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					  <div class="myTitle1" >Regulation of killing ability</div>
                                          <div class="myTitle2" >I Introduction</div>
					  <div class="myPara" > &nbsp;&nbsp;&nbsp;&nbsp;Even though we have orthogonal gene circuit, the killing ability of two warriors may have great difference. (Fig.1)<br></div>
					   <div class="myPic1" ><img src="https://static.igem.org/mediawiki/2017/8/8d/Tsinghua-a-r1.jpg"width="650px"></div>
					  <div class="myPara" >&nbsp;&nbsp;&nbsp;&nbsp;Sometimes, to mimic situation we investigate in real world, it may be necessary to regulate killing ability to an appropriate level, for example, to make two warriors have same killing ability. What can we do to regulate the killing ability of warriors?<br>
&nbsp;&nbsp;&nbsp;&nbsp;Intuitively, many potential factors can influence the killing ability, like the catalysis rate of AHL synthase, cross-membrane rate between AHL molecules. Then what factors can we change to regulate the killing ability of warriors? Our model suggest that it is possible to regulate the killing ability of two warriors by just changing the promoter intensity of some proteins.
<br><br></div>
					  <div class="myTitle2" >II Evaluation of warrior’s killing ability</div>
					  <div class="myPara" >&nbsp;&nbsp;&nbsp;&nbsp;First, to simplify the evaluation, we will have some hypothesis at first:<br>
1. The increasing rate of two warriors are the same.<br>
2. The environmental capacity (it is mentioned in the famous Logistic model) of two warriors are the same if the environmental conditions and the initial amount of resources are the same.(By these hypothesis, we can ignore influence of growth rate on killing ability, which will make things more complicated.)<br>
3. Also, we could only consider the condition that there is glucose inside the medium and there is only warrior lived in the medium.<br>
4. RFP intensity is used to indicate the amount of LacI, so it can indicate the killing effect of the warrior. This means in our model that the warrior cannot actually kill another warrior, but can activate the RFP expression of another warrior. And more RFP inside bacteria means the bacteria is killed more by warrior in other side.<br>
&nbsp;&nbsp;&nbsp;&nbsp;In conclusion, we can say that killing ability of warrior I is larger than that of warrior II when they are co-cultured together, RFP inside warrior II increase faster or increase to a higher state level. (The initial amounts of two warriors are same.)
<br><br></div>
					  <div class="myTitle2" >III Gene circuit of warrior in the model</div>
					  <div class="myPic1" ><img src="https://static.igem.org/mediawiki/2017/0/00/Tsinghua-a-r2.jpg"width="650px"></div>
						
					  <div class="myPara" ><b>Note: Here for simplicity, we hypothesize that this gene circuit is orthogonal.</b><br><br></div>
					  <div class="myTitle2" >IV ODE parameters and its resource</div>
					  <div class="myPara" > Note: This is part of fluid model to help us solve this specific problem. Detailed explanation can be seen at Fluid Model. Subscript 1 indicate warrior I, subscript 2 indicate warrior II. AHL1 is C4HSL and AHL2 is 3OC6HSL.<br>
<strong>1. Amount of population(cell/medium):</strong>
<br></div>
					   <div class="myPic2" ><img src="https://static.igem.org/mediawiki/2017/0/0e/Tsinghua-a-r3.jpg"width="700px"></div>
					  <div class="myPara" ><strong>2. <i>AHL1</i> (Produced by warrior I) molecule concentration inside cell (molecule/cell)<br></strong></div>
					   <div class="myPic2" ><img src="https://static.igem.org/mediawiki/2017/6/60/Tsinghua-a-r4.jpg"width="700px"></div>
					  <div class="myPara" ><strong> 3. <i>AHL2</i> (Produced by warrior II) molecule concentration inside cell (molecule/cell)<br></strong></div>
					   <div class="myPic2" ><img src="https://static.igem.org/mediawiki/2017/1/17/Tsinghua-a-r5.jpg"width="700px"></div>
					  <div class="myPara" ><strong>4. <i>AHL1</i> (Produced by warrior I) molecule concentration inside environment (molecule/medium)<br></strong></div>
					   <div class="myPic2" ><img src="https://static.igem.org/mediawiki/2017/0/0f/Tsinghua-a-r6.jpg"width="700px"></div>
					  <div class="myPara" ><strong>5. <i>AHL2</i> (Produced by warrior II) molecule concentration inside environment (molecule/medium)<br></strong></div>
					   <div class="myPic2" ><img src="https://static.igem.org/mediawiki/2017/1/14/Tsinghua-a-r7.jpg"width="700px"></div>
					  <div class="myPara" ><strong>6. Amount of RFP inside the cell (a.u/cell)<br></strong></div>
					   <div class="myPic2" ><img src="https://static.igem.org/mediawiki/2017/9/9a/Tsinghua-a-r8.jpg"width="700px"></div>
					  <div class="myPara" ><strong>7. Amount of <i>AHLI1</i> inside warrior I (molecule/cell)<br></strong></div>
					   <div class="myPic2" ><img src="https://static.igem.org/mediawiki/2017/f/fd/Tsinghua-a-r9.jpg"width="700px"></div>
					  <div class="myPara" ><strong>8. Amount of <i>AHLI2</i> inside warrior II (molecule/cell)：<br></strong></div>
					   <div class="myPic2" ><img src="https://static.igem.org/mediawiki/2017/1/18/Tsinghua-a-r10.jpg"width="700px"></div>
                       <div class="myPara" ><br></div>

