Difference between revisions of "Team:UCL/Model"

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UCL SETA - Model

Models

Overview Cell Growth and Expression Protein Polymerisation W.O.R.M. Mechanical Simulation Bibliography

Overview

One of the challenges for biomaterial application is to manufacture large scale protein production economically and efficiently. In our model, we integrated several levels of modelling to optimise the productivity of the whole system.

For our biomaterial production, we use split intein technology instead of genetic modification to gain longer protein polymers. Split inteins have a specific working temperature and pH condition for sufficient reaction rate. In our models, in addition to basic cell growth and protein expression model, we introduce intein polymerisation model (WORM) and fiber mechanical property simulation. Integrating these models, we can estimate the time required for desired protein polymerisation and thus provide the fiber with strong mechanical properties.

Cell Growth and Expression

Verhulst and Pearl and Reed played a huge part in developing a theory on logistic population growth. The rate considers an additional inhibition factor proportional to x² from the Malthus population model.

$\frac{dy}{dx}=kx(1-bx)$ where $x(0)=x_{{o}}$

This Riccati equation can then be solved analytically into:

$x=\frac{x_{{o}}e^{kt}}{1-bx_{{o}}(1-e^{kt})}$

We fit our data into the first-order ordinary differential equation model, and the sigmoidal curve that we get has a property of stationary area which is determined by 1/b.

Symbols	Values and Units	Descriptions
t	hr	Time
x	dimensionless	Optical density of a cell at 600 nm wavelength
b	dimensionless	Inhibiting factor, which is inversely proportional to maximum cell population size.
k	$hr^{-1}$	Population growth rate

Table 1.

Additional information.

We determined the better growth media from the two broth we had available, which were Terrific Broth (TB) and Lysogeny Broth (LB).

Figure 1.

Comparing TB and LB as E. coli growth medium.

The fitted equations tells us the inhibiting factor b for TB and LB, which are 0.3 and 0.4077, respectively. This is as predicted because TB has less inhibition compared to LB because of the nutrient abundance, carrier capacity, and toxicity level caused by the waste side-products of the cells. The same trend is also observed in maximum population, that TB provides a better environment for larger cell population in longer cultivations. Meanwhile for earlier growth rate k, cells in LB grow quicker before achieving stationary phase, which is 0.44hr^-1 compared with 0.24hr^-1 in TB.

Figure 2.

Optical density measurements of each cell induction.

Figure 3.

Population growth rate (k) for each IPTG concentration.

IPTG Conc.	400mM	500mM	600mM	700mM	800mM
k	0.4215	0.4649	0.4735	0.4773	0.4759
b	0.4361	0.4227	0.4208	0.4097	0.4069

Table 2.

the growth rate (k) and inhibitor factor (b) corresponding to different IPTG induction concentration.

We then build up the kinetics inside a cell and integrate it with lab data analysis to understand protein production and get an effective lab plan for our E. coli system. GFP, RFP, and Intein Passenger fluorescence data of 544/590 nm filtering wavelengths were then extracted and processed. Each dependent value is described as protein-fold change post induction, which is normalised with the same system but uninduced, based on the fraction below:

$p=\frac{F}{F_{{o}}}$

The first-order system of linear differential equations are:

$\frac{dm}{dt}=\alpha x-\beta m$ $\frac{dp}{dt}=\gamma m-\delta p$

The first equation represents the mRNA system in a cell with the assumption of constant gene concentration in each cell. Meanwhile the latter equation is the protein system which is dependent on the previous equation, and fitted to the data from the plate reader, using the Trust Region Algorithm with non-negative floating number as the boundary in each parameter.

Figure 4.

Fluorescence of Intein Passenger induced with 400 mM IPTG and normalised in each E. coli cell. The rest of induced cultures are fitted in the same equation.

IPTG Conc.(mM)	400	500	600	700	800
x	200	200	200	200	200
α	0.02073	0.02332	0.02348	0.02156	0.02264
β	0.2482	0.2644	0.2642	0.2665	0.2777
γ	0.6564	0.6799	0.6668	0.7346	0.7449
δ	0.2563	0.2744	0.2719	0.2738	0.2827

Table 3.

Parameter values estimation using Trust Region Algorithm of curve fitting.

Symbols	Units	Descriptions
t	hours	Time
x	200	Plasmid copy number of pSB1C3 in each cell
m	dimensionless	Respective normalised mRNA concentration in each cell
p	dimensionless	Respective normalised protein concentration in each cell
α	hr^-1	Gene transcription rate or mRNA production rate
β	hr^-1	mRNA degradation rate and dilution factor
γ	hr^-1	Protein translation rate
δ	hr^-1	Protein degradation rate and dilution factor

Table 4.

Additional information.

The α and β terms generally stay constant without a trend between IPTG concentrations which is explainable even though IPTG affects cell transcription activity. Since mRNA has a high rate of synthesis and degradation, it reaches equilibrium immediately as its stability closely follows inversely proportional to the mRNA concentration in the cell. The parameter γ and δ give a small yet noticeable increasing trend in higher IPTG level, this is related to protein concentration equilibrium, but not to the extent of mRNA degradation rate since protein relatively ‘lives’ longer than mRNA.

Figure 5.

Fluorescence model of Intein Passenger induced with IPTG 400 mM. Fold-change in protein concentration reaches maximum at 42.8 ratio in longer duration of cell cultivation.

Generally IPTG does not give significant differences in high concentrations, so we chose 500 mM of IPTG which the model suggests as the most efficient with the similar expression level compared to the cells with higher concentration of IPTG. Further improvements of this model might include larger choices of medium and wider range of IPTG concentrations, together with cost analysis as it is an important factor in the larger scale production.

Protein Polymerisation

To better accommodate the integrated model and identify bottlenecks, we further develop a model entirely of in vitro polymerisation as it is indispensable to the overall process of biopolymer production.

$\frac{dP_{n}}{dt}=\sum_{i=1}^{n} \kappa\left [ P_{i} \right ]\left [ P_{n-1} \right ]-\sum_{i=1}^{n_{max}-n}\kappa\left [ P_{n} \right ]\left [ P_{i} \right ]$

Each single protein is interpreted as a monomer with an irreversible polymerising capacity until it reaches an assumed maximum size. Polymer formation rate of each species (P_n) were then modelled in Python and plotted. The first term describes the polymer species formation from the sum of all reactions in the range of its possible constituents, and the second term represents the further polymerisation of the respective species as a substrate, which build up into larger repeats.

Figure 6.

Monomer distribution in each species as a function of time for Npu DnaE(N) + Ssp DnaE(C) split inteins. Starting solution only consists of monomers, modeled as they merge and accumulate into larger species of polymer.

Figure 7.

Monomer distribution in each species as a function of time Ssp DnaE inteins. Starting solution only consists of monomers, modeled as they merge and accumulate into larger species of polymer.

Figure 8.

Ratio of monomer distribution species over time in Npu DnaE(N) + Ssp DnaE(C) split inteins combination. Frame rates do not represent real-time.

Figure 9.

Ratio of monomer distribution species over time in Ssp DnaE inteins. Frame rates do not represent real-time.

Symbols	Values and Units	Descriptions
t	s	Time
n	dimensionless	Number of repeats, defining a certain species of polymer
P_n	mol L^-1	Polymer species n
k	L mol^-1 S^-1>	Rate constants of Intein activity of a certain intein
n_max	dimensionless	Maximum repeat number in a polymer

Table 5.

Additional information.

Basically each intein rate of reaction (k) differs with each intein system we use.

Intein	Rate of reaction (s^-1)
Npu DNA-E	1.1x10^-2
SspDNA-E	3.3x10^-4
Npu DNA-E(N) + SspDNA-E	2.8x10^-5
SceVMA	2.0x10^-3
SspDNA-B	9.9x10^-4

Table 6.

Specific activity of each inteins. Rate of reaction are collected from the references mentioned in the bottom of the page.

The results we get from the example runs of Npu DnaE(N) and Ssp DnaE(C) intein combinations are after 10 minutes of the simulated experiment, only 3.45% of monomers are in the form of the top 25% longest polymer species. However for Ssp DnaE inteins, in the course of 10 minutes, 72.3% of the monomers are polymerised into the same top 25% of the longest polymer region. This really shows how the intein polymerisation will require much less time compared to the cell culture and protein production. Further improvements will be introducing more parameters into the estimation function to represent more accurate results.

Intein Polymerisation

W.O.R.M.

To further model the polymerisation of our proteins, we devised a simplified proof of concept of our project using reporter proteins RFP and GFP to predict the polymerisation capabilities of split inteins. Each reporter protein is flanked by orthogonal inteins, which match up to the other reporter’s inteins to allow RFP and GFP to continuously link up into various lengths of polymers.

Figure 10.

Polymerisation of intein flanked reporter proteins. Npu-N intein on GFP splices out with the Npu-C intein on RFP. Similarly, the TerL-N intein on the RFP splices out with the TerL-C intein on the GFP. This reaction results in the polymerisation of GFP and RFP.

We used the python libraries PyGame and PyMunk to create an animated and interactive agent-based model of intein polymerisation. Acronymed WORM, this model depicts a Wonderful Oligomerisation Reaction Model.

Depicted in the video is a snapshot of the model WORM that shows intein polymerisation. The yellow and orange rectangles represent an split intein pair, as do the blue and cyan rectangles. The red and green rectangles represent the reporter proteins, RFP and GFP, respectively.

As an agent-based mode, WORM was created based on the following rules:

Particles diffuse with random angular and lateral velocity;
Velocity increases with temperature (based on average kinetic energy): $(\frac{1}{2}mv^{2})=\frac{3}{2}kT$
Where:
- m= mass (kg)
- v= velocity
- k= Boltzmann constant
- T= temperature (K)
Like reacts with unlike (RFP reacts with GFP);
Particles are polarised (Npu-N [A1]→ Npu-C [B3], TerL-N [B1]→ TerL-C [A3]);
Splicing removes 1+3 on reaction (removes the inteins);
Steric hindrance by particles.

Figure 11.

Distribution of polymer lengths after 20 runs. WORM was run for 5 minutes, 20 times. The distribution of total polymers for each polymer lengths from 1-20, and lengths over 20, were plotted. The error was plotted as the square root of the total polymers in each polymer length.

