Difference between revisions of "Team:HK SSC/Model"

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<html>
 +
<div class="column full_size">
 +
<h1> Modelling</h1><br>
  
 +
<h3>Introduction</h3>
 +
<p>We would like to see how the transformation of this plasmid affects the growth of cells. We transformed our part (BBa_K3219002) into E.coli BL21 and E.coli DH5α, and compared their growth rates to E.coli transformed with common plasmids. In this case, we can see how this large plasmid of 12kbp affects the growth rate of cells, and whether it should be modified in future studies.</p>
  
 +
<h3>Background:</h3>
 +
<p>It was found that plasmid sizes and different origin of replications have distinct effects on the growth of E.coli <ref>U. EONG CHEAH, WILLIAM A. WEIGAND, BENJAMIN C. STARK. “Effects of Recombinant Plasmid Size on Cellular Processes in Escherichia coli.” Plasmid (1987): 127-134 . Journal.</ref>. Generally, larger plasmid sizes result in a slower growth rate in E.coli. While our plasmid is larger than 12 kbp, we would like to know whether this large plasmid size will affect the growth rate of E.coli transformed with this plasmid. If the transformation of this plasmid results in a very low cell yield or a slow growth curve, protocols may have to be modified in order to achieve ideal plasmid yield (for conducting plasmid purification) or protein expression.</p> <br>
  
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<div class="column full_size judges-will-not-evaluate">
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<h3>Methods: </h3>
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<h3>★  ALERT! </h3>
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<h5>1. Preparation of Cells</h5><br>
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<p>This page is used by the judges to evaluate your team for the <a href="https://2019.igem.org/Judging/Medals">medal criterion</a> or <a href="https://2019.igem.org/Judging/Awards"> award listed below</a>. </p>
+
<p>E.coli competent cells were prepared using Inoue Method<ref> Im, H. (2011). The Inoue Method for Preparation and Transformation of Competent E. coli: "Ultra Competent" Cells. Bio-101: e143. DOI: 10.21769/BioProtoc.143.</ref> .<br></p>
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<p> Delete this box in order to be evaluated for this medal criterion and/or award. See more information at <a href="https://2019.igem.org/Judging/Pages_for_Awards"> Instructions for Pages for awards</a>.</p>
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−
</div>
+
  
 +
<h5>2. Calibration</h5>
 +
<p>We followed iGEM 2019 Plate Reader Abs600 (OD) Calibration protocol, so that we can estimate our number cells.<br></p>
  
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<div class="clear"></div>
+
<h5>3. Design and Cloning of BBa_K1894002</h5></br>
 +
<p>Please see the <a href="https://2019.igem.org/Team:HK_SSC/Design"> Design page</a> for details. </p>
 +
<p>The cloning methods could be summarized below:<p/><br>
 +
<div class="cen"><img style="width:70%; height:70%; margin: auto;" src="https://static.igem.org/mediawiki/parts/e/e1/T--HK_SSC--Gibson.png"/></div>
  
 +
<p>Our Sanger sequencing results have shown no undesired mutations in the junctions.<p/><br>
  
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<div class="column full_size">
+
<h5>4. Transformation</h5>
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<h1> Modeling</h1>
+
<p>We transformed this plasmid into E.coli DH5α and E.coli BL21 (DE3) using heat shock (42°C for 45 seconds). In order to compare the growth curves of plasmid BBa_K1894002 with other plasmids including part BBa_K1894001, Psb1c3 and pUC19, we also transformed these plasmids into E.coli DH5α and E.coli BL21 (DE3). Transformants were spread onto agar plates with respective antibiotics.</p><br>
  
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<p>Mathematical models and computer simulations provide a great way to describe the function and operation of BioBrick Parts and Devices. Synthetic Biology is an engineering discipline, and part of engineering is simulation and modeling to determine the behavior of your design before you build it. Designing and simulating can be iterated many times in a computer before moving to the lab. This award is for teams who build a model of their system and use it to inform system design or simulate expected behavior in conjunction with experiments in the wetlab.</p>
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<h5>5. Inoculation</h5>
  
