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Why Do We Design This Experiment

B.subtilis has been widely applied as engineered bacteria, especially in food industry and pharmaceutical industry, for its safety and excellent secretion capacity. Therefore, after comparing characters of distinct mutants we selected B.subtilis WB800N mutant as our engineered bacteria and looked up plenty of papers to select the optimal conditions for our experiment. To our disappointment, very few experiments have been done on WB800N mutant, and most optimization experiments regarding B.subtilis focus solely on the optimization of production of specific proteins produced by B.subtilis. Consider the final goal of our project, it is imperative to design this experiment on our own to find out the best condition for B.subtilis WB800N.

Methodology

Any optimization design will inevitably involve the ideology of Design of Experiment (DOE), which includes several dependent plots. Among them Orthogonal Design and Response Surface Design(RSM) are the most common two in biological experiments. Generally, Orthogonal Design consumes less time and has been used more widely, yet it is not logically rigorous in mathematics, and sometimes it overlooks interactions and alias between or among factors. In contrast, RSM is constructed on rigorous mathematical theories and excels in data analysis. Having weighed the features of the two methods carefully, we finally chose RSM.

Sweeping Factors

The first step of any methods of DOE is to investigate all variables that affect the results and select controllable factors for the experiment. In terms of this experiment, all factors can be categorized into two kinds: environment factors, like temperature, the rotation speed of the shaker, and the components of the medium. We have looked up several papers about the optimization experiments on B.subtilis, finding the rotation speed of shakers ranging from 100 r/min to 250 r/min, and generally rotation speed only plays a tiny role. Additionally, our lab has only two shakers. While we can place twenty different mediums into one shaker at a time, we must run the shakers every time we alert the speed, which surely consumes longer time. Thus, we fixed the rotation speed of shakers at 200r/min. However, temperature and inoculation time are both vital environment factors whose effects cannot be ignored.
Inoculation amount and pack amount are also two factors that affect results slightly. We fixed them at 5 percent and 30mL/500mL respectively according to earlier authentic experiments.
A typical medium consists of carbon source, nitrogen source and inorganic salt, all of which are essential to ensure the regular metabolism of engineered bacteria. Finally in light of convenience, we infered the components of typical LB medium and determined three independent medium factors: peptone, yeast extract and sodium chloride (NaCl). Peptone provides nitrogen and carbon for the colonies, while yeast extract contains most required inorganic salt, therefore we did not list any inorganic salt except NaCl. We had no idea why NaCl is listed alone, and we suspected the influence of NaCl as yeast extract had already contains sodium.
Thus, we had five independent factors: temperature, inoculation time, peptone, yeast extract and NaCl. We further investigated some papers and defined their ranges. The following table displays their levels, and the unit of peptone, yeast extract and NaCl is g/L:

Factor	Low	High
Temperature	25℃	35℃
25℃	12h	24h
Peptone	5	15
Yeast Extract	2.5	7.5
NaCl	5	15

Table 1. Factors and their values of our design

Designs&Results

The methodology of RSM can be divided into two subplots: Central Composite Design (CCD) and Box-Behnken Design. Generally the overall runs of Box-Behnken Design is fewer when factors are fixed, but Central composite design is often recommended when the sequential experimentation is required, because it incorporates information from a properly planned factorial experiment. In our experiment, time is more precious than reagents, and as time itself is also an independent factor, Box-Behnken Design would not have saved any time if adopted. Thus we selected CCD.
CCD itself can also be classified into three subplots: Central Composite Circumscribed Design(CCC), Central Composite Inscribed design(CCI) and Central Composite Face-centered Design(CCF). The alpha value of CCC is related to the number of factors, whereas in CCF α is fixed at 1, and only CCC is rotatable. The rotational invariance empowers CCC to be mathematically preferred, but the value of alpha in a five-factor-CCC is over 2. In other words, if we adopted CCC, we would get some absurd treatments where the concentration of some specific actual material were negative. If we narrowed down the ranges to ensure the concentration of all medium component are positive in every treatment, the ranges would be too narrow to yield cogent results. Therefore, we finally selected CCF.
We conducted our experiments according to the following table, which was calculated by Minitab, and the results measured by OD value, were also included:

No.	Temperature	Time	Peptone	Yeast extract	NaCl	OD
1	25	12	5	2.5	5	0.511
2	35	24	5	2.5	5	1.625
3	35	12	15	2.5	5	2.783
4	25	24	15	2.5	5	1.74
5	35	12	5	7.5	5	2.317
6	25	24	5	7.5	5	2.4
7	25	12	15	7.5	5	0.912
8	35	24	15	7.5	5	3
9	35	12	5	2.5	15	2.169
10	25	24	5	2.5	15	1.77
11	25	12	15	2.5	15	0.371
12	35	24	15	2.5	15	2.7
13	25	12	5	7.5	15	0.754
14	35	24	5	7.5	15	2.58
15	35	12	15	7.5	15	3.128
16	25	24	15	7.5	15	2.38
17	30	18	10	5	10	2.908
18	30	18	10	5	10	2.908
19	30	18	10	5	10	1.75
20	30	18	10	5	10	2.908
21	35	12	5	2.5	5	2.082
22	25	24	5	2.5	5	1.75
23	25	12	15	2.5	5	0.508
24	35	24	15	2.5	5	2.6
25	25	12	5	7.5	5	0.989
26	35	24	5	7.5	5	2.8
27	35	12	15	7.5	5	2.782
28	25	24	15	7.5	5	1.7
29	25	12	5	2.5	15	0.508
30	35	24	5	2.5	15	1.338
31	35	12	15	2.5	15	3.061
32	25	24	15	2.5	15	2.2
33	35	12	5	7.5	15	2.167
34	25	24	5	7.5	15	1.53
35	25	12	15	7.5	15	0.555
36	35	24	15	7.5	15	2.9
37	30	18	10	5	10	2.908
38	30	18	10	5	10	2.908
39	30	18	10	5	10	2.908
40	30	18	10	5	10	2.957
41	25	18	10	5	10	1.907
42	35	18	10	5	10
43	30	12	10	5	10	2.652
44	30	24	10	5	10	2.908
45	30	18	5	5	10	2.726
46	30	18	15	5	10	3.042
47	30	18	10	2.5	10	2.598
48	30	18	10	7.5	10	3.124
49	30	18	10	5	5	2.999
50	30	18	10	5	15	2.834
51	30	18	10	5	10	2.908
52	30	18	10	5	10	2.908
53	30	18	10	5	10	2.908
54	30	18	10	5	10	2.908

Table 2. Treatments and results of our experiment

The result of No.42 medium is destroyed due to some unknown reasons. Additionally, multiple center points, which means conducting multiple experiments at the center points with identical treatments, is a very common phenomenon in DOE, although we dis only experiment at the center point and reuse its result, as a result of our limited time and reagents.
Estimated Regression Coefficients for OD
S = 0.295758 PRESS = 7.78904
R-Sq = 92.25% R-Sq(pred) = 78.45% R-Sq(adj) = 87.41%

Term	Coef	SE Coef	T	P
Constant	2.87625	0.07126	40.361	0.000
Temperature	0.60225	0.05210	11.560	0.000
Time	0.28447	0.05072	5.608	0.000
Peptone	0.18665	0.05072	3.680	0.001
Yeast Extract	0.16776	0.05072	3.308	0.002
NaCl	-0.01626	0.05072	-0.321	0.751
Temperature*Temperature	-0.54900	0.24585	-2.233	0.033
Time*Time	-0.18725	0.19289	-0.971	0.339
Peptone*Peptone	-0.08325	0.19289	-0.432	0.669
Yeast Extract*Yeast Extract	-0.10625	0.19289	-0.551	0.586
NaCl*NaCl	-0.05075	0.19289	-0.263	0.794
Temperature*Time	-0.358338	0.05228	-6.579	0.000
Temperature*Peptone	0.17881	0.05228	3.420	0.002
Temperature*Yeast Extract	0.04544	0.05228	0.869	0.391
Temperature*NaCl	0.01550	0.05228	0.296	0.769
Time*Peptone	0.02575	0.05228	0.493	0.626
Time*Yeast Extract	.06112	0.05228	1.169	0.261
Time*NaCl	-0.00144	0.05228	-0.027	0.978
Peptone*Yeast Extract	-0.07469	0.05228	-1.429	0.163
Peptone*NaCl	0.09150	0.05228	1.750	0.090
Yeast Extract*NaCl	-0.04450	0.05228	-0.851	0.401