The interactive components of the WORM model include user’s choice of temperature and protein mass to selectively model the reaction conditions and functionalized protein of interest. These applications can be used by running the code found in our GitHub. The combination of predictive experimental data and the visually appealing computational model makes a user friendly platform for the public to better understand biology and modelling.

Mechanical Simulation

In this part, we also modeled the biomaterials’ micro-structure of the coarse-grained system. We predicted the structure of different spider silk repeats (tetramer, octamer and dodecamer) both after stretching and shearing. This method is a deterministic approach to foresee the network structure of spider silk repeats prior to the actual lab experiment and helps us to understand the design of the actual protein that we are going to produce afterwards.

Here is our initial designed sequence, a spider silk protein in repeats of four:


                                                GQGGYGGLGSQGAGRGGLGGQGAGAAAAAAAAGGAGQGGYGGLGSQGAGRGGLGGQGAGAAAAAAAAAGGYGGLGSQGAGRGGLGGQGAGAAAAAAAAGGAGQGGYGGLGSQGAGRGGLGGQGAGAAAAAAAAGGA

The respective sequence is then quantitatively analysed for their hydrophobicity and hydrophilicity and plotted.

Figure 12.

Kyte-Doolittle-Hydropathy plot. The vertical axis is the measure of hydrophobicity in each amino acid in the sequence, with higher score means greater degree of hydrophobicity. With the help of ProtScale from ExPASy.

In this particular sequence, we can observe that the hydrophobic and hydrophilic parts alternate inside the spider silk fiber, which will form aggregates and bridge connections to achieve desired tensile and toughness due to the hydrophobic interactions.

This alternating pattern seen in the figure allows us to simplify proteins as the hydrophobic domains and hydrophilic domains. These domains are then represented as a collection of the so-called ‘beads’, and depending on relative molecular volume of each domain, the coarse-grained model is then scaled and measured into an estimate of three amino acid residues per bead.

This system is composed from 3 components: water, hydrophilic and hydrophobic beads. Every 9 water molecules is represented as a single bead, so its 270 Å size (each water molecule is about 30 Å) is proportional to the protein beads.

We then introduce LAMMPS as our software, which is an abbreviation of Large-scale Atomic/Molecular Massively Parallel Simulator, to simulate the behaviour of our ‘beads’ collection in a single compartment and thus gave us an insight into the physical property of the recombinant spider silk.

Simulations

Hover to compare the 2 sides.

Equal Stretch

Tetramer

Octamer

Dodecamer

For these simulation results, the configurations of spider silk fiber after applying a constant engineering strain rate (7.5 × 10^-6 τ^-1 along the x-axis) and at equilibrium after shear flow (0.01 τ^-1 along x axis for 420000 shear steps). The red nodes show hydrophobic aggregated protein clusters and the blue lines are the bridge connecting two clusters. Uniform distribution and high density of clusters and bridges represent the better mechanical property, higher tensile and toughness. For stretch testing. we can analyse the tetramer structure which consists of large and frequent cavity in the fiber. The octamer and dodecamer is predicted to be able to tolerate this strain, which means they have a considerable threshold of mechanical strain. In order to examine which structure is the best, we apply shear flow. In this case, it is observed that only the dodecamer still keeps its original structure but others break at different levels. In conclusion, dodecamer is the best configuration required in making desired fibers.

Bibliography

Verhulst PF. Notice sur la loi que la population poursuit dans son accroissement. Correspondance mathématique et physique. 1838;10:113–21.
Pearl R, Reed L. On the Rate of Growth of the Population of the United States since 1790 and Its Mathematical Representation. Proceedings of the National Academy of Sciences. 1920;6(6):275-288.
Bailey J, Ollis D. Biochemical engineering fundamentals. 2nd ed. New York: McGraw-Hill; 1944.
Part:pSB1C3 - parts.igem.org [Internet]. Parts.igem.org. 2008. Available from: http://parts.igem.org/Part:pSB1C3
Zettler J, Schütz V, Mootz HD. The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans-splicing reaction. FEBS Letters 2009;583:909–14.
Nichols NM, Benner JS, Martin DD, Evans TC. Zinc Ion Effects on Individual Ssp DnaE Intein Splicing Steps: Regulating Pathway Progression. Biochemistry 2003;42:5301–11.
Al-Ali H, Ragan TJ, Gao X, Harris TK. Reconstitution of Modular PDK1 Functions on Trans-Splicing of the Regulatory PH and Catalytic Kinase Domains. Bioconjugate Chemistry 2007;18:1294–302.
Brenzel S, Kurpiers T, Mootz HD. Engineering Artificially Split Inteins for Applications in Protein Chemistry: Biochemical Characterization of the Split Ssp DnaB Intein and Comparison to the Split Sce VMA Intein. Biochemistry 2006;45:1571–8.
Kyte J, Doolittle RFA. simple method for displaying the hydropathic character of a protein. Journal of Molecular Biology. 1982;157(1):105–32.
ExPASy - ProtScale [Internet]. Web.expasy.org. Available from: https://web.expasy.org/protscale/
Rim N, Roberts E, Ebrahimi D, Dinjaski N, Jacobsen M, Martín-Moldes Z, Buehler M, Kaplan D and Wong J. Predicting Silk Fiber Mechanical Properties through Multiscale Simulation and Protein Design. ACS Biomaterials Science & Engineering. 2017;3(8), pp.1542-1556.
Lin S, Ryu S, Tokareva O, Gronau G, Jacobsen M, Huang W, Rizzo D, Li D, Staii C, Pugno N, Wong J, Kaplan D and Buehler M. Predictive modelling-based design and experiments for synthesis and spinning of bioinspired silk fibres. Nature Communications. 2015;6(1).