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</div>
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<p>Single colonies from each plate were picked. They are inoculated in 3mL of LB with antibiotics for 16 hours at 37°C shaking at 250r.p.m. </p><br>
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<div class="clear"></div>
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<div class="column full_size">
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<h5>6. Measurement</h5>
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<h3> Gold Medal Criterion #3</h3>
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<p>OD600 of the cell cultures were measured and diluted to OD600 ~ 0.1. Then, the diluted culture was inoculated at 37°C shaking at 250r.p.m. OD600 was taken exactly every 30 minutes interval. E.coli BL21 (DE3) with inserts of Psb1c3 (2070) and shuttle vector BBa_K1894001 (6913bp), and plasmid BBa_K1894002 (12280bp) are compared. E.coli DH5α with inserts Psb1c3 (2070 bp), PUC19 (2686 bp), shuttle vector BBa_K1894001 (6913 bp) and plasmid BBa_K1894002 (12280bp) are compared. The experiments are performed in triplicates. </p>
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<p>
+
 
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Convince the judges that your project's design and/or implementation is based on insight you have gained from modeling. This could be either a new model you develop or the implementation of a model from a previous team. You must thoroughly document your model's contribution to your project on your team's wiki, including assumptions, relevant data, model results, and a clear explanation of your model that anyone can understand.  
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<table class="wikitable">
−
<br><br>
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<caption>Assumptions made:
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The model should impact your project design in a meaningful way. Modeling may include, but is not limited to, deterministic, exploratory, molecular dynamic, and stochastic models. Teams may also explore the physical modeling of a single component within a system or utilize mathematical modeling for predicting function of a more complex device.
+
</caption>
 +
<tr>
 +
<td>Assumption
 +
</td>
 +
<td>Justification
 +
</td></tr>
 +
<tr>
 +
<td>1. The OD<sub>600</sub> = 0.1 is equivalent to 9.3x10<sup>6</sup>
 +
</td>
 +
<td>This is according to the calibration curve performed using iGEM standard protocols: Calibration Protocol - Plate Reader Abs600 (OD) Calibration with Microsphere Particles V.2
 +
</td></tr>
 +
<tr>
 +
<td>2. The OD<sub>600</sub> of the overnight culture is the maximum OD<sub>600</sub>.
 +
</td>
 +
<td>OD<sub>600</sub> remains constant staring from the stationary phase  and 16 hours of incubation takes the E.coli to stationary phase .
 +
</td></tr></table> <br />
 +
<h4><span class="mw-headline" id="Results">Results</span></h4>
 +
<p>The below graph shows the results of the optical density measurements. The data shown is the average of three replicates.
 