Table 3. Estimated Regression Coefficients for OD

Suppose we redefine the factors according to the following table:

Term	Mark
OD	F
Temperature	T
Time	T
Peptone	P
Yeast Extract	Y
NaCl	C

Table 4. Mark for each term

According to the ANOVA calculated by Mnitab, we got the expression of OD:

P represents confidence coefficient, which is a key judgment to check the reliability of the fitting function. In other words, if P=0.05, the probability that this term is wrong is 5%. The coefficient of determination (R) was calculated to be 0.9225, indicating that the model could explain 92% of the variability. From the above table we can identify eight statistically significant and reliable terms:

Constant;
Temperature;
Time;
Yeast Extract;
Peptone;
Temperature*Temperature;
Temperature*Time;
Temperature*Yeast Extract;

The influences of linear terms predominated, except NaCl, which substantiated our suspicion, whereas most square terms and interaction terms were ignorable and statistically unreliable. Temperature and time and two most influential factors.
As the intact response surface is six-dimensional, it is impossible to draw the intact surface in our three-dimensional world. Therefore we had to fix some factors to lower the dimensions, draw contours and surfaces, and e can extrapolate this super surface by combining these pictures:

Figure 1. Surface plots of OD vs time and temperature.

Figure 2. Contour plots of OD vs time and temperature.

Figure 3. Surface plots of OD vs time and peptone.

Figure 4. Contour plots of OD vs time and peptone.

Figure 5. Surface plots of OD vs peptone and temperature.

Figure 6. Contour plots of OD vs peptone and temperature.

Figure 7. Conyour plots of OD vs yeast extract and temperature.

Figure 8. Contour plots of OD vs yeast extract and temperature.

The following four pictures illustrate the distribution of residual error:

Figure 9. Residual error vs order

Figure 10. Histogram of residual error

Figure 11. Residual error vs fits

Figure 12. Normal probability plot of residual error

Optimization

One remarkable character of CCD is that it is sequential, which is also the essence of RSM. Since we had got the fitting function, the next step is to calculate the gradient of the function, and define a small number as step length. Further experiments are supposed to be conducted from the beginning point according to the gradient and step length, and the final maximal treatment would be made sure. The methodology of RSM seems like climbing a mountain whose peak is unknown, and we are supposed to adjust our orientation according to the topography. The fitting surface, which can be often a super surface in higher dimensional spaces, can be likened to the mountain without clear peaks, and calculating gradient to orientating.
Unfortunately our remaining time is not enough to support further experiments, and as we looked up other researches utilizing RSM, none of which did second round experiment, and we realized perhaps that was the difference between a scientific research and a real industrial procedure. Yet the analytical methodology of response surface still acted as a powerful tool for ANOVA. Roughly, we could consider the treatment of No. 15 medium (Temperature 35℃, Time 12h, Peptone 15, Yeast Extract 7.5, NaCl 15)as the maximal condition for B.subtilis.