Our Location

University College London
Gower St, Bloomsbury
London
WC1E 6BT

Contact Us

ucl.igem@ucl.ac.uk

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-        <a href="https://2018.igem.org/Team:UCL/Model#Intein">Intein</a>
-        <a href="https://2018.igem.org/Team:UCL/Model#Intein_Polymerisation">Intein Polymerisation</a>
-        <a href="https://2018.igem.org/Team:UCL/Model#LAMMPS">LAMMPS</a>
-        <a href="https://2018.igem.org/Team:UCL/Model#Scale_Up">Scale Up</a>
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          <a href="https://2018.igem.org/Team:UCL/Human_Practices">Human Practices</a>
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                              <div class="mmmm col-sm-10">
                                  <p class="card-text card-text_igem text-justify">
-                                  One of the challenges for biomaterial application is to manufacture large scale protein production economically and efficiently. In our model, we integrated several levels of modelling to optimise the productivity of the whole system.
+                                    One of the challenges for biomaterial application is to manufacture large scale protein production economically and efficiently. In our model, we integrated several levels of modelling to optimise the productivity of the whole system.
-<br><br>
+                                </p>
-For our biomaterial production, we use split intein technology instead of genetic modification to gain longer protein polymers. Split inteins have a specific working temperature and pH condition for sufficient reaction rate. In our models, in addition to basic cell growth and protein expression model, we introduce intein polymerisation model (WORM) and fiber mechanical property simulation. Integrating these models, we can estimate the time required for desired protein polymerisation and thus provide the fiber with strong mechanical properties.
+                                <p class="card-text card-text_igem text-justify">
+                                    For our biomaterial production, we use split intein technology instead of genetic modification to gain longer protein polymers. Split inteins have a specific working temperature and pH condition for sufficient reaction rate. In our models, in addition to basic cell growth and protein expression model, we introduce intein polymerisation model (WORM) and fiber mechanical property simulation. Integrating these models, we can estimate the time required for desired protein polymerisation and thus provide the fiber with strong mechanical properties.
                                  </p>
                              </div>
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                                  <p class="card-text card-text_igem text-justify">
-                              <p>
                                  Verhulst and Pearl and Reed played a huge part in developing a theory on logistic population growth. The rate considers an additional inhibition factor proportional to <i>x<sup>2</sup></i> from the Malthus population model.
+                                </p>
-</p>
+                                <center>
-                                    <center>
+                                    <img class="model_img" src="https://latex.codecogs.com/gif.latex?\frac{dy}{dx}=kx(1-bx)" title="\frac{dy}{dx}=kx(1-bx)" />
+                                </center>
+                                <center>
-<br />
+                                    where
-                              <img src="https://latex.codecogs.com/gif.latex?\frac{dy}{dx}=kx(1-bx)" title="\frac{dy}{dx}=kx(1-bx)" /></center>
+                                </center>
-<br />
+                                 <center>
-                              <center>
+                                    <img class="model_img" src="https://latex.codecogs.com/gif.latex?x(0)=x_{{o}}" title="x(0)=x_{{o}}" />
-                                 where
+                                </center>
-                                <img src="https://latex.codecogs.com/gif.latex?x(0)=x_{{o}}" title="x(0)=x_{{o}}" />
+                                <p class="card-text card-text_igem text-justify">
-</center>
+                                    This Riccati equation can then be solved analytically into:
-<br /><br /><br />
+                                </p>
-                            <p>
+                                <center>
+                                    <img class="model_img" src="https://latex.codecogs.com/gif.latex?x=\frac{x_{{o}}e^{kt}}{1-bx_{{o}}(1-e^{kt})}" title="x=\frac{x_{{o}}e^{kt}}{1-bx_{{o}}(1-e^{kt})}" />
-                       This Riccati equation can then be solved analytically into:
+                                </center>
-                     </p>
+                                <p class="card-text card-text_igem text-justify">
-                              <br />
+                                    We fit our data into the first-order ordinary differential equation model, and the sigmoidal curve that we get has a property of stationary area which is determined by 1/b.
-                              <center>
+                                </p>
-                              <img src="https://latex.codecogs.com/gif.latex?x=\frac{x_{{o}}e^{kt}}{1-bx_{{o}}(1-e^{kt})}" title="x=\frac{x_{{o}}e^{kt}}{1-bx_{{o}}(1-e^{kt})}" />                        </center>
-                              <br /><br /><br /><p>
-                              We fit our data into the first-order ordinary differential equation model, and the sigmoidal curve that we get has a property of stationary area which is determined by 1/b.</p>
-                               <table class="table ooooo">
+                               <table class="table ooooo table_text_igem">
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                                            <td class="tablecenter">dimensionless</td>
                                            <td class="tablecenter">
-Inhibiting factor, which is inversely proportional to maximum cell population size.
+                                              Inhibiting factor, which is inversely proportional to maximum cell population size.
-</td>
+                                          </td>
                                        </tr>
                                        <tr>
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                                            <img src="https://latex.codecogs.com/gif.latex?hr^{-1}" title="hr^{-1}" /></td>
                                            <td class="tablecenter">
-Population growth rate
+                                              Population growth rate
-</td>
+                                          </td>
                                        </tr>
                                    </tbody>
                                </table>
                                <center>
-                               <figcaption>
+                               <figcaption class="card-text_igem2">
-                                 Table 01: Additional information.
+                                 <div class="model_caption_title_igem">Table 1.</div> Additional information.
                                </figcaption>
                                </center>
-<br /><br>
+                              <p class="card-text card-text_igem text-justify">
-<p>We determined the better growth media from the two broth we had available, which were Terrific Broth (TB) and Lysogeny Broth (LB).
+                                  We determined the better growth media from the two broth we had available, which were Terrific Broth (TB) and Lysogeny Broth (LB).
+                              </p>
-</p>
+                              <center>
-<br>
+                                  <img class="model_img" class="m1"src="https://static.igem.org/mediawiki/2018/a/a0/T--UCL--m1.png"alt="oops! You can't see our image.">
-<center>
+                              </center>
-<img class="m1"src="https://static.igem.org/mediawiki/2018/a/a0/T--UCL--m1.png"alt="oops! You can't see our image.">
+                              <center>
-</center>
+                                  <figcaption class="card-text_igem2">
-<center>
+                                      <div class="model_caption_title_igem">Figure 1.</div>Comparing TB and LB as E. coli growth medium.
+                                  </figcaption>
+                              </center>
+                              <p class="card-text card-text_igem text-justify">
+                                  The fitted equations tells us the inhibiting factor <i>b</i> for TB and LB, which are 0.3 and 0.4077, respectively. This is as predicted because TB has less inhibition compared to LB because of the nutrient abundance, carrier capacity, and toxicity level caused by the waste side-products of the cells. The same trend is also observed in maximum population, that TB provides a better environment for larger cell population in longer cultivations. Meanwhile for earlier growth rate <i>k</i>, cells in LB grow quicker before achieving stationary phase, which is 0.44hr<sup>-1</sup> compared with 0.24hr<sup>-1</sup> in TB.
+                              </p>
+                              <center>
+                                  <img class="m1 model_img"src="https://static.igem.org/mediawiki/2018/4/44/T--UCL--m2.png"alt="oops! You can't see our image.">
+                              </center>
+                              <center>
+                                  <figcaption class="card-text_igem2">
+                                      <div class="model_caption_title_igem">Figure 2.</div> Optical density measurements of each cell induction.
+                                  </figcaption>
+                              </center>
+                              <center>
+                                  <img class="m1 model_img"src="https://static.igem.org/mediawiki/2018/6/64/T--UCL--m3.png"alt="oops! You can't see our image.">
+                              </center>
+                              <center>
+                                  <figcaption class="card-text_igem2">
+                                      <div class="model_caption_title_igem">Figure 3.</div>Population growth rate (<i>k</i>) for each IPTG concentration.
+                                  </figcaption>
+                              </center>
+                              <table class="table ooooo table_text_igem">
+                                  <thead>
+                                      <tr>
+                                          <th class="tableleft">IPTG Conc.</th>
+                                          <th class="tablecenter">400mM</th>
+                                          <th class="tablecenter">500mM</th>
+                                          <th class="tablecenter">600mM</th>
+                                          <th class="tablecenter">700mM</th>
+                                          <th class="tablecenter">800mM</th>
+                                      </tr>
+                                  </thead>
+                                  <tbody>
+                                      <tr>
+                                          <td class="tableleft"><i>k</i></td>
+                                          <td class="tablecenter">0.4215</td>
+                                          <td class="tablecenter">0.4649</td>
+                                          <td class="tablecenter">0.4735</td>
+                                          <td class="tablecenter">0.4773</td>
+                                          <td class="tablecenter">0.4759</td>
+                                      </tr>
+                                      <tr>
+                                          <td class="tableleft"><i>b</i></td>
+                                          <td class="tablecenter">0.4361</td>
+                                          <td class="tablecenter">0.4227</td>
+                                          <td class="tablecenter">0.4208</td>
+                                          <td class="tablecenter">0.4097</td>
+                                          <td class="tablecenter">0.4069</td>
+                                      </tr>
+                                  </tbody>
+                              </table>
+                              <center>
+                                  <figcaption class="card-text_igem2">
+                                     <div class="model_caption_title_igem">Table 2.</div> the growth rate (k) and inhibitor factor (b) corresponding to different IPTG induction concentration.
+                                  </figcaption>
+                              </center>
+                              <p class="card-text card-text_igem text-justify">
+                                  We then build up the kinetics inside a cell and integrate it with lab data analysis to understand protein production and get an effective lab plan for our E. coli system. GFP, RFP, and Intein Passenger fluorescence data of 544/590 nm filtering wavelengths were then extracted and processed. Each dependent value is described as protein-fold change post induction, which is normalised with the same system but uninduced, based on the fraction below:
+                              </p>
+                              <center>
+                                  <img class="model_img" src="https://latex.codecogs.com/gif.latex?p=\frac{F}{F_{{o}}}" title="p=\frac{F}{F_{{o}}}" />
+                              </center>
+                              <p class="card-text card-text_igem text-justify">
+                                  The first-order system of linear differential equations are:
+                              </p>
+                              <center>