</p>
 
</p>
 +
<table class="wikitable">
 +
<caption>E.coli DH5α
 +
</caption>
 +
<tr>
 +
<td>Time (h)
 +
</td>
 +
<td>Psb1c3  (OD<sub>600</sub>)
 +
</td>
 +
<td>PUC19  (OD<sub>600</sub>)
 +
</td>
 +
<td>BBa_K1894001 (OD<sub>600</sub>)
 +
</td>
 +
<td><b>BBa_K1894002</b>  (OD<sub>600</sub>)
 +
</td></tr>
 +
<tr>
 +
<td>0
 +
</td>
 +
<td>0.172
 +
</td>
 +
<td>0.119
 +
</td>
 +
<td>0.122
 +
</td>
 +
<td><b>0.141</b>
 +
</td></tr>
 +
<tr>
 +
<td>0.5
 +
</td>
 +
<td>0.198
 +
</td>
 +
<td>0.155
 +
</td>
 +
<td>0.151
 +
</td>
 +
<td><b>0.184</b>
 +
</td></tr>
 +
<tr>
 +
<td>1.0
 +
</td>
 +
<td>0.387
 +
</td>
 +
<td>0.337
 +
</td>
 +
<td>0.332
 +
</td>
 +
<td><b>0.283</b>
 +
</td></tr>
 +
<tr>
 +
<td>1.5
 +
</td>
 +
<td>0.681
 +
</td>
 +
<td>0.572
 +
</td>
 +
<td>0.635
 +
</td>
 +
<td><b>0.460</b>
 +
</td></tr>
 +
<tr>
 +
<td>2.0
 +
</td>
 +
<td>1.219
 +
</td>
 +
<td>0.972
 +
</td>
 +
<td>0.932
 +
</td>
 +
<td><b>0.592</b>
 +
</td></tr>
 +
<tr>
 +
<td>2.5
 +
</td>
 +
<td>1.604
 +
</td>
 +
<td>1.165
 +
</td>
 +
<td>1.383
 +
</td>
 +
<td><b>0.765</b>
 +
</td></tr>
 +
<tr>
 +
<td>3.0
 +
</td>
 +
<td>2.166
 +
</td>
 +
<td>1.399
 +
</td>
 +
<td>1.683
 +
</td>
 +
<td><b>0.991</b>
 +
</td></tr>
 +
<tr>
 +
<td>3.5
 +
</td>
 +
<td>2.430
 +
</td>
 +
<td>1.584
 +
</td>
 +
<td>1.851
 +
</td>
 +
<td><b>1.166</b>
 +
</td></tr>
 +
<tr>
 +
<td>4.0
 +
</td>
 +
<td>2.437
 +
</td>
 +
<td>1.608
 +
</td>
 +
<td>1.621
 +
</td>
 +
<td><b>1.369</b>
 +
</td></tr>
 +
<tr>
 +
<td>4.5
 +
</td>
 +
<td>2.731
 +
</td>
 +
<td>1.734
 +
</td>
 +
<td>2.194
 +
</td>
 +
<td><b>1.542</b>
 +
</td></tr>
 +
<tr>
 +
<td>16.0
 +
</td>
 +
<td>3.249
 +
</td>
 +
<td>1.902
 +
</td>
 +
<td>2.481
 +
</td>
 +
<td><b>1.815</b>
 +
</td></tr></table>
 +
<table class="wikitable">
 +
<caption>E.coli BL21 (DE3)
 +
</caption>
 +
<tr>
 +
<td>Time (h)
 +
</td>
 +
<td>Psb1c3  (OD<sub>600</sub>)
 +
</td>
 +
<td>BBa_K1894001    (OD<sub>600</sub>)
 +
</td>
 +
<td><b>BBa_K1894002</b>  (OD<sub>600</sub>)
 +
</td></tr>
 +
<tr>
 +
<td>0
 +
</td>
 +
<td>0.108
 +
</td>
 +
<td>0.128
 +
</td>
 +
<td><b>0.197</b>
 +
</td></tr>
 +
<tr>
 +
<td>0.5
 +
</td>
 +
<td>0.162
 +
</td>
 +
<td>0.178
 +
</td>
 +
<td><b>0.304</b>
 +
</td></tr>
 +
<tr>
 +
<td>1
 +
</td>
 +
<td>0.377
 +
</td>
 +
<td>0.377
 +
</td>
 +
<td><b>0.548</b>
 +
</td></tr>
 +
<tr>
 +
<td>1.5
 +
</td>
 +
<td>0.677
 +
</td>
 +
<td>0.662
 +
</td>
 +
<td><b>0.979</b>
 +
</td></tr>
 +
<tr>
 +
<td>2
 +
</td>
 +
<td>1.213
 +
</td>
 +
<td>1.133
 +
</td>
 +
<td><b>1.321</b>
 +
</td></tr>
 +
<tr>
 +
<td>2.5
 +
</td>
 +
<td>1.406
 +
</td>
 +
<td>1.342
 +
</td>
 +
<td><b>1.61</b>
 +
</td></tr>
 +
<tr>
 +
<td>3.0
 +
</td>
 +
<td>1.746
 +
</td>
 +
<td>1.703
 +
</td>
 +
<td><b>2.031</b>
 +
</td></tr>
 +
<tr>
 +
<td>3.5
 +
</td>
 +
<td>1.911
 +
</td>
 +
<td>1.820
 +
</td>
 +
<td><b>2.218</b>
 +
</td></tr>
 +
<tr>
 +
<td>4.0
 +
</td>
 +
<td>2.063
 +
</td>
 +
<td>1.847
 +
</td>
 +
<td><b>2.262</b>
 +
</td></tr>
 +
<tr>
 +
<td>4.5
 +
</td>
 +
<td>2.266
 +
</td>
 +
<td>2.098
 +
</td>
 +
<td><b>2.391</b>
 +
</td></tr>
 +
<tr>
 +
<td>16.0
 +
</td>
 +
<td>2.836
 +
</td>
 +
<td>3.138
 +
</td>
 +
<td><b>2.618</b>
 +
</td></tr></table>
 +
<p><br />
 +
</p></br>
  
−
</div>
+
<p>We further converted these data to the estimated number of cell counts using the iGEM standard protocols: Calibration Protocol - Plate Reader Abs600 (OD) Calibration with Microsphere Particles V.2. </p></br>
  