Kill Switch

B.Subtilis Culture

Designs of Immune Experiments

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-<h1>【Why Do We Design This Experiment】</h1>
+<h1>Why Do We Design This Experiment</h1>
-<p>Bacillus subtilis has been widely applied as engineered bacteria, especially in food industry and pharmaceutical industry, for its safety and excellent secretion capacity. Therefore, after comparing characters of distinct mutants we selected Bacillus subtilis WB800N mutant as our engineered bacteria and looked up plenty of papers to select the optimal conditions for our experiment. To our disappointment, very few experiments have been done on WB800N mutant, and most optimization experiments regarding Bacillus subtilis focus solely on the optimization of production of specific proteins produced by Bacillus subtilis. Consider the final goal of our project, it is imperative to design this experiment on our own to find out the best condition for Bacillus subtilis WB800N.</p>
+<p align="justify"><i>B.subtilis</i> has been widely applied as engineered bacteria, especially in food industry and pharmaceutical industry, for its safety and excellent secretion capacity. Therefore, after comparing characters of distinct mutants we selected <i>B.subtilis</i> WB800N mutant as our engineered bacteria and looked up plenty of papers to select the optimal conditions for our experiment. To our disappointment, very few experiments have been done on WB800N mutant, and most optimization experiments regarding <i>B.subtilis</i> focus solely on the optimization of production of specific proteins produced by <i>B.subtilis</i>. Consider the final goal of our project, it is imperative to design this experiment on our own to find out the best condition for <i>B.subtilis</i> WB800N.</p>
 </div>
 <div>
-<h1>【Methodology】</h1>
+<h1>Methodology</h1>
-Any optimization designs will inevitably involve the ideology of Design of Experiment (DOE), which includes several dependent plots. Among them Orthogonal Design and Response Surface Design, RSM for short, are the most common two in biological experiments. Generally, Orthogonal Design consumes less time and has been used more widely, yet it is not logically rigorous in mathematics, and sometimes it overlooks interactions and alias between or among factors. In contrast, RSM is constructed on rigorous mathematical theories and excels in data analysis. Having weighing the features of the two methods carefully, we finally chose RSM.
+<p align="justify">
+Any optimization design will inevitably involve the ideology of Design of Experiment (DOE), which includes several dependent plots. Among them Orthogonal Design and Response Surface Design(RSM) are the most common two in biological experiments. Generally, Orthogonal Design consumes less time and has been used more widely, yet it is not logically rigorous in mathematics, and sometimes it overlooks interactions and alias between or among factors. In contrast, RSM is constructed on rigorous mathematical theories and excels in data analysis. Having weighed the features of the two methods carefully, we finally chose RSM.</p>
 </div>
 <div>
-<h1>【Sweeping Factors】</h1>
+<h1>Sweeping Factors</h1>
-The first step of any methods of DOE is to investigate all variables that affect the results and select controllable factors for the experiment. In terms of this experiment, all factors can be categorized into two kinds: environment factors, like temperature, the rotation speed of the shaker, and the components of the medium. We have looked up several papers about the optimization experiments on Bacillus subtilis, finding the rotation speed of shakers ranging from 100 r/min to 250 r/min, and generally rotation speed only plays a tiny role. Additionally, our lab has only two shakers. While we can place twenty different mediums into one shaker at a time, we must run the shakers every time we alert the speed, which surely consumes longer time. Thus, we fixed the rotation speed of shakers at 200r/min.However, temperature and inoculation time are both vital environment factors whose effects cannot be ignored.</br>
+<p align="justify">
+The first step of any methods of DOE is to investigate all variables that affect the results and select controllable factors for the experiment. In terms of this experiment, all factors can be categorized into two kinds: environment factors, like temperature, the rotation speed of the shaker, and the components of the medium. We have looked up several papers about the optimization experiments on <i>B.subtilis</i>, finding the rotation speed of shakers ranging from 100 r/min to 250 r/min, and generally rotation speed only plays a tiny role. Additionally, our lab has only two shakers. While we can place twenty different mediums into one shaker at a time, we must run the shakers every time we alert the speed, which surely consumes longer time. Thus, we fixed the rotation speed of shakers at 200r/min. However, temperature and inoculation time are both vital environment factors whose effects cannot be ignored.</br>
 Inoculation amount and pack amount are also two factors that affect results slightly. We fixed them at 5 percent and 30mL/500mL respectively according to earlier authentic experiments.</br>
 A typical medium consists of carbon source, nitrogen source and inorganic salt, all of which are essential to ensure the regular metabolism of engineered bacteria. Finally in light of convenience, we infered the components of typical LB medium and determined three independent medium factors: peptone, yeast extract and sodium chloride (NaCl). Peptone provides nitrogen and carbon for the colonies, while yeast extract contains most required inorganic salt, therefore we did not list any inorganic salt except NaCl. We had no idea why NaCl is listed alone, and we suspected the influence of NaCl as yeast extract had already contains sodium.</br>
 Thus, we had five independent factors: temperature, inoculation time, peptone, yeast extract and NaCl. We further investigated some papers and defined their ranges. The following table displays their levels, and the unit of peptone, yeast extract and NaCl is g/L:
-</br>
+</p>
-<table width="580" border="2">
+<div align="center">
-   <tr>
+<table border="1" cellspacing="0" cellpadding="0" width="577" backgroud-color="transparent" align="center">
-     <td>Factor</td>
+   <tr align="center">
+     <td align="center">Factor</td>
      <td>Low</td>
      <td>High</td>
    </tr>
-   <tr>
+   <tr align="center">
      <td>Temperature</td>
      <td>25℃</td>
      <td>35℃</td>
    </tr>
-   <tr>
+   <tr align="center">
      <td>25℃</td>
      <td>12h</td>
      <td>24h</td>
    </tr>
-   <tr>
+   <tr align="center">
      <td>Peptone</td>
      <td>5</td>
      <td>15</td>
    </tr>
-   <tr>
+   <tr align="center">
      <td>Yeast Extract</td>
      <td>2.5</td>
      <td>7.5</td>
    </tr>
-   <tr>
+   <tr align="center">
      <td>NaCl</td>
      <td>5</td>
@@ Line 105: / Line 112: @@
    </tr>
 </table>
+</div>
+<p align="center"><b>Table 1.</b> Factors and their values of our design</p>
 </div>
 <div>
-<h1>【Designs&Results】</h1>
+<h1>Designs&Results</h1>
-The methodology of RSM can be divided into two subplots: Central Composite Designs (CCD) and Box-Behnken Designs. Generally the overall runs of Box-Behnken Designs is fewer when the factors are fixed, but Central composite designs are often recommended when the design plan calls for sequential experimentation because these designs can incorporate information from a properly planned factorial experiment. In our experiment, time is more precious than reagents, and as time itself is also an independent factor, Box-Behnken Designs would not have saved any time if adopted. Thus we selected CCD.</br>
+<p align="justify">
-CCD itself can also be classified into three subplots: Central Composite Circumscribed Design (CCC), Central Composite Inscribed design(CCI) and Central Composite Face-centered Design(CCF). The alpha value of CCC is related to the number of factors, whereas in CCF  α is fixed at 1, and only CCC is rotatable. The rotational invariance empowers CCC to be mathematically preferred, yet the value of alpha in a five-factor-CCC is over 2. In other words, if we adopted CCC, we would get some absurd treatments where the concentration of some specific actual material were negative. If we narrowed down the range to ensure any concentration is positive, the ranges of all three medium factors would be too narrow to yield cogent results. Therefore, we finally selected CCF.</br>
+The methodology of RSM can be divided into two subplots: Central Composite Design (CCD) and Box-Behnken Design. Generally the overall runs of Box-Behnken Design is fewer when factors are fixed, but Central composite design is often recommended when the sequential experimentation is required, because it incorporates information from a properly planned factorial experiment. In our experiment, time is more precious than reagents, and as time itself is also an independent factor, Box-Behnken Design would not have saved any time if adopted. Thus we selected CCD.</br>
-We conducted our experiments according to the following table, which was calculated by Minitab, and the results, which were measure by OD value, were also included:</br>
+CCD itself can also be classified into three subplots: Central Composite Circumscribed Design(CCC), Central Composite Inscribed design(CCI) and Central Composite Face-centered Design(CCF). The alpha value of CCC is related to the number of factors, whereas in CCF  α is fixed at 1, and only CCC is rotatable. The rotational invariance empowers CCC to be mathematically preferred, but the value of alpha in a five-factor-CCC is over 2. In other words, if we adopted CCC, we would get some absurd treatments where the concentration of some specific actual material were negative. If we narrowed down the ranges to ensure the concentration of all medium component are positive in every treatment, the ranges would be too narrow to yield cogent results. Therefore, we finally selected CCF.</br>