+                                  <img class="model_img" src="https://latex.codecogs.com/gif.latex?\frac{dm}{dt}=\alpha&space;x-\beta&space;m" title="\frac{dm}{dt}=\alpha x-\beta m" />
+                              </center>
+                              <center>
+                                  <img class="model_img" src="https://latex.codecogs.com/gif.latex?\frac{dp}{dt}=\gamma&space;m-\delta&space;p" title="\frac{dp}{dt}=\gamma m-\delta p" />
+                              </center>
+                              <p class="card-text card-text_igem text-justify">
+                                  The first equation represents the mRNA system in a cell with the assumption of constant gene concentration in each cell. Meanwhile the latter equation is the protein system which is dependent on the previous equation, and fitted to the data from the plate reader, using the Trust Region Algorithm with non-negative floating number as the boundary in each parameter.
+                              </p>
+                              <center>
+                                  <img class="m1 model_img" src="https://static.igem.org/mediawiki/2018/3/33/T--UCL--f4.png"alt="oops! You can't see our image.">
+                              </center>
+                              <center>
+                                  <figcaption class="card-text_igem2">
+                                      <div class="model_caption_title_igem">Figure 4.</div> Fluorescence of Intein Passenger induced with 400 mM IPTG and normalised in each  E. coli cell. The rest of induced cultures are fitted in the same equation.
+                                  </figcaption>
+                              </center>
+                              <table class="table ooooo table_text_igem">
+                                  <tdead>
+                                      <tr>
+                                          <th class="tableleft">IPTG Conc.(mM)</th>
+                                          <th class="tablecenter">400</th>
+                                          <th class="tablecenter">500</th>
+                                          <th class="tablecenter">600</th>
+                                          <th class="tablecenter">700</th>
+                                          <th class="tablecenter">800</th>
+                                      </tr>
+                                  </tdead>
+                                  <tbody>
+                                      <tr>
+                                          <td class="tableleft"><i>x</i></td>
+                                          <td class="tablecenter">200</td>
+                                          <td class="tablecenter">200</td>
+                                          <td class="tablecenter">200</td>
+                                          <td class="tablecenter">200</td>
+                                          <td class="tablecenter">200</td>
+                                      </tr>
+                                      <tr>
+                                          <td class="tableleft"><i>&alpha;</i></td>
+                                          <td class="tablecenter">0.02073</td>
+                                          <td class="tablecenter">0.02332</td>
+                                          <td class="tablecenter">0.02348</td>
+                                          <td class="tablecenter">0.02156</td>
+                                          <td class="tablecenter">0.02264</td>
+                                      </tr>
+                                      <tr>
+                                          <td class="tableleft"><i>&beta;</i></td>
+                                          <td class="tablecenter">0.2482</td>
+                                          <td class="tablecenter">0.2644</td>
+                                          <td class="tablecenter">0.2642</td>
+                                          <td class="tablecenter">0.2665</td>
+                                          <td class="tablecenter">0.2777</td>
+                                      </tr>
+                                      <tr>
+                                          <td class="tableleft"><i>&gamma;</i></td>
+                                          <td class="tablecenter">0.6564</td>
+                                          <td class="tablecenter">0.6799</td>
+                                          <td class="tablecenter">0.6668</td>
+                                          <td class="tablecenter">0.7346</td>
+                                          <td class="tablecenter">0.7449</td>
+                                      </tr>
+                                      <tr>
+                                          <td class="tableleft"><i>&delta;</i></td>
+                                          <td class="tablecenter">0.2563</td>
+                                          <td class="tablecenter">0.2744</td>
+                                          <td class="tablecenter">0.2719</td>
+                                          <td class="tablecenter">0.2738</td>
+                                          <td class="tablecenter">0.2827</td>
+                                      </tr>
+                                  </tbody>
+                              </table>
+                              <center>
+                                  <figcaption class="card-text_igem2">
+                                      <div class="model_caption_title_igem">Table 3.</div> Parameter values estimation using Trust Region Algorithm of curve fitting.
+                                  </figcaption>
+                              </center>
+                              <table class="table ooooo table_text_igem">
+                                  <thead>
+                                      <tr>
+                                          <th class="tableleft">Symbols</th>
+                                          <th class="tablecenter">Units</th>
+                                          <th class="tablecenter">Descriptions</th>
+                                      </tr>
+                                  </thead>
+                                  <tbody>
+                                      <tr>
+                                          <td class="tableleft"><i>t</i></td>
+                                          <td class="tablecenter">hours</td>
+                                          <td class="tablecenter">Time</td>
+                                      </tr>
+                                      <tr>
+                                          <td class="tableleft"><i>x</i></td>
+                                          <td class="tablecenter">200</td>
+                                          <td class="tablecenter">
+                                              Plasmid copy number of pSB1C3 in each cell
+                                          </td>
+                                      </tr>
+                                      <tr>
+                                          <td class="tableleft"><i>m</i></td>
+                                          <td class="tablecenter">dimensionless</td>
+                                          <td class="tablecenter">
+                                              Respective normalised mRNA concentration in each cell
+                                          </td>
+                                      </tr>
+                                      <tr>
+                                          <td class="tableleft"><i>p</i></td>
+                                          <td class="tablecenter">
+                                              dimensionless</td>
+                                              <td class="tablecenter">
+                                                  Respective normalised protein concentration in each cell
+                                              </td>
+                                          </tr>
+                                          <tr>
+                                              <td class="tableleft">&alpha;</td>
+                                              <td class="tablecenter"><i>hr<sup>-1</sup></i></td>
+                                              <td class="tablecenter">
+                                                  Gene transcription rate or mRNA production rate
-<figcaption>
+                                              </td>
-  Figure 01. Comparing TB and LB as E. coli growth medium.
+                                          </tr>
-</figcaption>
+                                          <tr>
-</center>
+                                              <td class="tableleft"><i>&beta;</i></td>
-<br><br />
+                                              <td class="tablecenter"><i>hr<sup>-1</sup></i></td>
-<p>The fitted equations tells us the inhibiting factor <i>b</i> for TB and LB, which are 0.3 and 0.4077, respectively. This is as predicted because TB has less inhibition compared to LB because of the nutrient abundance, carrier capacity, and toxicity level caused by the waste side-products of the cells. The same trend is also observed in maximum population, that TB provides a better environment for larger cell population in longer cultivations. Meanwhile for earlier growth rate <i>k</i>, cells in LB grow quicker before achieving stationary phase, which is 0.44hr<sup>-1</sup> compared with 0.24hr<sup>-1</sup> in TB.
+                                              <td class="tablecenter">
-</p>
+                                                  mRNA degradation rate and dilution factor
-<br><br>
-<center>
-<img class="m1"src="https://static.igem.org/mediawiki/2018/4/44/T--UCL--m2.png"alt="oops! You can't see our image."></center>
-<center>
-<figcaption>
-  Figure 02. Optical density measurements of each cell induction.
-</figcaption>
-</center>
-<br><br>
-<center>
-<img class="m1"src="https://static.igem.org/mediawiki/2018/6/64/T--UCL--m3.png"alt="oops! You can't see our image."></center>
-<center>
-<figcaption>
-  Figure 03. Population growth rate (<i>k</i>) for each IPTG concentration.
-</figcaption>
-</center>
-<br><br><br>
+                                              </td>
+                                          </tr>
+                                          <tr>
+                                              <td class="tableleft">&gamma;</td>
+                                              <td class="tablecenter"><i>hr<sup>-1</sup></i></td>
+                                              <td class="tablecenter">
+                                                  Protein translation rate
-<table class="table ooooo">
+                                              </td>
-    <thead>
+                                          </tr>
-        <tr>
+                                          <tr>
-            <th class="tableleft">IPTG Conc.</th>
+                                              <td class="tableleft">&delta;</td>
-            <th class="tablecenter">400mM</th>
+                                              <td class="tablecenter"><i>hr<sup>-1</sup></i></td>
-            <th class="tablecenter">500mM</th>
+                                              <td class="tablecenter">
-            <th class="tablecenter">600mM</th>
+                                                  Protein degradation rate and dilution factor
-            <th class="tablecenter">700mM</th>
+                                              </td>
-            <th class="tablecenter">800mM</th>
+                                          </tr>
-        </tr>
+                                      </tbody>
-    </thead>
+                                  </table>
-    <tbody>
+                                  <center>
-        <tr>
+                                      <figcaption class="card-text_igem2">
-            <td class="tableleft"><i>k</i></td>
+                                          <div class="model_caption_title_igem">Table 4.</div> Additional information.
-            <td class="tablecenter">0.4215</td>
+                                      </figcaption>
-            <td class="tablecenter">0.4649</td>
+                                  </center>
-            <td class="tablecenter">0.4735</td>
+                                  <p class="card-text card-text_igem text-justify">
-            <td class="tablecenter">0.4773</td>
+                                      The <i>&alpha;</i> and <i>&beta;</i> terms generally stay constant without a trend between IPTG concentrations which is explainable even though IPTG affects cell transcription activity. Since mRNA has a high rate of synthesis and degradation, it reaches equilibrium immediately as its stability closely follows inversely proportional to the mRNA concentration in the cell. The parameter <i>&gamma;</i> and <i>&delta;</i> give a small yet noticeable increasing trend in higher IPTG level, this is related to protein concentration equilibrium, but not to the extent of mRNA degradation rate since protein relatively ‘lives’ longer than mRNA.
-            <td class="tablecenter">0.4759</td>
+                                  </p>
-        </tr>
+                                  <center>
-        <tr>
+                                      <img class="m1 model_img" src="https://static.igem.org/mediawiki/2018/0/0e/T--UCL--f5.png"alt="oops! You can't see our image.">
-            <td class="tableleft"><i>b</i></td>
+                                  </center>
-            <td class="tablecenter">0.4361</td>
+                                  <center>
-            <td class="tablecenter">0.4227</td>
+                                      <figcaption class="card-text_igem2">
-            <td class="tablecenter">0.4208</td>
+                                          <div class="model_caption_title_igem">Figure 5.</div> Fluorescence model of Intein Passenger induced with IPTG 400 mM. Fold-change in protein concentration reaches maximum at 42.8 ratio in longer duration of cell cultivation.
-            <td class="tablecenter">0.4097</td>