					  <div class="myTitle2" >V Original Parameter set of the model</div>
					   <div class="myPic2" ><img src="https://static.igem.org/mediawiki/2017/a/ae/Tsinghua-a-r11.jpg"width="700px"></div>
                       <div class="myPara" ><br></div>

					  <div class="myTitle2" >VI Results of model</div>
					  <div class="myPara" > Note: <br>
1. Since we have assumed that the increasing rate and carrying capacity of two warriors are same, so in the model, the amount of two warriors are same at all the time.<br>
2. In the following description, we will show how to regulate killing ability of warriors by showing how to regulate killing abilities of two warriors to the same level!<br>
3.Killing effect means the real time effect of one warrior on the other, but killing ability is a global property that partly describe how killing effect of one warrior change as time.<br>
<b>1. Results under original parameter sets</b>
<br></div>
					   <div class="myPic1" ><img src="https://static.igem.org/mediawiki/2017/2/22/Tsinghua-a-r12.jpg"width="600px"></div>
					  <div class="myPara" >&nbsp;&nbsp;&nbsp;&nbsp;As stated above, RFP intensity is used to indicate the killing effect of the warrior. From this original parameter set and result, we can see that since the maximal expression rate of RFP of two warriors (Indicating the amount of LacI) have large difference, the maximal killing abilities are different largely. In the real situation, this means that warrior I has higher amount of LacI and thus less CmR so it has less resistance to chloramphenicol and slower growth rate.<br>
&nbsp;&nbsp;&nbsp;&nbsp;The first step we can do to regulate maximal killing effect is to increase/decrease the maximal expression rate of warrior I/II.<b>In the experiment, this can be achieved by changing the RBS after Prhl in warrior. For example, we can increase the maximal expression level of RFP inside warrior II to increase the killing ability of warrior I.</b>
<br><b>2. Increasing the maximal expression rate of RFP inside warrior II</b><br></div>
					   <div class="myPic1" ><img src="https://static.igem.org/mediawiki/2017/3/35/Tsinghua-a-r13.jpg"width="600px"></div>
					  <div class="myPara" > &nbsp;&nbsp;&nbsp;&nbsp;From this, Even the maximal expression intensity of AHL promoter of two warriors are the same, we will think warrior II still have much higher killing ability than that of warrior I. This is because its killing effect is high at the beginning and warrior I may be killed at a very fast speed at the beginning. Sometimes we may not only want to regulate their maximal killing effect, but also want to regulate their killing effect in real time. (You can imagine that in a war, warrior II accumulates strength at a very fast speed and kills warrior I immediately, though their maximal strength is the same. What you want to do here is actually equivalent to regulating their speed of accumulating strength.) <br>
&nbsp;&nbsp;&nbsp;&nbsp;Therefore, what could we do? Intuitively, we may think that if warrior II expresses less LuxI so that 3OC6HSL accumulates slower, the speed of accumulating strength is slower.These can both be demonstrated by our model.<br>
<b>3. Decreasing the expression rate of LuxI inside warrior II</b>
<br></div>
					   <div class="myPic1" ><img src="https://static.igem.org/mediawiki/2017/3/3f/Tsinghua-a-r14.jpg"width="600px"></div>
					  <div class="myPara" >&nbsp;&nbsp;&nbsp;&nbsp;By doing this, we can regulate the accumulating strength speed of warrior successfully! <b>In the experiment, this can be achieved by changing the constant promoter or RBS of LuxI in warrior II.</b> <br>
&nbsp;&nbsp;&nbsp;&nbsp;In conclusion, the core is that we can regulate the maximal killing effect by regulating the amount of LacI (In this model, RFP)and the speed of accumulating strength by regulating amount of AHL-production enzyme inside cell. All these can be regulated by thechange the promoter/RBS of the protein or adding LVA tag after the protein experimentally. <br>
&nbsp;&nbsp;&nbsp;&nbsp;<b>According to the results obtained from our model, what we should do in the experiment to achieve the goal is quite clear now.</b>
<br><br></div>
					  <div class="myTitle2" >VII Reference</div>
					  <div class="myPara" >[1]Pai A, You L. Optimal tuning of bacterial sensing potential. Mol Syst Biol, 2009, 5:286<br>
[2] Ron M, Rob P. Cell Biology by the numbers. United States: Garland Science, 2015<br>
[3] ETH_Zurich 2014: https://2014.igem.org/Team:ETH_Zurich/expresults
<br></div>
	<div class="myPara" ><br><br></div>
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