-<table border="1" cellspacing="0" cellpadding="0" width="577">
+We conducted our experiments according to the following table, which was calculated by Minitab, and the results measured by OD value, were also included:</p>
-   <tr>
+<table border="1" cellspacing="0" cellpadding="0" width="577" align="center">
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="left">No.</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="left">Temperature</p></td>
@@ Line 122: / Line 132: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="left">OD</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">1</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">25</p></td>
@@ Line 131: / Line 141: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">0.511</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">2</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">35</p></td>
@@ Line 140: / Line 150: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">1.625</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">3</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">35</p></td>
@@ Line 149: / Line 159: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">2.783</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">4</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">25</p></td>
@@ Line 158: / Line 168: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">1.74</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">5</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">35</p></td>
@@ Line 167: / Line 177: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">2.317</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">6</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">25</p></td>
@@ Line 176: / Line 186: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">2.4</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">7</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">25</p></td>
@@ Line 185: / Line 195: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">0.912</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">8</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">35</p></td>
@@ Line 194: / Line 204: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">3</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">9</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">35</p></td>
@@ Line 203: / Line 213: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">2.169</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">10</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">25</p></td>
@@ Line 212: / Line 222: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">1.77</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">11</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">25</p></td>
@@ Line 221: / Line 231: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">0.371</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">12</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">35</p></td>
@@ Line 230: / Line 240: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">2.7</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">13</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">25</p></td>
@@ Line 239: / Line 249: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">0.754</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">14</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">35</p></td>
@@ Line 248: / Line 258: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">2.58</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">15</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">35</p></td>
@@ Line 257: / Line 267: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">3.128</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">16</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">25</p></td>
@@ Line 266: / Line 276: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">2.38</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">17</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">30</p></td>
@@ Line 275: / Line 285: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">2.908</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">18</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">30</p></td>
@@ Line 284: / Line 294: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">2.908</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">19</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">30</p></td>
@@ Line 293: / Line 303: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">1.75</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">20</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">30</p></td>
@@ Line 302: / Line 312: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">2.908</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">21</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">35</p></td>
@@ Line 311: / Line 321: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">2.082</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">22</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">25</p></td>
@@ Line 320: / Line 330: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">1.75</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">23</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">25</p></td>
@@ Line 329: / Line 339: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">0.508</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">24</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">35</p></td>
@@ Line 338: / Line 348: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">2.6</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">25</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">25</p></td>
@@ Line 347: / Line 357: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">0.989</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">26</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">35</p></td>
@@ Line 356: / Line 366: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">2.8</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">27</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">35</p></td>
@@ Line 365: / Line 375: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">2.782</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">28</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">25</p></td>
@@ Line 374: / Line 384: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">1.7</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">29</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">25</p></td>
@@ Line 383: / Line 393: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">0.508</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">30</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">35</p></td>
@@ Line 392: / Line 402: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">1.338</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">31</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">35</p></td>
@@ Line 401: / Line 411: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">3.061</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">32</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">25</p></td>
@@ Line 410: / Line 420: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">2.2</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">33</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">35</p></td>
@@ Line 419: / Line 429: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">2.167</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">34</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">25</p></td>
@@ Line 428: / Line 438: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">1.53</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">35</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">25</p></td>
@@ Line 437: / Line 447: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">0.555</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">36</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">35</p></td>
@@ Line 446: / Line 456: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">2.9</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">37</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">30</p></td>
@@ Line 455: / Line 465: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">2.908</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">38</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">30</p></td>
@@ Line 464: / Line 474: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">2.908</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">39</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">30</p></td>
@@ Line 473: / Line 483: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">2.908</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">40</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">30</p></td>
@@ Line 482: / Line 492: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">2.957</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">41</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">25</p></td>
@@ Line 491: / Line 501: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">1.907</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">42</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">35</p></td>
@@ Line 500: / Line 510: @@
      <td width="72" nowrap="nowrap" valign="bottom"></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">43</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">30</p></td>
@@ Line 509: / Line 519: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">2.652</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">44</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">30</p></td>
@@ Line 518: / Line 528: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">2.908</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">45</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">30</p></td>