+                                      </figcaption>
-            <td class="tablecenter">0.4069</td>
+                                  </center>
-        </tr>
+                                  <p class="card-text card-text_igem text-justify">
-    </tbody>
+                                          Generally IPTG does not give significant differences in high concentrations, so we chose 500 mM of IPTG which the model suggests as the most efficient with the similar expression level compared to the cells with higher concentration of IPTG. Further improvements of this model might include larger choices of medium and wider range of IPTG concentrations, together with cost analysis as it is an important factor in the larger scale production.
-</table>
+                                      </p>
-<center>
-<figcaption>
-  Table 02: the growth rate (k) and inhibitor factor (b) corresponding to different IPTG induction concentration.
-</figcaption>
-</center>
-<br><br><p>
-  We then build up the kinetics inside a cell and integrate it with lab data analysis to understand protein production and get an effective lab plan for our E. coli system. GFP, RFP, and Intein Passenger fluorescence data of 544/590 nm filtering wavelengths were then extracted and processed. Each dependent value is described as protein-fold change post induction, which is normalised with the same system but uninduced, based on the fraction below:
-</p>
-<center>
-<br /><img src="https://latex.codecogs.com/gif.latex?p=\frac{F}{F_{{o}}}" title="p=\frac{F}{F_{{o}}}" />
-</center>
-<br><br><br>
-<p>The first-order system of linear differential equations are:
-</p>
-<br>
-<center>
-<img src="https://latex.codecogs.com/gif.latex?\frac{dm}{dt}=\alpha&space;x-\beta&space;m" title="\frac{dm}{dt}=\alpha x-\beta m" />
-</center>
-<br><br>
-<center><img src="https://latex.codecogs.com/gif.latex?\frac{dp}{dt}=\gamma&space;m-\delta&space;p" title="\frac{dp}{dt}=\gamma m-\delta p" /></center>
-<br><br><br>
-<p>The first equation represents the mRNA system in a cell with the assumption of constant gene concentration in each cell. Meanwhile the latter equation is the protein system which is dependent on the previous equation, and fitted to the data from the plate reader, using the Trust Region Algorithm with non-negative floating number as the boundary in each parameter.
-</p>
-<br>
-<center>
-<img class="m1"src="https://static.igem.org/mediawiki/2018/3/33/T--UCL--f4.png"alt="oops! You can't see our image."></center>
-<center>
-<figcaption>
-  Figure 04: Fluorescence of Intein Passenger induced with 400 mM IPTG and normalised in each  E. coli cell. The rest of induced cultures are fitted in the same equation.
-</figcaption>
-</center>
-<br><br><br>
-<table class="table ooooo">
-    <tdead>
-        <tr>
-            <th class="tableleft">IPTG Conc.(mM)</th>
-            <th class="tablecenter">400</th>
-            <th class="tablecenter">500</th>
-            <th class="tablecenter">600</th>
-            <th class="tablecenter">700</th>
-            <th class="tablecenter">800</th>
-        </tr>
-    </tdead>
-    <tbody>
-      <tr>
-          <td class="tableleft"><i>x</i></td>
-          <td class="tablecenter">200</td>
-          <td class="tablecenter">200</td>
-          <td class="tablecenter">200</td>
-          <td class="tablecenter">200</td>
-          <td class="tablecenter">200</td>
-      </tr>
-      <tr>
-          <td class="tableleft"><i>&alpha;</i></td>
-          <td class="tablecenter">0.02073</td>
-          <td class="tablecenter">0.02332</td>
-          <td class="tablecenter">0.02348</td>
-          <td class="tablecenter">0.02156</td>
-          <td class="tablecenter">0.02264</td>
-      </tr>
-      <tr>
-          <td class="tableleft"><i>&beta;</i></td>
-          <td class="tablecenter">0.2482</td>
-          <td class="tablecenter">0.2644</td>
-          <td class="tablecenter">0.2642</td>
-          <td class="tablecenter">0.2665</td>
-          <td class="tablecenter">0.2777</td>
-      </tr>
-      <tr>
-          <td class="tableleft"><i>&gamma;</i></td>
-          <td class="tablecenter">0.6564</td>
-          <td class="tablecenter">0.6799</td>
-          <td class="tablecenter">0.6668</td>
-          <td class="tablecenter">0.7346</td>
-          <td class="tablecenter">0.7449</td>
-      </tr>
-      <tr>
-          <td class="tableleft"><i>&delta;</i></td>
-          <td class="tablecenter">0.2563</td>
-          <td class="tablecenter">0.2744</td>
-          <td class="tablecenter">0.2719</td>
-          <td class="tablecenter">0.2738</td>
-          <td class="tablecenter">0.2827</td>
-      </tr>
-    </tbody>
-</table>
-<center>
-<figcaption>
-  Table 03: Parameter values estimation using Trust Region Algorithm of curve fitting.
-</figcaption>
-</center>
-<br><br><br>
-<table class="table ooooo">
-    <thead>
-        <tr>
-            <th class="tableleft">Symbols</th>
-            <th class="tablecenter">Units</th>
-            <th class="tablecenter">Descriptions</th>
-        </tr>
-    </thead>
-    <tbody>
-        <tr>
-            <td class="tableleft"><i>t</i></td>
-            <td class="tablecenter">hours</td>
-            <td class="tablecenter">Time</td>
-        </tr>
-        <tr>
-            <td class="tableleft"><i>x</i></td>
-            <td class="tablecenter">200</td>
-            <td class="tablecenter">
-Plasmid copy number of pSB1C3 in each cell
-</td>
-        </tr>
-        <tr>
-            <td class="tableleft"><i>m</i></td>
-            <td class="tablecenter">dimensionless</td>
-            <td class="tablecenter">
-Respective normalised mRNA concentration in each cell
-</td>
-        </tr>
-        <tr>
-            <td class="tableleft"><i>p</i></td>
-            <td class="tablecenter">
-            dimensionless</td>
-            <td class="tablecenter">
-            Respective normalised protein concentration in each cell
-</td>
-        </tr>
-        <tr>
-            <td class="tableleft">&alpha;</td>
-            <td class="tablecenter"><i>hr<sup>-1</sup></i></td>
-            <td class="tablecenter">
-Gene transcription rate or mRNA production rate
-</td>
-        </tr>
-        <tr>
-            <td class="tableleft"><i>&beta;</i></td>
-            <td class="tablecenter"><i>hr<sup>-1</sup></i></td>
-            <td class="tablecenter">
-mRNA degradation rate and dilution factor
-</td>
-        </tr>
-        <tr>
-            <td class="tableleft">&gamma;</td>
-            <td class="tablecenter"><i>hr<sup>-1</sup></i></td>
-            <td class="tablecenter">
-Protein translation rate
-</td>
-        </tr>
-        <tr>
-            <td class="tableleft">&delta;</td>
-            <td class="tablecenter"><i>hr<sup>-1</sup></i></td>
-            <td class="tablecenter">
-Protein degradation rate and dilution factor
-</td>
-        </tr>
-    </tbody>
-</table>
-<center>
-<figcaption>
-  Table 03: Additional information.
-</figcaption>
-</center>
-<br><br>
-<p>
-  The <i>&alpha;</i> and <i>&beta;</i> terms generally stay constant without a trend between IPTG concentrations which is explainable even though IPTG affects cell transcription activity. Since mRNA has a high rate of synthesis and degradation, it reaches equilibrium immediately as its stability closely follows inversely proportional to the mRNA concentration in the cell. The parameter <i>&gamma;</i> and <i>&delta;</i> give a small yet noticeable increasing trend in higher IPTG level, this is related to protein concentration equilibrium, but not to the extent of mRNA degradation rate since protein relatively ‘lives’ longer than mRNA.
-</p>
-<br>
-<center>
-<img class="m1"src="https://static.igem.org/mediawiki/2018/0/0e/T--UCL--f5.png"alt="oops! You can't see our image."></center>
-<center>
-<figcaption>
-  Figure 05: Fluorescence model of Intein Passenger induced with IPTG 400 mM. Fold-change in protein concentration reaches maximum at 42.8 ratio in longer duration of cell cultivation.
-</figcaption>
-</center>
-<br><br>
-<p>Generally IPTG does not give significant differences in high concentrations, so we chose 500 mM of IPTG which the model suggests as the most efficient with the similar expression level compared to the cells with higher concentration of IPTG. Further improvements of this model might include larger choices of medium and wider range of IPTG concentrations, together with cost analysis as it is an important factor in the larger scale production.
-</p>
-                                </p>
                              </div>
                          </div>
@@ Line 568: / Line 533: @@
              </div>
          </section>
          <section id="Protein_Polymerisation">
              <div class="card card_igem wow fadeInUp" data-wow-duration="2s" data-wow-delay="0.1s">
@@ Line 581: / Line 542: @@
                          <div class="row">
                              <div class="mmmm col-sm-10 mrfont">
-                              <br><br>
+                                <p class="card-text card-text_igem text-justify">
-<p>To better accommodate the integrated model and identify bottlenecks, we further develop a model entirely of in vitro polymerisation as it is indispensable to the overall process of biopolymer production.
+                                    To better accommodate the integrated model and identify bottlenecks, we further develop a model entirely of in vitro polymerisation as it is indispensable to the overall process of biopolymer production.
+                                </p>
-</p><br>
+                                <center>
-<center>
+                                    <img class="model_img" src="https://latex.codecogs.com/gif.latex?\frac{dP_{n}}{dt}=\sum_{i=1}^{n} \kappa\left [ P_{i} \right ]\left [ P_{n-1} \right ]-\sum_{i=1}^{n_{max}-n}\kappa\left [ P_{n} \right ]\left [ P_{i} \right ]" title="\frac{dP_{n}}{dt}=\sum_{i=1}^{n} \kappa\left [ P_{i} \right ]\left [ P_{n-1} \right ]-\sum_{i=1}^{n_{max}-n}\kappa\left [ P_{n} \right ]\left [ P_{i} \right ]" />
-<img src="https://latex.codecogs.com/gif.latex?\frac{dP_{n}}{dt}=\sum_{i=1}^{n} \kappa\left [ P_{i} \right ]\left [ P_{n-1} \right ]-\sum_{i=1}^{n_{max}-n}\kappa\left [ P_{n} \right ]\left [ P_{i} \right ]" title="\frac{dP_{n}}{dt}=\sum_{i=1}^{n} \kappa\left [ P_{i} \right ]\left [ P_{n-1} \right ]-\sum_{i=1}^{n_{max}-n}\kappa\left [ P_{n} \right ]\left [ P_{i} \right ]" />
+                                </center>
-</center>
+                                <p class="card-text card-text_igem text-justify">
-<br><br>
+                                    Each single protein is interpreted as a monomer with an irreversible polymerising capacity until it reaches an assumed maximum size. Polymer formation rate of each species (P<sub>n</sub>) were then modelled in Python and plotted. The first term describes the polymer species formation from the sum of all reactions in the range of its possible constituents, and the second term represents the further polymerisation of the respective species as a substrate, which build up into larger repeats.
-<p>Each single protein is interpreted as a monomer with an irreversible polymerising capacity until it reaches an assumed maximum size. Polymer formation rate of each species (P<sub>n</sub>) were then modelled in Python and plotted. The first term describes the polymer species formation from the sum of all reactions in the range of its possible constituents, and the second term represents the further polymerisation of the respective species as a substrate, which build up into larger repeats.
+                                </p>
-</p>
+                                <center>
-<br><br>
+                                    <img class="m1 model_img" src="https://static.igem.org/mediawiki/2018/7/78/T--UCL--m11.png"alt="oops!You can't load our image.">
-<center>
+                                </center>
-  <img class="m1"src="https://static.igem.org/mediawiki/2018/7/78/T--UCL--m11.png"alt="oops!You can't load our image.">
+                                <center>
-</center>
+                                    <figcaption class="card-text_igem2">
-<center>
+                                        <div class="model_caption_title_igem">Figure 6.</div> Monomer distribution in each species as a function of time for <i>Npu</i> DnaE(N) + <i>Ssp</i> DnaE(C) split inteins. Starting solution only consists of monomers, modeled as they merge and accumulate into larger species of polymer.
-  <figcaption>
+                                    </figcaption>
-    Figure 06. Monomer distribution in each species as a function of time for <i>Npu</i> DnaE(N) + <i>Ssp</i> DnaE(C) split inteins. Starting solution only consists of monomers, modeled as they merge and accumulate into larger species of polymer.
+                                </center>
-  </figcaption>
+                                <center>
-</center>
+                                    <img class="m1 model_img"src="https://static.igem.org/mediawiki/2018/4/40/T--UCL--m12.png"alt="oops!You can't load our image.">
-<br><br>
+                                </center>
-<center>
+                                <center>
-  <img class="m1"src="https://static.igem.org/mediawiki/2018/4/40/T--UCL--m12.png"alt="oops!You can't load our image.">
+                                    <figcaption class="card-text_igem2">
-</center>
+                                        <div class="model_caption_title_igem">Figure 7.</div> Monomer distribution in each species as a function of time <i>Ssp</i> DnaE inteins. Starting solution only consists of monomers, modeled as they merge and accumulate into larger species of polymer.
-<center>
+                                    </figcaption>
-  <figcaption>
+                                </center>
-    Figure 07. Monomer distribution in each species as a function of time <i>Ssp</i> DnaE inteins. Starting solution only consists of monomers, modeled as they merge and accumulate into larger species of polymer.