−
<div class="column two_thirds_size">
+
<table class="wikitable">
−
<h3>Best Model Special Prize</h3>
+
<caption>E.coli DH5α
 +
</caption>
 +
<tr>
 +
<td>Time (h)
 +
</td>
 +
<td>Psb1c3  (No. of Cells)
 +
</td>
 +
<td>PUC19  (No. of Cells)
 +
</td>
 +
<td>BBa_K1894001 (No. of Cells)
 +
</td>
 +
<td><b>BBa_K1894002</b>  (No. of Cells)
 +
</td></tr>
 +
<tr>
 +
<td>0
 +
</td>
 +
<td>1.83x10<sup>7</sup>
 +
</td>
 +
<td>1.11x10<sup>7</sup>
 +
</td>
 +
<td>1.14x10<sup>7</sup>
 +
</td>
 +
<td><b>1.31x10<sup>7</sup></b>
 +
</td></tr>
 +
<tr>
 +
<td>0.5
 +
</td>
 +
<td>1.84x10<sup>7</sup>
 +
</td>
 +
<td>1.44x10<sup>7</sup>
 +
</td>
 +
<td>1.41x10<sup>7</sup>
 +
</td>
 +
<td><b>1.71x10<sup>7</sup></b>
 +
</td></tr>
 +
<tr>
 +
<td>1.0
 +
</td>
 +
<td>3.61x10<sup>7</sup>
 +
</td>
 +
<td>3.14x10<sup>7</sup>
 +
</td>
 +
<td>3.09x10<sup>7</sup>
 +
</td>
 +
<td><b>2.63x10<sup>7</sup></b>
 +
</td></tr>
 +
<tr>
 +
<td>1.5
 +
</td>
 +
<td>6.35x10<sup>7</sup>
 +
</td>
 +
<td>5.33x10<sup>7</sup>
 +
</td>
 +
<td>5.92x10<sup>7</sup>
 +
</td>
 +
<td><b>4.29x10<sup>7</sup></b>
 +
</td></tr>
 +
<tr>
 +
<td>2.0
 +
</td>
 +
<td>11.4x10<sup>7</sup>
 +
</td>
 +
<td>9.06x10<sup>7</sup>
 +
</td>
 +
<td>8.69x10<sup>7</sup>
 +
</td>
 +
<td><b>5.52x10<sup>7</sup></b>
 +
</td></tr>
 +
<tr>
 +
<td>2.5
 +
</td>
 +
<td>15x10<sup>7</sup>
 +
</td>
 +
<td>10.9x10<sup>7</sup>
 +
</td>
 +
<td>12.9x10<sup>7</sup>
 +
</td>
 +
<td><b>7.13x10<sup>7</sup></b>
 +
</td></tr>
 +
<tr>
 +
<td>3.0
 +
</td>
 +
<td>20.2x10<sup>7</sup>
 +
</td>
 +
<td>13.0x10<sup>7</sup>
 +
</td>
 +
<td>15.7x10<sup>7</sup>
 +
</td>
 +
<td><b>9.24x10<sup>7</sup></b>
 +
</td></tr>
 +
<tr>
 +
<td>3.5
 +
</td>
 +
<td>22.7x10<sup>7</sup>
 +
</td>
 +
<td>14.8x10<sup>7</sup>
 +
</td>
 +
<td>17.3x10<sup>7</sup>
 +
</td>
 +
<td><b>10.9x10<sup>7</sup></b>
 +
</td></tr>
 +
<tr>
 +
<td>4.0
 +
</td>
 +
<td>23.9x10<sup>7</sup>
 +
</td>
 +
<td>15.0x10<sup>7</sup>
 +
</td>
 +
<td>18.4x10<sup>7</sup>
 +
</td>
 +
<td><b>12.8x10<sup>7</sup></b>
 +
</td></tr>
 +
<tr>
 +
<td>4.5
 +
</td>
 +
<td>25.5x10<sup>7</sup>
 +
</td>
 +
<td>16.2x10<sup>7</sup>
 +
</td>
 +
<td>20.4x10<sup>7</sup>
 +
</td>
 +
<td><b>14.4x10<sup>7</sup></b>
 +
</td></tr>
 +
<tr>
 +
<td>16.0
 +
</td>
 +
<td>30.3x10<sup>7</sup>
 +
</td>
 +
<td>17.7x10<sup>7</sup>
 +
</td>
 +
<td>23.1x10<sup>7</sup>
 +
</td>
 +
<td><b>16.9x10<sup>7</sup></b>
  
