts were obtained from the Acetobacter Xylinum bacterial cellulose synthase, as they were suspected to function together to regulate the expression and secretion of bacterial cellulose.

This bcs Z(BBa_K2559000) is coding an endoglucanase in the process of cellulose hydrolysis, bcs H (BBa_K2559001) can express cellulose complement protein A which affects the expression levels of bcsB and bcsC, interacts with bcsD. bglX (BBa_K2559003) is a β-glucosidase.

(3) BBa_K2559003/ BBa_K2559004/ BBa_K2559011

This three parts BBa_K2559000, BBa_K2559001 and BBa_K2559002 were obtained from E. coli, and but the promoters (PrplJ, PdapA, PcaiF) are totally different in expressional intensity.

(4) BBa_K2559007/ BBa_K2559008/ BBa_K2559012/ BBa_K2559013

This four parts BBa_K2559007, BBa_K2559008, BBa_K2559012 and BBa_K2559013 were used for the construction of our synergistic recombination and CRISPR/cas9 kit

School's name:SCAU

Member's name:SCAU

Designed by:SCAU