+                                <center>
+                                    <video class="video_igem" width="100%" controls>
-  </figcaption>
+                                        <source src="https://static.igem.org/mediawiki/2018/3/30/T--UCL--abc.mp4" type="video/mp4" />
-</center>
+                                        Your browser does not support this video.
-<br><br>
+                                    </video>
-<center>
+                                </center>
-  <video width="100%" controls>
+                                <center>
-    <source src="https://static.igem.org/mediawiki/2018/3/30/T--UCL--abc.mp4" type="video/mp4" />
+                                    <figcaption class="card-text_igem2">
-    Your browser does not support this video.
+                                        <div class="model_caption_title_igem">Figure 8.</div> Ratio of monomer distribution species over time in Npu DnaE(N) + <i>Ssp</i> DnaE(C) split inteins combination. Frame rates do not represent real-time.
-  </video>
+                                    </figcaption>
-</center>
+                                </center>
-<center>
+                                <center>
-  <figcaption>
+                                    <video class="video_igem" width="100%" controls>
-    Figure 8. Ratio of monomer distribution species over time in Npu DnaE(N) + <i>Ssp</i> DnaE(C) split inteins combination. Frame rates do not represent real-time.
+                                        <source src="https://static.igem.org/mediawiki/2018/3/31/T--UCL--cbb.mp4" type="video/mp4" />
+                                        Your browser does not support this video.
-  </figcaption>
+                                    </video>
-</center>
+                                </center>
-<br><br>
+                                <center>
-<center>
+                                    <figcaption class="card-text_igem2">
-  <video width="100%" controls>
+                                        <div class="model_caption_title_igem">Figure 9.</div> Ratio of monomer distribution species over time in <i>Ssp</i> DnaE inteins. Frame rates do not represent real-time.
-    <source src="https://static.igem.org/mediawiki/2018/3/31/T--UCL--cbb.mp4" type="video/mp4" />
+                                    </figcaption>
-    Your browser does not support this video.
+                                </center>
-  </video>
+                                <table class="table ooooo table_text_igem">
-</center>
+                                    <thead>
-<center>
+                                        <tr>
-  <figcaption>
+                                            <th class="tableleft">Symbols</th>
-    Figure 9. Ratio of monomer distribution species over time in <i>Ssp</i> DnaE inteins. Frame rates do not represent real-time.
+                                            <th class="tablecenter">Values and Units</th>
+                                            <th class="tablecenter">Descriptions</th>
-  </figcaption>
+                                        </tr>
-</center>
+                                    </thead>
-<br><br>
+                                    <tbody>
+                                        <tr>
-<table class="table ooooo">
+                                            <td class="tableleft"><i>t</i></td>
-    <thead>
+                                            <td class="tablecenter">s</td>
-        <tr>
+                                            <td class="tablecenter">Time</td>
-            <th class="tableleft">Symbols</th>
+                                        </tr>
-            <th class="tablecenter">Values and Units</th>
+                                        <tr>
-            <th class="tablecenter">Descriptions</th>
+                                            <td class="tableleft"><i>n</i></td>
-        </tr>
+                                            <td class="tablecenter">dimensionless</td>
-    </thead>
+                                            <td class="tablecenter">
-    <tbody>
+                                                Number of repeats, defining a certain species of polymer
-        <tr>
+                                            </td>
-            <td class="tableleft"><i>t</i></td>
+                                        </tr>
-            <td class="tablecenter">s</td>
+                                        <tr>
-            <td class="tablecenter">Time</td>
+                                            <td class="tableleft"><i>P<sub>n</sub></i></td>
-        </tr>
+                                            <td class="tablecenter">mol L<sup>-1</sup></td>
-        <tr>
+                                            <td class="tablecenter">
-            <td class="tableleft"><i>n</i></td>
+                                                Polymer species <i>n</i>
-            <td class="tablecenter">dimensionless</td>
+                                            </td>
-            <td class="tablecenter">
+                                        </tr>
-Number of repeats, defining a certain species of polymer
+                                        <tr>
-</td>
+                                            <td class="tableleft"><i>k</i></td>
-        </tr>
+                                            <td class="tablecenter">
-        <tr>
+                                                L mol<sup>-1</sup> S<sup>-1></sup>
-            <td class="tableleft"><i>P<sub>n</sub></i></td>
+                                            </td>
-            <td class="tablecenter">mol L<sup>-1</sup></td>
+                                            <td class="tablecenter">
-            <td class="tablecenter">
+                                                Rate constants of Intein activity of a certain intein
-              Polymer species <i>n</i>
+                                            </td>
-</td>
+                                        </tr>
-        </tr>
+                                        <tr>
-        <tr>
+                                            <td class="tableleft"><i>n<sub>max</sub></i></td>
-            <td class="tableleft"><i>k</i></td>
+                                            <td class="tablecenter">dimensionless</td>
-            <td class="tablecenter">
+                                            <td class="tablecenter">
-          L mol<sup>-1</sup> S<sup>-1></sup></td>
+                                                Maximum repeat number in a polymer</td>
-            <td class="tablecenter">
+                                        </tr>
-              Rate constants of Intein activity of a certain intein
+                                    </tbody>
-</td>
+                                </table>
-        </tr>
+                                <center>
-        <tr>
+                                <figcaption class="card-text_igem2">
-            <td class="tableleft"><i>n<sub>max</sub></i></th>
+                                    <div class="model_caption_title_igem">Table 5.</div> Additional information.
-            <td class="tablecenter">dimensionless</th>
+                                </figcaption>
-            <td class="tablecenter">
+                                </center>
-Maximum repeat number in a polymer</th>
+                                <p class="card-text card-text_igem text-justify">
-        </tr>
+                                    Basically each intein rate of reaction (<i>k</i>) differs with each intein system we use.
-    </tbody>
+                                </p>
-</table>
+                                <table class="table ooooo table_text_igem">
-<center>
+                                    <thead>
-<figcaption>
+                                        <tr>
-  Table 04: Additional information.
+                                            <th class="tableleft">Intein</th>
-</figcaption>
+                                            <th class="tablecenter">Rate of reaction (s<sup>-1</sup>)</th>
-</center>
+                                        </tr>
-<br><br><br>
+                                    </thead>
-<p>Basically each intein rate of reaction (<i>k</i>) differs with each intein system we use.</p>
+                                    <tbody>
-<br />
+                                        <tr>
-<table class="table ooooo">
+                                            <td class="tableleft"><i>Npu</i> DNA-E</td>
-    <thead>
+                                            <td class="tablecenter">1.1x10<sup>-2</sup></td>
-        <tr>
-            <th class="tableleft">Intein</th>
-            <th class="tablecenter">Rate of reaction (s<sup>-1</sup>)</th>
-        </tr>
-    </thead>
-    <tbody>
-        <tr>
-            <td class="tableleft"><i>Npu</i> DNA-E</td>
-            <td class="tablecenter">1.1x10<sup>-2</sup></td>
-        </tr>
-        <tr>
-            <td class="tableleft"><i>Ssp</i>DNA-E</td>
-            <td class="tablecenter">3.3x10<sup>-4</sup></td>
-</td>
-        </tr>
-        <tr>
-            <td class="tableleft"><i>Npu</i> DNA-E(N) + <i>Ssp</i>DNA-E</td>
-            <td class="tablecenter">2.8x10<sup>-5</sup></td>
-</td>
-        </tr>
-        <tr>
-            <td class="tableleft"><i>Sce</i>VMA</td>
-            <td class="tablecenter">
-.0x10<sup>-3</sup></td>
-        </tr>
-        <tr>
-            <td class="tableleft"><i>Ssp</i>DNA-B</th>
-            <td class="tablecenter">9.9x10<sup>-4</sup></th>
-        </tr>
-    </tbody>
-</table>
-<center>
-<figcaption>
-  Table 05. Specific activity of each inteins. Rate of reaction are collected from the references mentioned in the bottom of the page.
-</figcaption>
-</center>
-<br><br>
-<p>
-  The results we get from the example runs of <i>Npu</i> DnaE(N) and <i>Ssp</i> DnaE(C) intein combinations are after 10 minutes of the simulated experiment, only 3.45% of monomers are in the form of the top 25% longest polymer species. However for <i>Ssp</i> DnaE inteins, in the course of 10 minutes, 72.3% of the monomers are polymerised into the same top 25% of the longest polymer region. This really shows how the intein polymerisation will require much less time compared to the cell culture and protein production. Further improvements will be introducing more parameters into the estimation function to represent more accurate results.
-</p>
-<br><br><br>
-<div id="WORM"class="card-title text-center">
-    <h4>Intein Polymerisation</h4>
-    <br>
-    <h3 class="orange mrfont2">W.O.R.M.</h3>
-</div>
-<br><br>
-<p>
-  To further model the polymerisation of our proteins, we devised a simplified proof of concept of our project using reporter proteins RFP and GFP to predict the polymerisation capabilities of split inteins. Each reporter protein is flanked by orthogonal inteins, which match up to the other reporter’s inteins to allow RFP and GFP to continuously link up into various lengths of polymers.
-</p>
-<br>
-<center>
-  <img class="m1"src="https://static.igem.org/mediawiki/2018/c/c5/T--UCL--iii1.png"alt="oops!You can't load our image.">
-</center>
-<center>
-  <figcaption>
-    Figure 10: Polymerisation of intein flanked reporter proteins. <br>
-Npu-N intein on GFP splices out with the Npu-C intein on RFP. Similarly, the TerL-N intein on the RFP splices out with the TerL-C intein on the GFP. This reaction results in the polymerisation of GFP and RFP.
-  </figcaption>
-</center>
-<br>
-We used the python libraries PyGame and PyMunk to create an animated and interactive agent-based model of intein polymerisation. Acronymed WORM, this model depicts a Wonderful Oligomerisation Reaction Model.
-<br><br>
-<video width="100%" controls>
-  <source src="https://static.igem.org/mediawiki/2018/6/61/T--UCL--worm.mp4" type="video/mp4" />
-  Your browser does not support this video.
-</video>
-<br><br>
-<p>Depicted in the video is a snapshot of the model WORM that shows intein polymerisation. The yellow and orange rectangles represent an split intein pair, as do the blue and cyan rectangles. The red and green rectangles represent the reporter proteins, RFP and GFP, respectively. </p>
-<br>
-<p>As an agent-based mode, WORM was created based on the following rules: <br>
-)Particles diffuse with random angular and lateral velocity, <br>
-)Velocity increases with temperature (based on average kinetic energy)
-<br>
-<center><img src="https://latex.codecogs.com/gif.latex?(\frac{1}{2}mv^{2})=\frac{3}{2}kT" title="(\frac{1}{2}mv^{2})=\frac{3}{2}kT" /></center>
-<p>   Where: <br>
-		m= mass (kg) <br>
-		v= velocity <br>
-		k= Boltzmann constant <br>
-    T= temperature (K) <br>
-</p>
-<br><br>
-)Like reacts with unlike (RFP reacts with GFP), <br>
-)Particles are polarised (Npu-N [A1]→ Npu-C [B3], TerL-N [B1]→ TerL-C [A3]) <br>
-)Splicing removes 1+3 on reaction (removes the inteins), <br>
-)Steric hindrance by particles   <br>
-<br><br>
-<center>
-  <img class="m1"src="https://static.igem.org/mediawiki/2018/7/73/T--UCL--f111.png"alt="oops!You can't load our image.">
-</center>
-<center>
-  <figcaption>
-    FIGURE 11: Distribution of polymer lengths after 20 runs. <br>
-WORM was run for 5 minutes, 20 times. The distribution of total polymers for each polymer lengths from 1-20, and lengths over 20, were plotted. The error was plotted as the square root of the total polymers in each polymer length.
-  </figcaption>
-</center>
-<br><br>
-<p>The interactive components of the WORM model include user’s choice of temperature and protein mass to selectively model the reaction conditions and functionalized protein of interest. These applications can be used by running the code found in our GitHub. The combination of predictive experimental data and the visually appealing computational model makes a user friendly platform for the public to better understand biology and modelling.</p>
-<br><br>
+                                        </tr>
+                                        <tr>
+                                            <td class="tableleft"><i>Ssp</i>DNA-E</td>