@@ Line 527: / Line 537: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">2.726</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">46</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">30</p></td>
@@ Line 536: / Line 546: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">3.042</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">47</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">30</p></td>
@@ Line 545: / Line 555: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">2.598</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">48</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">30</p></td>
@@ Line 554: / Line 564: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">3.124</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">49</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">30</p></td>
@@ Line 563: / Line 573: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">2.999</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">50</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">30</p></td>
@@ Line 572: / Line 582: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">2.834</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">51</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">30</p></td>
@@ Line 581: / Line 591: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">2.908</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">52</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">30</p></td>
@@ Line 590: / Line 600: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">2.908</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">53</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">30</p></td>
@@ Line 599: / Line 609: @@
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">2.908</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="72" nowrap="nowrap" valign="bottom"><p align="right">54</p></td>
      <td width="83" nowrap="nowrap" valign="bottom"><p align="right">30</p></td>
@@ Line 609: / Line 619: @@
    </tr>
 </table>
+<p align="center"><b> Table 2.</b> Treatments and results of our experiment</p>
 <br />
-The result of No.42 medium is destroyed due to some unfortunate reason. Additionally, multiple center points, which means conducting multiple experiments at the center points with identical treatments, is a very common phenomenon in DOE, yet we decided to do only experiment at the center point and reuse its result due to our limited time and reagents.</br>
+The result of No.42 medium is destroyed due to some unknown reasons. Additionally, multiple center points, which means conducting multiple experiments at the center points with identical treatments, is a very common phenomenon in DOE, although we dis only experiment at the center point and reuse its result, as a result of our limited time and reagents.</br>
-Estimated Regression Coefficients for OD</br>
+<b>Estimated Regression Coefficients for OD</br></b>
 <table border="1" cellspacing="0" cellpadding="0" width="100%">
-   <tr>
+   <tr align="center">
      <td width="43%" valign="top"><p><a name="OLE_LINK9" id="OLE_LINK9"></a><a name="OLE_LINK8" id="OLE_LINK8">Term                            </a></p></td>
      <td width="16%" valign="top"><p>Coef</p></td>
@@ Line 620: / Line 631: @@
      <td width="11%" valign="top"><p>P</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="43%" valign="top"><p>Constant                     </p></td>
      <td width="16%" valign="top"><p> 2.87625  </p></td>
@@ Line 627: / Line 638: @@
      <td width="11%" valign="top"><p>0.000</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="43%" valign="top"><p>Temperature                  </p></td>
      <td width="16%" valign="top"><p> 0.60225  </p></td>
@@ Line 634: / Line 645: @@
      <td width="11%" valign="top"><p>0.000</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="43%" valign="top"><p>Time                         </p></td>
      <td width="16%" valign="top"><p> 0.28447  </p></td>
@@ Line 641: / Line 652: @@
      <td width="11%" valign="top"><p>0.000</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="43%" valign="top"><p>Peptone                      </p></td>
      <td width="16%" valign="top"><p> 0.18665  </p></td>
@@ Line 648: / Line 659: @@
      <td width="11%" valign="top"><p>0.001</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="43%" valign="top"><p>Yeast    Extract                  </p></td>
      <td width="16%" valign="top"><p>0.16776  </p></td>
@@ Line 655: / Line 666: @@
      <td width="11%" valign="top"><p>0.002</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="43%" valign="top"><p>NaCl                         </p></td>
      <td width="16%" valign="top"><p>-0.01626  </p></td>
@@ Line 662: / Line 673: @@
      <td width="11%" valign="top"><p>0.751</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="43%" valign="top"><p>Temperature*Temperature     </p></td>
      <td width="16%" valign="top"><p>-0.54900  </p></td>
@@ Line 669: / Line 680: @@
      <td width="11%" valign="top"><p>0.033</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="43%" valign="top"><p>Time*Time                    </p></td>
      <td width="16%" valign="top"><p>-0.18725  </p></td>
@@ Line 676: / Line 687: @@
      <td width="11%" valign="top"><p>0.339</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="43%" valign="top"><p>Peptone*Peptone             </p></td>
      <td width="16%" valign="top"><p> -0.08325  </p></td>
@@ Line 683: / Line 694: @@
      <td width="11%" valign="top"><p>0.669</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="43%" valign="top"><p>Yeast    Extract*Yeast Extract    </p></td>
      <td width="16%" valign="top"><p>-0.10625  </p></td>
@@ Line 690: / Line 701: @@
      <td width="11%" valign="top"><p>0.586</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="43%" valign="top"><p>NaCl*NaCl                   </p></td>
      <td width="16%" valign="top"><p> -0.05075  </p></td>
@@ Line 697: / Line 708: @@
      <td width="11%" valign="top"><p>0.794</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="43%" valign="top"><p>Temperature*Time             </p></td>
      <td width="16%" valign="top"><p>-0.358338</p></td>
@@ Line 704: / Line 715: @@
      <td width="11%" valign="top"><p>0.000</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="43%" valign="top"><p>Temperature*Peptone           </p></td>
      <td width="16%" valign="top"><p>0.17881</p></td>
@@ Line 711: / Line 722: @@
      <td width="11%" valign="top"><p>0.002</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="43%" valign="top"><p>Temperature*Yeast    Extract    </p></td>
      <td width="16%" valign="top"><p> 0.04544  </p></td>
@@ Line 718: / Line 729: @@
      <td width="11%" valign="top"><p>0.391</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="43%" valign="top"><p>Temperature*NaCl             </p></td>
      <td width="16%" valign="top"><p>0.01550</p></td>
@@ Line 725: / Line 736: @@
      <td width="11%" valign="top"><p>0.769</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="43%" valign="top"><p>Time*Peptone                 </p></td>
      <td width="16%" valign="top"><p> 0.02575  </p></td>
@@ Line 732: / Line 743: @@
      <td width="11%" valign="top"><p>0.626</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="43%" valign="top"><p>Time*Yeast    Extract            </p></td>
      <td width="16%" valign="top"><p>.06112  </p></td>
@@ Line 739: / Line 750: @@
      <td width="11%" valign="top"><p>0.261</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="43%" valign="top"><p>Time*NaCl                   </p></td>
      <td width="16%" valign="top"><p> -0.00144  </p></td>
@@ Line 746: / Line 757: @@
      <td width="11%" valign="top"><p>0.978</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="43%" valign="top"><p>Peptone*Yeast    Extract         </p></td>
      <td width="16%" valign="top"><p>-0.07469  </p></td>
@@ Line 753: / Line 764: @@
      <td width="11%" valign="top"><p>0.163</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="43%" valign="top"><p>Peptone*NaCl                 </p></td>
      <td width="16%" valign="top"><p>0.09150  </p></td>
@@ Line 760: / Line 771: @@
      <td width="11%" valign="top"><p>0.090</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="43%" valign="top"><p>Yeast    Extract*NaCl           </p></td>
      <td width="16%" valign="top"><p>-0.04450  </p></td>
@@ Line 767: / Line 778: @@
      <td width="11%" valign="top"><p>0.401</p></td>
    </tr>
+<big><b>S = 0.295758   PRESS = 7.78904</br>
+R-Sq = 92.25%  R-Sq(pred) = 78.45%  R-Sq(adj) = 87.41%</b></big></br>
+<br>
 </table>
+<p align="center"><b> Table 3.</b> Estimated Regression Coefficients for OD</p>
 <br />
-<table border="1" cellspacing="0" cellpadding="0" width="557">
+Suppose we redefine the factors according to the following table:</br>
-   <tr>
+<table border="1" cellspacing="0" cellpadding="0" width="557" align="center">
+   <tr align="center">
      <td width="279" valign="top"><p>Term</p></td>
      <td width="279" valign="top"><p>Mark</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="279" valign="top"><p>OD</p></td>
      <td width="279" valign="top"><p>F</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="279" valign="top"><p>Temperature</p></td>
      <td width="279" valign="top"><p>T</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="279" valign="top"><p>Time</p></td>
      <td width="279" valign="top"><p>T</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="279" valign="top"><p>Peptone</p></td>
      <td width="279" valign="top"><p>P</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="279" valign="top"><p>Yeast Extract</p></td>
      <td width="279" valign="top"><p>Y</p></td>
    </tr>
-   <tr>
+   <tr align="center">
      <td width="279" valign="top"><p>NaCl</p></td>
      <td width="279" valign="top"><p>C</p></td>
    </tr>
 </table>
-<br /><br />
+<br />
-<table border="1" cellspacing="0" cellpadding="0">
+<p align="center"><b> Table 4.</b> Mark for each term</p>