−
<p>
+
</td></tr></table></br>
−
To compete for the <a href="https://2019.igem.org/Judging/Awards">Best Model prize</a>, please describe your work on this page  and also fill out the description on the <a href="https://2019.igem.org/Judging/Judging_Form">judging form</a>. Please note you can compete for both the Gold Medal criterion #3 and the Best Model prize with this page.  
+
<table class="wikitable">
−
<br><br>
+
<caption>E.coli BL21 (DE3)
−
You must also delete the message box on the top of this page to be eligible for the Best Model Prize.
+
</caption>
−
</p>
+
<tr>
 +
<td>Time (h)
 +
</td>
 +
<td>Psb1c3  (No. of cells)
 +
</td>
 +
<td>BBa_K1894001    (No. of cells)
 +
</td>
 +
<td><b>BBa_K1894002</b>   (No. of cells)
 +
</td></tr>
 +
<tr>
 +
<td>0
 +
</td>
 +
<td>1x10<sup>7</sup>
 +
</td>
 +
<td>1.19x10<sup>7</sup>
 +
</td>
 +
<td><b>1.83x10<sup>7</sup></b>
 +
</td></tr>
 +
<tr>
 +
<td>0.5
 +
</td>
 +
<td>1.51x10<sup>7</sup>
 +
</td>
 +
<td>1.66x10<sup>7</sup>
 +
</td>
 +
<td><b>2.83x10<sup>7</sup></b>
 +
</td></tr>
 +
<tr>
 +
<td>1
 +
</td>
 +
<td>3.51x10<sup>7</sup>
 +
</td>
 +
<td>3.51x10<sup>7</sup>
 +
</td>
 +
<td><b>5.10x10<sup>7</sup></b>
 +
</td></tr>
 +
<tr>
 +
<td>1.5
 +
</td>
 +
<td>6.31x10<sup>7</sup>
 +
</td>
 +
<td>6.17x10<sup>7</sup>
 +
</td>
 +
<td><b>9.13x10<sup>7</sup></b>
 +
</td></tr>
 +
<tr>
 +
<td>2
 +
</td>
 +
<td>11.3x10<sup>7</sup>
 +
</td>
 +
<td>10.6x10<sup>7</sup>
 +
</td>
 +
<td><b>12.3x10<sup>7</sup></b>
 +
</td></tr>
 +
<tr>
 +
<td>2.5
 +
</td>
 +
<td>13.1x10<sup>7</sup>
 +
</td>
 +
<td>12.5x10<sup>7</sup>
 +
</td>
 +
<td><b>15.0x10<sup>7</sup></b>
 +
</td></tr>
 +
<tr>
 +
<td>3.0
 +
</td>
 +
<td>16.3x10<sup>7</sup>
 +
</td>
 +
<td>15.9x10<sup>7</sup>
 +
</td>
 +
<td><b>18.9x10<sup>7</sup></b>
 +
</td></tr>
 +
<tr>
 +
<td>3.5
 +
</td>
 +
<td>17.8x10<sup>7</sup>
 +
</td>
 +
<td>17.0x10<sup>7</sup>
 +
</td>
 +
<td><b>20.5x10<sup>7</sup></b>
 +
</td></tr>
 +
<tr>
 +
<td>4.0
 +
</td>
 +
<td>19.2x10<sup>7</sup>
 +
</td>
 +
<td>18.1x10<sup>7</sup>
 +
</td>
 +
<td><b>21.4x10<sup>7</sup></b>
 +
</td></tr>
 +
<tr>
 +
<td>4.5
 +
</td>
 +
<td>21.1x10<sup>7</sup>
 +
</td>
 +
<td>19.6x10<sup>7</sup>
 +
</td>
 +
<td><b>22.3x10<sup>7</sup></b>
 +
</td></tr>
 +
<tr>
 +
<td>16.0
 +
</td>
 +
<td>26.4x10<sup>7</sup>
 +
</td>
 +
<td>29.3x10<sup>7</sup>
 +
</td>
 +
<td><b>24.4x10<sup>7</sup></b>
 +
</td></tr></table>
 +
<p><br />
 +
</p></br>
 +
<h5>7. Curve Fitting</h5>
 +
<p>Gompertz model is the most frequently used sigmoid model to fit growth data in biology.</p>
 +
<p>We took the logarithm to base 10 value, and plotted a growth curve using the Grompertz model. Here are the results:</p></br>
 +
<h3> Equation of BBa_K3219002 transformed in E.coli</h3>
 +
<div class="cen"><img style="width:70%; height:70%; margin: auto;" src="https://static.igem.org/mediawiki/2019/b/b8/T--HK_SSC--aDH5a_equation.gif"/></div></br>
  
−
</div>
 
  
 +
<div class="center"><h3>Growth curve of E.coli DH5α Transfromants</h3></div></br>
 +
<div class="cen"><img style="width:70%; height:70%; margin: auto;" src="https://static.igem.org/mediawiki/2019/9/95/T--HK_SSC--Our_part_dh5a.png"/></div></br>
 +
<p><a href="https://www.desmos.com/calculator/urwlp5munf"> Link</a> to graph.</p></br/br>
  