+                                            <td class="tablecenter">3.3x10<sup>-4</sup></td>
+                                        </tr>
+                                        <tr>
+                                            <td class="tableleft"><i>Npu</i> DNA-E(N) + <i>Ssp</i>DNA-E</td>
+                                            <td class="tablecenter">2.8x10<sup>-5</sup></td>
+                                        </tr>
+                                        <tr>
+                                            <td class="tableleft"><i>Sce</i>VMA</td>
+                                                <td class="tablecenter">2.0x10<sup>-3</sup></td>
+                                        </tr>
+                                        <tr>
+                                            <td class="tableleft"><i>Ssp</i>DNA-B</td>
+                                            <td class="tablecenter">9.9x10<sup>-4</sup></td>
+                                        </tr>
+                                    </tbody>
+                                </table>
+                                <center>
+                                    <figcaption class="card-text_igem2">
+                                        <div class="model_caption_title_igem">Table 6.</div> Specific activity of each inteins. Rate of reaction are collected from the references mentioned in the bottom of the page.
+                                    </figcaption>
+                                </center>
+                                <p class="card-text card-text_igem text-justify">
+                                    The results we get from the example runs of <i>Npu</i> DnaE(N) and <i>Ssp</i> DnaE(C) intein combinations are after 10 minutes of the simulated experiment, only 3.45% of monomers are in the form of the top 25% longest polymer species. However for <i>Ssp</i> DnaE inteins, in the course of 10 minutes, 72.3% of the monomers are polymerised into the same top 25% of the longest polymer region. This really shows how the intein polymerisation will require much less time compared to the cell culture and protein production. Further improvements will be introducing more parameters into the estimation function to represent more accurate results.
+                                </p>
+                                <div id="WORM"class="card-title text-center">
+                                    <h4>Intein Polymerisation</h4>
+                                    <h3 class="orange mrfont2">W.O.R.M.</h3>
+                                </div>
+                                <p class="card-text card-text_igem text-justify">
+                                    To further model the polymerisation of our proteins, we devised a simplified proof of concept of our project using reporter proteins RFP and GFP to predict the polymerisation capabilities of split inteins. Each reporter protein is flanked by orthogonal inteins, which match up to the other reporter’s inteins to allow RFP and GFP to continuously link up into various lengths of polymers.
+                                </p>
+                                <center>
+                                    <img class="m1"src="https://static.igem.org/mediawiki/2018/c/c5/T--UCL--iii1.png"alt="oops!You can't load our image.">
+                                </center>
+                                <center>
+                                    <figcaption>
+                                        <div class="model_caption_title_igem">Figure 10.</div> Polymerisation of intein flanked reporter proteins. Npu-N intein on GFP splices out with the Npu-C intein on RFP. Similarly, the TerL-N intein on the RFP splices out with the TerL-C intein on the GFP. This reaction results in the polymerisation of GFP and RFP.
+                                    </figcaption>
+                                </center>
+                                <p class="card-text card-text_igem text-justify">
+                                    We used the python libraries PyGame and PyMunk to create an animated and interactive agent-based model of intein polymerisation. Acronymed WORM, this model depicts a Wonderful Oligomerisation Reaction Model.
+                                </p>
+                                <video class="video_igem" width="100%" controls>
+                                    <source src="https://static.igem.org/mediawiki/2018/6/61/T--UCL--worm.mp4" type="video/mp4" />
+                                        Your browser does not support this video.
+                                </video>
+                                <p class="card-text card-text_igem text-justify">
+                                    Depicted in the video is a snapshot of the model WORM that shows intein polymerisation. The yellow and orange rectangles represent an split intein pair, as do the blue and cyan rectangles. The red and green rectangles represent the reporter proteins, RFP and GFP, respectively.
+                                </p>
+                                <p class="card-text card-text_igem text-justify">
+                                    As an agent-based mode, WORM was created based on the following rules:
+                                </p>
+                                <ol>
+                                    <li>
+                                        Particles diffuse with random angular and lateral velocity;
+                                    </li>
+                                    <li>
+                                        Velocity increases with temperature (based on average kinetic energy):
+                                    <center>
+                                        <img class="model_img" src="https://latex.codecogs.com/gif.latex?(\frac{1}{2}mv^{2})=\frac{3}{2}kT" title="(\frac{1}{2}mv^{2})=\frac{3}{2}kT" />
+                                        <p class="card-text card-text_igem text-center">Where:</p>
+                                    </center>
+                                        <div class="row list_padding_igem">
+                                            <div class="col-sm-6">
+                                                <ul>
+                                                    <li>
+                                                        m= mass (kg)
+                                                    </li>
+                                                    <li>
+                                                        v= velocity
+                                                    </li>
+                                                </ul>
+                                            </div>
+                                            <div class="col-sm-6">
+                                                <ul>
+                                                    <li>
+                                                        k= Boltzmann constant
+                                                    </li>
+                                                    <li>
+                                                        T= temperature (K)
+                                                    </li>
+                                                </ul>
+                                            </div>
+                                        </div>
+                                    </li>
+                                    <li>
+                                        Like reacts with unlike (RFP reacts with GFP);
+                                    </li>
+                                    <li>
+                                        Particles are polarised (Npu-N [A1]→ Npu-C [B3], TerL-N [B1]→ TerL-C [A3]);
+                                    </li>
+                                    <li>
+                                        Splicing removes 1+3 on reaction (removes the inteins);
+                                    </li>
+                                    <li>
+                                        Steric hindrance by particles.
+                                    </li>
+                                <center>
+                                    <img class="m1 model_img"src="https://static.igem.org/mediawiki/2018/7/73/T--UCL--f111.png"alt="oops!You can't load our image.">
+                                </center>
+                                <center>
+                                    <figcaption class="card-text_igem2">
+                                        <div class="model_caption_title_igem">Figure 11.</div> Distribution of polymer lengths after 20 runs. WORM was run for 5 minutes, 20 times. The distribution of total polymers for each polymer lengths from 1-20, and lengths over 20, were plotted. The error was plotted as the square root of the total polymers in each polymer length.
+                                    </figcaption>
+                                </center>
+                                <p class="card-text card-text_igem text-justify">
+                                    The interactive components of the WORM model include user’s choice of temperature and protein mass to selectively model the reaction conditions and functionalized protein of interest. These applications can be used by running the code found in our GitHub. The combination of predictive experimental data and the visually appealing computational model makes a user friendly platform for the public to better understand biology and modelling.
+                                </p>
                              </div>
                          </div>
                      </div>
@@ Line 823: / Line 771: @@
              </div>
          </section>
          <section id="Mech">
              <div class="mrfont card card_igem wow fadeInUp" data-wow-duration="2s" data-wow-delay="0.1s">
@@ Line 838: / Line 780: @@
                          <div class="row">
                              <div class="col-sm-10 mmmm mrfont">
-                                <p class="card-text card-text_igem text-justify">
+                                    <p class="card-text card-text_igem text-justify">
-                                  <br>
+                                        In this part, we also modeled the biomaterials’ micro-structure of the coarse-grained system. We predicted the structure of different spider silk repeats (tetramer, octamer and dodecamer) both after stretching and shearing. This method is a deterministic approach to foresee the network structure of spider silk repeats prior to the actual lab experiment and helps us to understand the design of the actual protein that we are going to produce afterwards.
-                                    <p>In this part, we also modeled the biomaterials’ micro-structure of the coarse-grained system. We predicted the structure of different spider silk repeats (tetramer, octamer and dodecamer) both after stretching and shearing. This method is a deterministic approach to foresee the network structure of spider silk repeats prior to the actual lab experiment and helps us to understand the design of the actual protein that we are going to produce afterwards.
+                                    </p>
-</p>
+                                    <p class="card-text card-text_igem text-justify">
-<br>
+                                        Here is our initial designed sequence, a spider silk protein in repeats of four:
-<p>Here is our initial designed sequence, a spider silk protein in repeats of four:</p>
+                                    </p>
-<span class="nodec">
+                                    <center>
-GQGGYGGLGSQGAGRGGLGGQGAGAAAAAAAAGGAGQGGYGGLGSQGAGRGGLGGQGAGAAAAAAAAAGGYGGLGSQGAGRGGLGGQGAGAAAAAAAAGGAGQGGYGGLGSQGAGRGGLGGQGAGAAAAAAAAGGA</span>
+                                        <div class="col-sm-7">
-<br><br><br>
+                                            <code class="nodec">
-The respective sequence is then quantitatively analysed for their hydrophobicity and hydrophilicity and plotted.
+                                                GQGGYGGLGSQGAGRGGLGGQGAGAAAAAAAAGGAGQGGYGGLGSQGAGRGGLGGQGAGAAAAAAAAAGGYGGLGSQGAGRGGLGGQGAGAAAAAAAAGGAGQGGYGGLGSQGAGRGGLGGQGAGAAAAAAAAGGA
-<br>
+                                            </code>
-<center>
+                                        </div>
-  <img class="m1"src="https://static.igem.org/mediawiki/2018/1/19/T--UCL--m7.png"alt="oops!You can't load our image.">
+                                    </center>
-</center>
+                                    <p class="card-text card-text_igem text-justify">
-<center>
+                                        The respective sequence is then quantitatively analysed for their hydrophobicity and hydrophilicity and plotted.
-  <figcaption>
+                                    </p>
-    Figure 11. Kyte-Doolittle-Hydropathy plot. The vertical axis is the measure of hydrophobicity in each amino acid in the sequence, with higher score means greater degree of hydrophobicity. With the help of ProtScale from ExPASy.
+                                    <center>
-  </figcaption>
+                                        <img class="m1 model_img" src="https://static.igem.org/mediawiki/2018/1/19/T--UCL--m7.png"alt="oops!You can't load our image.">
-</center>
+                                    </center>
-<br><br>
+                                    <center>
-<p>In this particular sequence, we can observe that the hydrophobic and hydrophilic parts alternate inside the spider silk fiber, which will form aggregates and bridge connections to achieve desired tensile and toughness due to the hydrophobic interactions.
+                                        <figcaption class="card-text_igem2">
-</p>
+                                            <div class="model_caption_title_igem">Figure 12.</div> Kyte-Doolittle-Hydropathy plot. The vertical axis is the measure of hydrophobicity in each amino acid in the sequence, with higher score means greater degree of hydrophobicity. With the help of ProtScale from ExPASy.
-<br>