-  <tr>
+<p align="justify">
-    <td width="75" valign="top"><p>StdOrder</p></td>
+According to the ANOVA calculated by Mnitab, we got the expression of OD:</br>
-    <td width="57" valign="top"><p>OD</p></td>
+<img src="https://static.igem.org/mediawiki/2013/9/96/BS_formation.png"></br>
-    <td width="59" valign="top"><p>Fit</p></td>
+P represents confidence coefficient, which is a key judgment to check the reliability of the fitting function. In other words, if P=0.05, the probability that this term is wrong is 5%. The coefficient of determination (R) was calculated to be 0.9225, indicating that the model could explain 92% of the variability. From the above table we can identify eight statistically significant and reliable terms:</p>
-    <td width="59" valign="top"><p>SE</p></td>
+<ul>
-    <td width="59" valign="top"><p>Fit</p></td>
+<li>Constant;</li>
-    <td width="71" valign="top"><p>Residual</p></td>
+<li>Temperature;</li>
-    <td width="58" valign="top"><p>StResid</p></td>
+<li>Time;</li>
-  </tr>
+<li>Yeast Extract;</li>
-  <tr>
+<li>Peptone;</li>
-    <td width="75" valign="top"><p>1</p></td>
+<li>Temperature*Temperature;</li>
-    <td width="57" valign="top"><p>1</p></td>
+<li>Temperature*Time;</li>
-    <td width="59" valign="top"><p>1.740</p></td>
+<li>Temperature*Yeast Extract;</li>
-    <td width="59" valign="top"><p>1.757</p></td>
+</ul>
-    <td width="59" valign="top"><p>0.207</p></td>
-    <td width="71" valign="top"><p>-0.017</p></td>
-    <td width="58" valign="top"><p>-0.08</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>2</p></td>
-    <td width="57" valign="top"><p>2</p></td>
-    <td width="59" valign="top"><p>0.912</p></td>
-    <td width="59" valign="top"><p>0.612</p></td>
-    <td width="59" valign="top"><p>0.207</p></td>
-    <td width="71" valign="top"><p>0.300</p></td>
-    <td width="58" valign="top"><p>1.42</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>3</p></td>
-    <td width="57" valign="top"><p>3</p></td>
-    <td width="59" valign="top"><p>1.770</p></td>
-    <td width="59" valign="top"><p>1.563</p></td>
-    <td width="59" valign="top"><p>0.207</p></td>
-    <td width="71" valign="top"><p>0.207</p></td>
-    <td width="58" valign="top"><p>0.98</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>4</p></td>
-    <td width="57" valign="top"><p>4</p></td>
-    <td width="59" valign="top"><p>2.317</p></td>
-    <td width="59" valign="top"><p>2.593</p></td>
-    <td width="59" valign="top"><p>0.208</p></td>
-    <td width="71" valign="top"><p>-0.276</p></td>
-    <td width="58" valign="top"><p>-1.31</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>5</p></td>
-    <td width="57" valign="top"><p>5</p></td>
-    <td width="59" valign="top"><p>2.169</p></td>
-    <td width="59" valign="top"><p>1.958</p></td>
-    <td width="59" valign="top"><p>0.208</p></td>
-    <td width="71" valign="top"><p>0.211</p></td>
-    <td width="58" valign="top"><p>1.00</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>6</p></td>
-    <td width="57" valign="top"><p>6</p></td>
-    <td width="59" valign="top"><p>3.000</p></td>
-    <td width="59" valign="top"><p>2.979</p></td>
-    <td width="59" valign="top"><p>0.208</p></td>
-    <td width="71" valign="top"><p>0.021</p></td>
-    <td width="58" valign="top"><p>0.10</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>7</p></td>
-    <td width="57" valign="top"><p>7</p></td>
-    <td width="59" valign="top"><p>1.750</p></td>
-    <td width="59" valign="top"><p>2.876</p></td>
-    <td width="59" valign="top"><p>0.071</p></td>
-    <td width="71" valign="top"><p>-1.126</p></td>
-    <td width="58" valign="top"><p>-3.92R</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>8</p></td>
-    <td width="57" valign="top"><p>8</p></td>
-    <td width="59" valign="top"><p>0.511</p></td>
-    <td width="59" valign="top"><p>0.619</p></td>
-    <td width="59" valign="top"><p>0.207</p></td>
-    <td width="71" valign="top"><p>-0.108</p></td>
-    <td width="58" valign="top"><p>-0.51</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>9</p></td>
-    <td width="57" valign="top"><p>9</p></td>
-    <td width="59" valign="top"><p>2.580</p></td>
-    <td width="59" valign="top"><p>2.253</p></td>
-    <td width="59" valign="top"><p>0.208</p></td>
-    <td width="71" valign="top"><p>0.327</p></td>
-    <td width="58" valign="top"><p>1.55</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>10</p></td>
-    <td width="57" valign="top"><p>10</p></td>
-    <td width="59" valign="top"><p>3.128</p></td>
-    <td width="59" valign="top"><p>3.036</p></td>
-    <td width="59" valign="top"><p>0.208</p></td>
-    <td width="71" valign="top"><p>0.092</p></td>
-    <td width="58" valign="top"><p>0.44</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>11</p></td>
-    <td width="57" valign="top"><p>11</p></td>
-    <td width="59" valign="top"><p>2.380</p></td>
-    <td width="59" valign="top"><p>2.091</p></td>
-    <td width="59" valign="top"><p>0.207</p></td>
-    <td width="71" valign="top"><p>0.289</p></td>
-    <td width="58" valign="top"><p>1.37</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>12</p></td>
-    <td width="57" valign="top"><p>12</p></td>
-    <td width="59" valign="top"><p>2.783</p></td>
-    <td width="59" valign="top"><p>2.697</p></td>
-    <td width="59" valign="top"><p>0.208</p></td>
-    <td width="71" valign="top"><p>0.086</p></td>
-    <td width="58" valign="top"><p>0.41</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>13</p></td>
-    <td width="57" valign="top"><p>13</p></td>
-    <td width="59" valign="top"><p>2.700</p></td>
-    <td width="59" valign="top"><p>2.758</p></td>
-    <td width="59" valign="top"><p>0.208</p></td>
-    <td width="71" valign="top"><p>-0.058</p></td>
-    <td width="58" valign="top"><p>-0.28</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>14</p></td>
-    <td width="57" valign="top"><p>14</p></td>
-    <td width="59" valign="top"><p>2.908</p></td>
-    <td width="59" valign="top"><p>2.876</p></td>
-    <td width="59" valign="top"><p>0.071</p></td>
-    <td width="71" valign="top"><p>0.032</p></td>
-    <td width="58" valign="top"><p>0.11</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>15</p></td>
-    <td width="57" valign="top"><p>15</p></td>
-    <td width="59" valign="top"><p>2.908</p></td>
-    <td width="59" valign="top"><p>2.876</p></td>
-    <td width="59" valign="top"><p>0.071</p></td>
-    <td width="71" valign="top"><p>0.032</p></td>
-    <td width="58" valign="top"><p>0.11</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>16</p></td>
-    <td width="57" valign="top"><p>16</p></td>
-    <td width="59" valign="top"><p>0.371</p></td>
-    <td width="59" valign="top"><p>0.761</p></td>
-    <td width="59" valign="top"><p>0.207</p></td>
-    <td width="71" valign="top"><p>-0.390</p></td>
-    <td width="58" valign="top"><p>-1.85</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>17</p></td>
-    <td width="57" valign="top"><p>17</p></td>
-    <td width="59" valign="top"><p>2.400</p></td>
-    <td width="59" valign="top"><p>2.329</p></td>
-    <td width="59" valign="top"><p>0.207</p></td>
-    <td width="71" valign="top"><p>0.071</p></td>
-    <td width="58" valign="top"><p>0.34</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>18</p></td>
-    <td width="57" valign="top"><p>18</p></td>
-    <td width="59" valign="top"><p>1.625</p></td>
-    <td width="59" valign="top"><p>1.742</p></td>
-    <td width="59" valign="top"><p>0.208</p></td>
-    <td width="71" valign="top"><p>-0.117</p></td>
-    <td width="58" valign="top"><p>-0.56</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>19</p></td>
-    <td width="57" valign="top"><p>19</p></td>
-    <td width="59" valign="top"><p>0.754</p></td>
-    <td width="59" valign="top"><p>0.647</p></td>
-    <td width="59" valign="top"><p>0.207</p></td>
-    <td width="71" valign="top"><p>0.107</p></td>
-    <td width="58" valign="top"><p>0.51</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>20</p></td>
-    <td width="57" valign="top"><p>20</p></td>
-    <td width="59" valign="top"><p>2.908</p></td>
-    <td width="59" valign="top"><p>2.876</p></td>
-    <td width="59" valign="top"><p>0.071</p></td>
-    <td width="71" valign="top"><p>0.032</p></td>
-    <td width="58" valign="top"><p>0.11</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>21</p></td>
-    <td width="57" valign="top"><p>21</p></td>
-    <td width="59" valign="top"><p>3.124</p></td>
-    <td width="59" valign="top"><p>2.938</p></td>
-    <td width="59" valign="top"><p>0.203</p></td>
-    <td width="71" valign="top"><p>0.186</p></td>
-    <td width="58" valign="top"><p>0.87</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>22</p></td>
-    <td width="57" valign="top"><p>22</p></td>
-    <td width="59" valign="top"><p>2.652</p></td>
-    <td width="59" valign="top"><p>2.405</p></td>
-    <td width="59" valign="top"><p>0.203</p></td>
-    <td width="71" valign="top"><p>0.247</p></td>
-    <td width="58" valign="top"><p>1.15</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>23</p></td>
-    <td width="57" valign="top"><p>23</p></td>
-    <td width="59" valign="top"><p>2.908</p></td>
-    <td width="59" valign="top"><p>2.876</p></td>
-    <td width="59" valign="top"><p>0.071</p></td>
-    <td width="71" valign="top"><p>0.032</p></td>
-    <td width="58" valign="top"><p>0.11</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>25</p></td>
-    <td width="57" valign="top"><p>25</p></td>
-    <td width="59" valign="top"><p>2.908</p></td>
-    <td width="59" valign="top"><p>2.876</p></td>
-    <td width="59" valign="top"><p>0.071</p></td>
-    <td width="71" valign="top"><p>0.032</p></td>
-    <td width="58" valign="top"><p>0.11</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>26</p></td>
-    <td width="57" valign="top"><p>26</p></td>
-    <td width="59" valign="top"><p>2.598</p></td>
-    <td width="59" valign="top"><p>2.602</p></td>
-    <td width="59" valign="top"><p>0.203</p></td>