−
<div class="column third_size">
+
<h3>Equation of BBa_K3219002 transformed to E.coli BL21 (DE3)</h3>
−
<div class="highlight decoration_A_full">
+
<div class="cen"><img style="width:70%; height:70%; margin: auto;" src="https://static.igem.org/mediawiki/2019/e/ee/T--HK_SSC--Bl21_equation.png"/></div></br>
−
<h3> Inspiration </h3>
+
 
−
<p>
+
 
−
Here are a few examples from previous teams:
+
<div class="center"><h3>Growth curve of E.coli BL21 (DE3) Transfromants</h3></div></br>
 +
<div class="cen"><img style="width:70%; height:70%; margin: auto;" src="https://static.igem.org/mediawiki/2019/2/28/T--HK_SSC--Our_part_bl21.png"/></div></br>
 +
<p><a href="https://www.desmos.com/calculator/u4cau02pv0"> Link</a> to graph.</p>
 +
 
 +
https://static.igem.org/mediawiki/2019/9/95/T--HK_SSC--Our_part_dh5a.png
 +
 
 +
<h3>Conculsion</h3>
 +
<p>In DH5α, the growth rate of BBa_K1894002 seems to be distinctively slower than that of other plasmids. The cell yield is also very low, almost half of the cell yield of Psb1c3 transformants. We believe that this result fits our hypothesis that this large plasmid affects cell growth. Protocols have to be optimized when using BBa_K1894002. For example, the inoculation time before plasmid purification can be increased just to achieve the ideal DNA yield, and it may take a longer time for colonies to form after transformation due to the low replication rate. <p>
 +
 
 +
<h3>References </h3>
 +
<p>↑ Dittmann, Elke. “Insertional mutagenesis of a peptide synthetase gene that is responsible for hepatotoxin production in the cyanobacterium Microcystis Aeruginosa PCC 7806.” Molecular Microbiology (1997): 779–787. Journal.</p>
 +
<p>↑ http://parts.igem.org/Part:BBa_K1894001</p>
 +
<p>↑ http://parts.igem.org/Part:BBa_K1689013</p>
 +
<p>↑ 章军, 徐虹, 楼士林, 欧阳青. Blue-green alga shuttle plasmid expression vector and method for expressing thymison 'alpha' 1. Thesis. Xia Men: Xia Men University, 1999.</p>
 +
<p>↑ Semary, Nermin Adel El. “Optimized electroporation-induced transformation in Microcystis aeruginosa PCC7806.” Biotechnol. Agron. Soc. Environ (2010): 149-152 . Journal.</p>
 +
<p>↑ Dittmann, Elke. “Insertional mutagenesis of a peptide synthetase gene that is responsible for hepatotoxin production in the cyanobacterium Microcystis Aeruginosa PCC 7806.” Molecular Microbiology (1997): 779–787. Journal.</p>
 +
<p>↑ U. EONG CHEAH, WILLIAM A. WEIGAND, BENJAMIN C. STARK. “Effects of Recombinant Plasmid Size on Cellular Processes in Escherichia coli.” Plasmid (1987): 127-134 . Journal.</p>
 +
<p>↑ Im, H. (2011). The Inoue Method for Preparation and Transformation of Competent E. coli: "Ultra Competent" Cells. Bio-101: e143. DOI: 10.21769/BioProtoc.143.</p>
 +
 
 +
</p><p><br />
 
</p>
 
</p>
−
<ul>
+
 
−
<li><a href="https://2018.igem.org/Team:GreatBay_China/Model">2018 GreatBay China</a></li>
+
 
−
<li><a href="https://2018.igem.org/Team:Leiden/Model">2018 Leiden</a></li>
+
 
−
<li><a href="https://2016.igem.org/Team:Manchester/Model">2016 Manchester</a></li>
+
 
−
<li><a href="https://2016.igem.org/Team:TU_Delft/Model">2016 TU Delft</li>
+
 
−
<li><a href="https://2014.igem.org/Team:ETH_Zurich/modeling/overview">2014 ETH Zurich</a></li>
+
 
−
<li><a href="https://2014.igem.org/Team:Waterloo/Math_Book">2014 Waterloo</a></li>
+
−
</ul>
+
−
</div>
+
−
</div>
+
  
 
</html>
 
</html>

Latest revision as of 02:39, 22 October 2019

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JUDGING FORM ⇗

Modelling


Introduction

We would like to see how the transformation of this plasmid affects the growth of cells. We transformed our part (BBa_K3219002) into E.coli BL21 and E.coli DH5α, and compared their growth rates to E.coli transformed with common plasmids. In this case, we can see how this large plasmid of 12kbp affects the growth rate of cells, and whether it should be modified in future studies.

Background:

It was found that plasmid sizes and different origin of replications have distinct effects on the growth of E.coli U. EONG CHEAH, WILLIAM A. WEIGAND, BENJAMIN C. STARK. “Effects of Recombinant Plasmid Size on Cellular Processes in Escherichia coli.” Plasmid (1987): 127-134 . Journal.. Generally, larger plasmid sizes result in a slower growth rate in E.coli. While our plasmid is larger than 12 kbp, we would like to know whether this large plasmid size will affect the growth rate of E.coli transformed with this plasmid. If the transformation of this plasmid results in a very low cell yield or a slow growth curve, protocols may have to be modified in order to achieve ideal plasmid yield (for conducting plasmid purification) or protein expression.


Methods:

1. Preparation of Cells

E.coli competent cells were prepared using Inoue Method Im, H. (2011). The Inoue Method for Preparation and Transformation of Competent E. coli: "Ultra Competent" Cells. Bio-101: e143. DOI: 10.21769/BioProtoc.143. .

2. Calibration

We followed iGEM 2019 Plate Reader Abs600 (OD) Calibration protocol, so that we can estimate our number cells.

3. Design and Cloning of BBa_K1894002

Please see the Design page for details.

The cloning methods could be summarized below:


Our Sanger sequencing results have shown no undesired mutations in the junctions.


4. Transformation

We transformed this plasmid into E.coli DH5α and E.coli BL21 (DE3) using heat shock (42°C for 45 seconds). In order to compare the growth curves of plasmid BBa_K1894002 with other plasmids including part BBa_K1894001, Psb1c3 and pUC19, we also transformed these plasmids into E.coli DH5α and E.coli BL21 (DE3). Transformants were spread onto agar plates with respective antibiotics.


5. Inoculation

Single colonies from each plate were picked. They are inoculated in 3mL of LB with antibiotics for 16 hours at 37°C shaking at 250r.p.m.


6. Measurement

OD600 of the cell cultures were measured and diluted to OD600 ~ 0.1. Then, the diluted culture was inoculated at 37°C shaking at 250r.p.m. OD600 was taken exactly every 30 minutes interval. E.coli BL21 (DE3) with inserts of Psb1c3 (2070) and shuttle vector BBa_K1894001 (6913bp), and plasmid BBa_K1894002 (12280bp) are compared. E.coli DH5α with inserts Psb1c3 (2070 bp), PUC19 (2686 bp), shuttle vector BBa_K1894001 (6913 bp) and plasmid BBa_K1894002 (12280bp) are compared. The experiments are performed in triplicates.

Assumptions made:
Assumption Justification
1. The OD600 = 0.1 is equivalent to 9.3x106 This is according to the calibration curve performed using iGEM standard protocols: Calibration Protocol - Plate Reader Abs600 (OD) Calibration with Microsphere Particles V.2
2. The OD600 of the overnight culture is the maximum OD600. OD600 remains constant staring from the stationary phase and 16 hours of incubation takes the E.coli to stationary phase .

Results

The below graph shows the results of the optical density measurements. The data shown is the average of three replicates.

E.coli DH5α
Time (h) Psb1c3 (OD600) PUC19 (OD600) BBa_K1894001 (OD600) BBa_K1894002 (OD600)
0 0.172 0.119 0.122 0.141
0.5 0.198 0.155 0.151 0.184
1.0 0.387 0.337 0.332 0.283
1.5 0.681 0.572 0.635 0.460
2.0 1.219 0.972 0.932 0.592
2.5 1.604 1.165 1.383 0.765
3.0 2.166 1.399 1.683 0.991
3.5 2.430 1.584 1.851 1.166
4.0 2.437 1.608 1.621 1.369
4.5 2.731 1.734 2.194 1.542
16.0 3.249 1.902 2.481 1.815
E.coli BL21 (DE3)
Time (h) Psb1c3 (OD600) BBa_K1894001 (OD600) BBa_K1894002 (OD600)
0 0.108 0.128 0.197
0.5 0.162 0.178 0.304
1 0.377 0.377 0.548
1.5 0.677 0.662 0.979
2 1.213 1.133 1.321
2.5 1.406 1.342 1.61
3.0 1.746 1.703 2.031
3.5 1.911 1.820 2.218
4.0 2.063 1.847 2.262
4.5 2.266 2.098 2.391
16.0 2.836 3.138 2.618



We further converted these data to the estimated number of cell counts using the iGEM standard protocols: Calibration Protocol - Plate Reader Abs600 (OD) Calibration with Microsphere Particles V.2.