-    <td width="71" valign="top"><p>-0.004</p></td>
-    <td width="58" valign="top"><p>-0.02</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>27</p></td>
-    <td width="57" valign="top"><p>27</p></td>
-    <td width="59" valign="top"><p>2.908</p></td>
-    <td width="59" valign="top"><p>2.876</p></td>
-    <td width="59" valign="top"><p>0.071</p></td>
-    <td width="71" valign="top"><p>0.032</p></td>
-    <td width="58" valign="top"><p>0.11</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>28</p></td>
-    <td width="57" valign="top"><p>28</p></td>
-    <td width="59" valign="top"><p>1.907</p></td>
-    <td width="59" valign="top"><p>1.725</p></td>
-    <td width="59" valign="top"><p>0.260</p></td>
-    <td width="71" valign="top"><p>0.182</p></td>
-    <td width="58" valign="top"><p>1.29</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>29</p></td>
-    <td width="57" valign="top"><p>29</p></td>
-    <td width="59" valign="top"><p>2.908</p></td>
-    <td width="59" valign="top"><p>2.876</p></td>
-    <td width="59" valign="top"><p>0.071</p></td>
-    <td width="71" valign="top"><p>0.032</p></td>
-    <td width="58" valign="top"><p>0.11</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>30</p></td>
-    <td width="57" valign="top"><p>30</p></td>
-    <td width="59" valign="top"><p>2.999</p></td>
-    <td width="59" valign="top"><p>2.842</p></td>
-    <td width="59" valign="top"><p>0.203</p></td>
-    <td width="71" valign="top"><p>0.157</p></td>
-    <td width="58" valign="top"><p>0.73</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>31</p></td>
-    <td width="57" valign="top"><p>31</p></td>
-    <td width="59" valign="top"><p>2.726</p></td>
-    <td width="59" valign="top"><p>2.606</p></td>
-    <td width="59" valign="top"><p>0.203</p></td>
-    <td width="71" valign="top"><p>0.120</p></td>
-    <td width="58" valign="top"><p>0.56</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>32</p></td>
-    <td width="57" valign="top"><p>32</p></td>
-    <td width="59" valign="top"><p>3.042</p></td>
-    <td width="59" valign="top"><p>2.980</p></td>
-    <td width="59" valign="top"><p>0.203</p></td>
-    <td width="71" valign="top"><p>0.062</p></td>
-    <td width="58" valign="top"><p>0.29</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>33</p></td>
-    <td width="57" valign="top"><p>33</p></td>
-    <td width="59" valign="top"><p>2.834</p></td>
-    <td width="59" valign="top"><p>2.809</p></td>
-    <td width="59" valign="top"><p>0.203</p></td>
-    <td width="71" valign="top"><p>0.025</p></td>
-    <td width="58" valign="top"><p>0.12</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>34</p></td>
-    <td width="57" valign="top"><p>34</p></td>
-    <td width="59" valign="top"><p>2.908</p></td>
-    <td width="59" valign="top"><p>2.973</p></td>
-    <td width="59" valign="top"><p>0.203</p></td>
-    <td width="71" valign="top"><p>-0.065</p></td>
-    <td width="58" valign="top"><p>-0.30</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>35</p></td>
-    <td width="57" valign="top"><p>35</p></td>
-    <td width="59" valign="top"><p>0.508</p></td>
-    <td width="59" valign="top"><p>0.550</p></td>
-    <td width="59" valign="top"><p>0.207</p></td>
-    <td width="71" valign="top"><p>-0.042</p></td>
-    <td width="58" valign="top"><p>-0.20</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>36</p></td>
-    <td width="57" valign="top"><p>36</p></td>
-    <td width="59" valign="top"><p>0.989</p></td>
-    <td width="59" valign="top"><p>0.980</p></td>
-    <td width="59" valign="top"><p>0.207</p></td>
-    <td width="71" valign="top"><p>0.009</p></td>
-    <td width="58" valign="top"><p>0.04</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>37</p></td>
-    <td width="57" valign="top"><p>37</p></td>
-    <td width="59" valign="top"><p>1.338</p></td>
-    <td width="59" valign="top"><p>1.644</p></td>
-    <td width="59" valign="top"><p>0.208</p></td>
-    <td width="71" valign="top"><p>-0.306</p></td>
-    <td width="58" valign="top"><p>-1.45</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>38</p></td>
-    <td width="57" valign="top"><p>38</p></td>
-    <td width="59" valign="top"><p>2.908</p></td>
-    <td width="59" valign="top"><p>2.876</p></td>
-    <td width="59" valign="top"><p>0.071</p></td>
-    <td width="71" valign="top"><p>0.032</p></td>
-    <td width="58" valign="top"><p>0.11</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>39</p></td>
-    <td width="57" valign="top"><p>39</p></td>
-    <td width="59" valign="top"><p>2.908</p></td>
-    <td width="59" valign="top"><p>2.876</p></td>
-    <td width="59" valign="top"><p>0.071</p></td>
-    <td width="71" valign="top"><p>0.032</p></td>
-    <td width="58" valign="top"><p>0.11</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>40</p></td>
-    <td width="57" valign="top"><p>40</p></td>
-    <td width="59" valign="top"><p>2.082</p></td>
-    <td width="59" valign="top"><p>2.051</p></td>
-    <td width="59" valign="top"><p>0.208</p></td>
-    <td width="71" valign="top"><p>0.031</p></td>
-    <td width="58" valign="top"><p>0.15</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>41</p></td>
-    <td width="57" valign="top"><p>41</p></td>
-    <td width="59" valign="top"><p>1.750</p></td>
-    <td width="59" valign="top"><p>1.724</p></td>
-    <td width="59" valign="top"><p>0.207</p></td>
-    <td width="71" valign="top"><p>0.026</p></td>
-    <td width="58" valign="top"><p>0.12</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>42</p></td>
-    <td width="57" valign="top"><p>42</p></td>
-    <td width="59" valign="top"><p>2.167</p></td>
-    <td width="59" valign="top"><p>2.323</p></td>
-    <td width="59" valign="top"><p>0.208</p></td>
-    <td width="71" valign="top"><p>-0.156</p></td>
-    <td width="58" valign="top"><p>-0.74</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>43</p></td>
-    <td width="57" valign="top"><p>43</p></td>
-    <td width="59" valign="top"><p>2.957</p></td>
-    <td width="59" valign="top"><p>2.876</p></td>
-    <td width="59" valign="top"><p>0.071</p></td>
-    <td width="71" valign="top"><p>0.081</p></td>
-    <td width="58" valign="top"><p>0.28</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>44</p></td>
-    <td width="57" valign="top"><p>44</p></td>
-    <td width="59" valign="top"><p>2.800</p></td>
-    <td width="59" valign="top"><p>2.529</p></td>
-    <td width="59" valign="top"><p>0.208</p></td>
-    <td width="71" valign="top"><p>0.271</p></td>
-    <td width="58" valign="top"><p>1.29</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>45</p></td>
-    <td width="57" valign="top"><p>45</p></td>
-    <td width="59" valign="top"><p>0.555</p></td>
-    <td width="59" valign="top"><p>0.645</p></td>
-    <td width="59" valign="top"><p>0.207</p></td>
-    <td width="71" valign="top"><p>-0.090</p></td>
-    <td width="58" valign="top"><p>-0.43</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>46</p></td>
-    <td width="57" valign="top"><p>46</p></td>
-    <td width="59" valign="top"><p>2.900</p></td>
-    <td width="59" valign="top"><p>3.069</p></td>
-    <td width="59" valign="top"><p>0.208</p></td>
-    <td width="71" valign="top"><p>-0.169</p></td>
-    <td width="58" valign="top"><p>-0.80</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>47</p></td>
-    <td width="57" valign="top"><p>47</p></td>
-    <td width="59" valign="top"><p>3.061</p></td>
-    <td width="59" valign="top"><p>2.970</p></td>
-    <td width="59" valign="top"><p>0.208</p></td>
-    <td width="71" valign="top"><p>0.091</p></td>
-    <td width="58" valign="top"><p>0.43</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>48</p></td>
-    <td width="57" valign="top"><p>48</p></td>
-    <td width="59" valign="top"><p>2.600</p></td>
-    <td width="59" valign="top"><p>2.491</p></td>
-    <td width="59" valign="top"><p>0.208</p></td>
-    <td width="71" valign="top"><p>0.109</p></td>
-    <td width="58" valign="top"><p>0.52</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>49</p></td>
-    <td width="57" valign="top"><p>49</p></td>
-    <td width="59" valign="top"><p>2.782</p></td>
-    <td width="59" valign="top"><p>2.940</p></td>
-    <td width="59" valign="top"><p>0.208</p></td>
-    <td width="71" valign="top"><p>-0.158</p></td>
-    <td width="58" valign="top"><p>-0.75</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>50</p></td>
-    <td width="57" valign="top"><p>50</p></td>
-    <td width="59" valign="top"><p>2.200</p></td>
-    <td width="59" valign="top"><p>1.963</p></td>
-    <td width="59" valign="top"><p>0.207</p></td>
-    <td width="71" valign="top"><p>0.237</p></td>
-    <td width="58" valign="top"><p>1.12</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>51</p></td>
-    <td width="57" valign="top"><p>51</p></td>
-    <td width="59" valign="top"><p>0.508</p></td>
-    <td width="59" valign="top"><p>0.464</p></td>
-    <td width="59" valign="top"><p>0.207</p></td>
-    <td width="71" valign="top"><p>0.044</p></td>
-    <td width="58" valign="top"><p>0.21</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>52</p></td>
-    <td width="57" valign="top"><p>52</p></td>
-    <td width="59" valign="top"><p>1.530</p></td>
-    <td width="59" valign="top"><p>1.991</p></td>
-    <td width="59" valign="top"><p>0.207</p></td>
-    <td width="71" valign="top"><p>-0.461</p></td>
-    <td width="58" valign="top"><p>-2.18R</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>53</p></td>
-    <td width="57" valign="top"><p>53</p></td>
-    <td width="59" valign="top"><p>1.700</p></td>
-    <td width="59" valign="top"><p>2.064</p></td>
-    <td width="59" valign="top"><p>0.207</p></td>
-    <td width="71" valign="top"><p>-0.364</p></td>
-    <td width="58" valign="top"><p>-1.73</p></td>
-  </tr>
-  <tr>
-    <td width="75" valign="top"><p>54</p></td>
-    <td width="57" valign="top"><p>54</p></td>
-    <td width="59" valign="top"><p>2.908</p></td>