E.coli DH5α
Time (h) Psb1c3 (No. of Cells) PUC19 (No. of Cells) BBa_K1894001 (No. of Cells) BBa_K1894002 (No. of Cells)
0 1.83x107 1.11x107 1.14x107 1.31x107
0.5 1.84x107 1.44x107 1.41x107 1.71x107
1.0 3.61x107 3.14x107 3.09x107 2.63x107
1.5 6.35x107 5.33x107 5.92x107 4.29x107
2.0 11.4x107 9.06x107 8.69x107 5.52x107
2.5 15x107 10.9x107 12.9x107 7.13x107
3.0 20.2x107 13.0x107 15.7x107 9.24x107
3.5 22.7x107 14.8x107 17.3x107 10.9x107
4.0 23.9x107 15.0x107 18.4x107 12.8x107
4.5 25.5x107 16.2x107 20.4x107 14.4x107
16.0 30.3x107 17.7x107 23.1x107 16.9x107

E.coli BL21 (DE3)
Time (h) Psb1c3 (No. of cells) BBa_K1894001 (No. of cells) BBa_K1894002 (No. of cells)
0 1x107 1.19x107 1.83x107
0.5 1.51x107 1.66x107 2.83x107
1 3.51x107 3.51x107 5.10x107
1.5 6.31x107 6.17x107 9.13x107
2 11.3x107 10.6x107 12.3x107
2.5 13.1x107 12.5x107 15.0x107
3.0 16.3x107 15.9x107 18.9x107
3.5 17.8x107 17.0x107 20.5x107
4.0 19.2x107 18.1x107 21.4x107
4.5 21.1x107 19.6x107 22.3x107
16.0 26.4x107 29.3x107 24.4x107



7. Curve Fitting

Gompertz model is the most frequently used sigmoid model to fit growth data in biology.

We took the logarithm to base 10 value, and plotted a growth curve using the Grompertz model. Here are the results:


Equation of BBa_K3219002 transformed in E.coli


Growth curve of E.coli DH5α Transfromants



Link to graph.


Equation of BBa_K3219002 transformed to E.coli BL21 (DE3)


Growth curve of E.coli BL21 (DE3) Transfromants



Link to graph.

https://static.igem.org/mediawiki/2019/9/95/T--HK_SSC--Our_part_dh5a.png

Conculsion

In DH5α, the growth rate of BBa_K1894002 seems to be distinctively slower than that of other plasmids. The cell yield is also very low, almost half of the cell yield of Psb1c3 transformants. We believe that this result fits our hypothesis that this large plasmid affects cell growth. Protocols have to be optimized when using BBa_K1894002. For example, the inoculation time before plasmid purification can be increased just to achieve the ideal DNA yield, and it may take a longer time for colonies to form after transformation due to the low replication rate.

References

↑ Dittmann, Elke. “Insertional mutagenesis of a peptide synthetase gene that is responsible for hepatotoxin production in the cyanobacterium Microcystis Aeruginosa PCC 7806.” Molecular Microbiology (1997): 779–787. Journal.

↑ http://parts.igem.org/Part:BBa_K1894001

↑ http://parts.igem.org/Part:BBa_K1689013

↑ 章军, 徐虹, 楼士林, 欧阳青. Blue-green alga shuttle plasmid expression vector and method for expressing thymison 'alpha' 1. Thesis. Xia Men: Xia Men University, 1999.

↑ Semary, Nermin Adel El. “Optimized electroporation-induced transformation in Microcystis aeruginosa PCC7806.” Biotechnol. Agron. Soc. Environ (2010): 149-152 . Journal.

↑ Dittmann, Elke. “Insertional mutagenesis of a peptide synthetase gene that is responsible for hepatotoxin production in the cyanobacterium Microcystis Aeruginosa PCC 7806.” Molecular Microbiology (1997): 779–787. Journal.

↑ U. EONG CHEAH, WILLIAM A. WEIGAND, BENJAMIN C. STARK. “Effects of Recombinant Plasmid Size on Cellular Processes in Escherichia coli.” Plasmid (1987): 127-134 . Journal.

↑ Im, H. (2011). The Inoue Method for Preparation and Transformation of Competent E. coli: "Ultra Competent" Cells. Bio-101: e143. DOI: 10.21769/BioProtoc.143.