-    <td width="59" valign="top"><p>2.876</p></td>
-    <td width="59" valign="top"><p>0.071</p></td>
-    <td width="71" valign="top"><p>0.032</p></td>
-    <td width="58" valign="top"><p>0.11</p></td>
-  </tr>
-</table>
-S = 0.295758   PRESS = 7.78904</br>
-R-Sq = 92.25%  R-Sq(pred) = 78.45%  R-Sq(adj) = 87.41%</br>
-Suppose we redefine the factors accoding to the following table:</br>
-According to the ANOVA calculated by minitab, we got the expression of OD:</br>
-f(T,t,p,y,c)=2.87625+0.60225T+0.28447t+0.18665p+0.16776y-0.01626c-0.549T^2-0.18725t^2-0.08325p^2-0.10625y^2-0.05075c^2-0.35558Tt+0.17881Tp+0.04544Ty+0.0155Tc+0.02575tp+0.06112ty-0.00144tc-0.07469py+0.09150pc-0.04450yc</br>
-P represents confidence coefficient, which is a key judgment to check the reliability of the fitting function. In other words, if P=0.05, the probability that this term is wrong is 5%. The coefficient of determination (R) was calculated to be 0.9225, indicating that the model could explain 92% of the variability .From the above table we can identify eight statistically significant and reliable terms:</br>
-Constant;</br>
-Temperature;</br>
-Time;</br>
-Yeast Extract;</br>
-Peptone;</br>
-Temperature*Temperature;</br>
-Temperature*Time;</br>
-Temperature*Yeast Extract;</br>
-The influences of linear terms predominated, except NaCl, which substantiated our suspicion whereas most square terms and interaction terms were ignorable and statistically unreliable. Temperature and time and two most influential factor.</br>
-As our world is three-dimensional but the intact response surface is six-dimensional, it is impossible to draw the intact surface. Yet we could fix some factors to lower the dimensional, which empowers us to imagine the full surface. Here are some surfaces and contours of our fitting surface, we can extrapolate this super surface by combining these pictures:</br>
+<p align="justify">
+The influences of linear terms predominated, except NaCl, which substantiated our suspicion, whereas most square terms and interaction terms were ignorable and statistically unreliable. Temperature and time and two most influential factors.</br>
+As the intact response surface is six-dimensional, it is impossible to draw the intact surface in our three-dimensional world. Therefore we had to fix some factors to lower the dimensions, draw contours and surfaces, and e can extrapolate this super surface by combining these pictures:</p>
+<img src="https://static.igem.org/mediawiki/2013/2/24/%E6%9E%AF%E8%8D%891.png"></br>
+<p align="center"><b> Figure 1.</b> Surface plots of OD vs time and temperature.</p></p></br></br></br>
+<img src="https://static.igem.org/mediawiki/2013/d/d0/%E6%9E%AF%E8%8D%892.png"></br>
+<p align="center"><b> Figure 2.</b> Contour plots of OD vs time and temperature.</p></br></br></br>
+<img src="https://static.igem.org/mediawiki/2013/1/1f/%E6%9E%AF%E8%8D%893.png"></br>
+<p align="center"><b> Figure 3.</b> Surface plots of OD vs time and peptone.</p></br></br></br>
+<img src="https://static.igem.org/mediawiki/2013/9/9f/%E6%9E%AF%E8%8D%894.png"></br>
+<p align="center"><b> Figure 4.</b> Contour plots of OD vs time and peptone.</p></br></br></br>
+<img src="https://static.igem.org/mediawiki/2013/2/22/%E6%9E%AF%E8%8D%895.png"></br>
+<p align="center"><b> Figure 5.</b> Surface plots of OD vs peptone and temperature.</p></br></br></br>
+<img src="https://static.igem.org/mediawiki/2013/5/54/%E6%9E%AF%E8%8D%896.png"></br>
+<p align="center"><b> Figure 6.</b> Contour plots of OD vs peptone and temperature.</p></br></br></br>
+<img src="https://static.igem.org/mediawiki/2013/4/40/%E6%9E%AF%E8%8D%897.png"></br>
+<p align="center"><b> Figure 7.</b> Conyour plots of OD vs yeast extract and temperature.</p></br>
+<img src="https://static.igem.org/mediawiki/2013/4/40/%E6%9E%AF%E8%8D%897.png"></br>
+<p align="center"><b> Figure 8.</b> Contour plots of OD vs yeast extract and temperature.</p></br></br></br>
+The following four pictures illustrate the distribution of residual error:</br></br>
+<img src="https://static.igem.org/mediawiki/2013/b/b1/%E6%9E%AF%E8%8D%899.png"></br>
+<p align="center"><b> Figure 9.</b> Residual error vs order</p></br></br></br>
+<img src="https://static.igem.org/mediawiki/2013/0/04/%E6%9E%AF%E8%8D%8910.png"></br>
+<p align="center"><b> Figure 10.</b> Histogram of residual error</p></br></br></br>
+<img src="https://static.igem.org/mediawiki/2013/2/29/%E6%9E%AF%E8%8D%8911.png"></br>
+<p align="center"><b> Figure 11.</b> Residual error vs fits</p></br></br></br>
+<img src="https://static.igem.org/mediawiki/2013/5/50/%E6%9E%AF%E8%8D%8912.png"></br>
+<p align="center"><b> Figure 12.</b> Normal probability plot of residual error</p></br></br></br>
 </div>
 <div>
-<h1>【Designs&Results】</h1>
+<h1>Optimization</h1>
-In our experiment , we discovered that the promoter P43 is too weak to express amilCP for a blue-color display. So we redesigned the circuits , aiming to find a more feasible plan, and test the part amilCP simultaneously. The new disign contains two circuits:</br>
+<p align="justify">
-<h2>1 Pgrac-amilCP-Terminator</h2>
+One remarkable character of CCD is that it is sequential, which is also the essence of RSM. Since we had got the fitting function, the next step is to calculate the gradient of the function, and define a small number as step length. Further experiments are supposed to be conducted from the beginning point according to the gradient and step length, and the final maximal treatment would be made sure. The methodology of RSM seems like climbing a mountain whose peak is unknown, and we are supposed to adjust our orientation according to the topography. The fitting surface, which can be often a super surface in higher dimensional spaces, can be likened to the mountain without clear peaks, and calculating gradient to orientating.</br>
+Unfortunately our remaining time is not enough to support further experiments, and as we looked up other researches utilizing RSM, none of which did second round experiment, and we realized perhaps that was the difference between a scientific research and a real industrial procedure. Yet the analytical methodology of response surface still acted as a powerful tool for ANOVA. Roughly, we could consider the treatment of No. 15 medium (Temperature 35℃, Time 12h, Peptone 15, Yeast Extract 7.5, NaCl 15)as the maximal condition for <i>B.subtilis</i>.
+</p>
-We use the Promoter grac , a promotor with lac promotor ,on the PHT vector to express amilCP, instead of Promoter 43. The Promoter grac is strong enough ,so we can easily see the results theoretically. </br>
-<img src="https://static.igem.org/mediawiki/2013/2/22/Reporter_2.tif" width="240" height="120" /> </br>
-pgrac-amilCP overlap PCR（Right three tracks） PSBC3 Double Digestion（Left four tracks）</br>
-<h2>2 P43-amilcp-SigB-Terminator</h2>
-We plan to use a positive feedback to magnify the expressing of amilCP. Because the P43 is a sigma B factor binding promotor, we designed a circuit, that the P43 is fused to the sigma B factor. We hope this could increase the response of P43.</br>
-Course of time limitation, we haven’t put this design to experiment.
-</div>
-<div>
-<h1>【designing of the suicide system】</h1>
-We design a circuit of killing switch based on its endogenous genetic system.</br>
-In B.subtilis, when it comes to the stationary phase, the environmental pressure increases and nutrition becomes limited, so B begin to produce spores. Now the community will be divided into two different parts. One of them are trying to kill others to get enough nutrient , delaying the production of  spores and achieving a competitive advantage. Killing is mediated by the exported toxic protein SdpC. SdpI will appear on the membrane surface to avoid itself from being damaged. SdpI could bind free SdpC and autopressor SdpR, to remove SdpR’s inhibition against I and R, to produce more SdpI to offset SdpC, finally guaranteeing the subgroup alive, thereby delaying the spores production.</br>
-https://static.igem.org/mediawiki/2013/2/2b/Reporter_3.png
-We transfer SdpC which is fused by promoter SdpI/R into high copy plasmids in order to damage the balance of the system, thereby killing whole colony. When SdpC appears, SdpI on the membrane will bind free SdpC and adsorb SdpR to cease its inhibition against SdpI P/R, trying to produce more SdpI. At the same time, it will activate the promoter SdpR/I in our circuits and generate more SdpC.The system would fall into an infinite loop, and according to our modeling ,the amount of SdpC increases beyond the ability of SdpI.Thus,the cells with protection mechanism will crack and die because of too much SdpC. All above forms the killing device.
-We Also designed a test circuit,which contains promotor grac and sdpABC only,aiming to determine the ability of SdpC.</br>
-<h2>Results</h2>
-https://static.igem.org/mediawiki/2013/e/ec/Reporter_4.tif</br>
-Colony PCR       E.coli  PHT43 + Promotor grac + SdpABC</br>
-https://static.igem.org/mediawiki/2013/9/92/Reporter_5.tif</br>
-PHT43 + Promotor SdpRI + SdpABC    Enzyme digestion</br>
-</div>
-<div>
-<h1>【References】</h1>
-Parallel pathways of repression and antirepression governing the transition to stationary phase in Bacillus subtilis
-AV Banse, A Chastanet, L Rahn-Lee…,PNAS ,2008 </br>
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-<div id="t1"><a href="https://2013.igem.org/Team:USTC_CHINA/Modeling/ReporterSystem">Reporter System</a></div>
+<div id="t1"><a href="https://2013.igem.org/Team:USTC_CHINA/Modeling/KillSwitch">Kill Switch</a></div>
 <div id="t1"><a class="active" href="https://2013.igem.org/Team:USTC_CHINA/Modeling/B.SubtilisCulture">B.Subtilis Culture</a></div>
-<div id="t1"><a href="https://2013.igem.org/Team:USTC_CHINA/Modeling/MiceModeling">Mice Modeling</a></div>
+<div id="t1"><a href="https://2013.igem.org/Team:USTC_CHINA/Modeling/DesignsofImmuneExperiments">Designs of Immune Experiments</a></div>
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Team:USTC CHINA/Modeling/B.SubtilisCulture