+                                        </figcaption>
-<p>
+                                    </center>
-  This alternating pattern seen in the figure allows us to simplify proteins as the hydrophobic domains and hydrophilic domains. These domains are then represented as a collection of the so-called ‘beads’, and depending on relative molecular volume of each domain, the coarse-grained model is then scaled and measured into an estimate of three amino acid residues per bead.
+                                    <p class="card-text card-text_igem text-justify">
-</p>
+                                        In this particular sequence, we can observe that the hydrophobic and hydrophilic parts alternate inside the spider silk fiber, which will form aggregates and bridge connections to achieve desired tensile and toughness due to the hydrophobic interactions.
-<br>
+                                    </p>
-<p>This system is composed from 3 components: water, hydrophilic and hydrophobic beads. Every 9 water molecules is represented as a single bead, so its 270 Å size (each water molecule is about 30 Å) is proportional to the protein beads. </p>
+                                    <p class="card-text card-text_igem text-justify">
-<br>
+                                        This alternating pattern seen in the figure allows us to simplify proteins as the hydrophobic domains and hydrophilic domains. These domains are then represented as a collection of the so-called ‘beads’, and depending on relative molecular volume of each domain, the coarse-grained model is then scaled and measured into an estimate of three amino acid residues per bead.
-<p>We then introduce LAMMPS as our software, which is an abbreviation of Large-scale Atomic/Molecular Massively Parallel Simulator, to simulate the behaviour of our ‘beads’ collection in a single compartment and thus gave us an insight into the physical property of the recombinant spider silk.</p>
+                                    </p>
-<br><br>
+                                    <p class="card-text card-text_igem text-justify">
+                                        This system is composed from 3 components: water, hydrophilic and hydrophobic beads. Every 9 water molecules is represented as a single bead, so its 270 Å size (each water molecule is about 30 Å) is proportional to the protein beads.
-<div class="card-title text-center mrfont2">
+                                    </p>
-  <h1 class="orange">Simulations</h1>
+                                    <p class="card-text card-text_igem text-justify">
-  <h5>Hover to compare the 2 sides.</h5>
+                                        We then introduce LAMMPS as our software, which is an abbreviation of Large-scale Atomic/Molecular Massively Parallel Simulator, to simulate the behaviour of our ‘beads’ collection in a single compartment and thus gave us an insight into the physical property of the recombinant spider silk.
-  <br><br>
+                                    </p>
-  <h6><span class="marrrgin"style="float:left">Equal</span> <span class="marrgin"style="float:right">Stretch</span></h6>
+                                        <div class="card-title text-center mrfont2">
-</div>
+                                            <h3 class="orange mrfont2">Simulations</h3>
-<br>
+                                            <h5 class="mrfont2">Hover to compare the 2 sides.</h5>
-<div class="col-sm-12">
+                                            <div class="model_h6">
-  <hr />
+                                                <span class="marrrgin"style="float:left">Equal</span>
-  <div class="card-title text-center mrfont2">
+                                                <span class="marrgin"style="float:right">Stretch</span>
-    <center><h5>Tetramer</h5></center>
+                                            </div>
-  </div>
+                                        </div>
-  <hr />
+                                        <div class="col-sm-12">
-<div class="row yyyyy">
+                                            <hr />
-<div class="col-sm-6">
+                                        <div class="card-title text-center mrfont2">
-    <p class="card-text card-text_igem text-justify">
+                                            <center>
-      <center>
+                                                <h5 class="mrfont2">
-        <img class="gifs"src="https://static.igem.org/mediawiki/2018/3/30/T--UCL--4e.gif" />
+                                                    Tetramer
-      </center>
+                                                </h5>
-    </p>
+                                            </center>
-</div>
+                                        </div>
-<div class="col-sm-6">
+                                        <hr />
-    <p class="card-text card-text_igem text-justify">
+                                        <div class="row yyyyy">
-      <center>
+                                            <div class="col-sm-6">
-        <img class="gifs"src="https://static.igem.org/mediawiki/2018/6/64/T--UCL--4s.gif" />
+                                                <p class="card-text card-text_igem text-justify">
-      </center>
+                                                    <center>
-    </p>
+                                                        <img class="gifs"src="https://static.igem.org/mediawiki/2018/3/30/T--UCL--4e.gif" />
-</div>
+                                                    </center>
-</div>
+                                                </p>
-<hr>
+                                            </div>
-<div class="card-title text-center mrfont2">
+                                            <div class="col-sm-6">
-  <center><h5>Octamer</h5></center>
+                                                <p class="card-text card-text_igem text-justify">
-</div>
+                                                    <center>
-<hr>
+                                                        <img class="gifs"src="https://static.igem.org/mediawiki/2018/6/64/T--UCL--4s.gif" />
-<div class="row yyyyy">
+                                                    </center>
-<div class="col-sm-6">
+                                                </p>
-    <p class="card-text card-text_igem text-justify">
+                                            </div>
-      <center>
+                                        </div>
-        <img class="gifs"src="https://static.igem.org/mediawiki/2018/d/da/T--UCL--8e.gif" />
+                                        <hr />
-      </center>
+                                        <div class="card-title text-center mrfont2">
-    </p>
+                                                <center>
-</div>
+                                                    <h5 class="mrfont2">
-<div class="col-sm-6">
+                                                        Octamer
-    <p class="card-text card-text_igem text-justify">
+                                                    </h5>
-      <center>
+                                                </center>
-        <img class="gifs"src="https://static.igem.org/mediawiki/2018/3/39/T--UCL--8s.gif" />
+                                        </div>
-      </center>
+                                        <hr />
-    </p>
+                                        <div class="row yyyyy">
-</div>
+                                            <div class="col-sm-6">
-</div>
+                                                <p class="card-text card-text_igem text-justify">
-<hr>
+                                                    <center>
-<div class="card-title text-center mrfont2">
+                                                        <img class="gifs"src="https://static.igem.org/mediawiki/2018/d/da/T--UCL--8e.gif" />
-  <center><h5>Dodecamer</h5></center>
+                                                    </center>
-</div>
+                                                </p>
-<hr>
+                                            </div>
-<div class="row yyyyy">
+                                            <div class="col-sm-6">
-<div class="col-sm-6">
+                                                <p class="card-text card-text_igem text-justify">
-    <p class="card-text card-text_igem text-justify">
+                                                    <center>
-      <center>
+                                                        <img class="gifs"src="https://static.igem.org/mediawiki/2018/3/39/T--UCL--8s.gif" />
-        <img class="gifs"src="https://static.igem.org/mediawiki/2018/9/9d/T--UCL--12e.gif" />
+                                                    </center>
-      </center>
+                                                </p>
-    </p>
+                                            </div>
-</div>
+                                        </div>
-<div class="col-sm-6">
+                                        <hr />
-    <p class="card-text card-text_igem text-justify">
+                                        <div class="card-title text-center mrfont2">
-      <center>
+                                                <center>
-        <img class="gifs"src="https://static.igem.org/mediawiki/2018/5/51/T--UCL--12s.gif" />
+                                                    <h5 class="mrfont2">
-      </center>
+                                                        Dodecamer
-    </p>
+                                                    </h5>
-</div>
+                                                </center>
-</div>
+                                        </div>
+                                        <hr />
-</div>
+                                        <div class="row yyyyy">
+                                            <div class="col-sm-6">
-<br><br>
+                                                <p class="card-text card-text_igem text-justify">
-<p>For these simulation results, the configurations of spider silk fiber after applying a constant engineering strain rate (7.5 × 10<sup>-6</sup> τ<sup>-1</sup> along the x-axis) and at equilibrium after shear flow (0.01 τ<sup>-1</sup> along x axis for 420000 shear steps). The red nodes show hydrophobic aggregated protein clusters and the blue lines are the bridge connecting two clusters. Uniform distribution and high density of clusters and bridges represent the better mechanical property, higher tensile and toughness. For stretch testing. we can analyse the tetramer structure which consists of large and frequent cavity in the fiber. The octamer and dodecamer is predicted to be able to tolerate this strain, which means they have a considerable threshold of mechanical strain. In order to examine which structure is the best, we apply shear flow. In this case, it is observed that only the dodecamer still keeps its original structure but others break at different levels. In conclusion, dodecamer is the best configuration required in making desired fibers.
+                                                    <center>
-</p>
+                                                        <img class="gifs"src="https://static.igem.org/mediawiki/2018/9/9d/T--UCL--12e.gif" />
+                                                    </center>
-                                 </p>
+                                                </p>
+                                            </div>
+                                            <div class="col-sm-6">
+                                                <p class="card-text card-text_igem text-justify">
+                                                    <center>
+                                                        <img class="gifs"src="https://static.igem.org/mediawiki/2018/5/51/T--UCL--12s.gif" />
+                                                    </center>
+                                                </p>
+                                            </div>
+                                        </div>
+                                    </div>
+                                        <p class="card-text card-text_igem text-justify">
+                                            For these simulation results, the configurations of spider silk fiber after applying a constant engineering strain rate (7.5 × 10<sup>-6</sup> τ<sup>-1</sup> along the x-axis) and at equilibrium after shear flow (0.01 τ<sup>-1</sup> along x axis for 420000 shear steps). The red nodes show hydrophobic aggregated protein clusters and the blue lines are the bridge connecting two clusters. Uniform distribution and high density of clusters and bridges represent the better mechanical property, higher tensile and toughness. For stretch testing. we can analyse the tetramer structure which consists of large and frequent cavity in the fiber. The octamer and dodecamer is predicted to be able to tolerate this strain, which means they have a considerable threshold of mechanical strain. In order to examine which structure is the best, we apply shear flow. In this case, it is observed that only the dodecamer still keeps its original structure but others break at different levels. In conclusion, dodecamer is the best configuration required in making desired fibers.
+                                        </p>
+                                 </div>
                              </div>
                          </div>
                      </div>
                  </div>
-             </div>
+             </section>
-        </section>
+            <section id="Bibliography">
+                <div class="card card_igem wow fadeInUp" data-wow-duration="2s" data-wow-delay="0.1s">
+                    <div class="card-title text-center">
+                        <h4>Bibliography</h4>
+                    </div>
+                    <div class="card-body text-justify">
+                        <div class="container">
+                            <div class="row">
+                                <div class="col-sm-10 mmmm mrfont">
+                                    <p class="card-text card-text_igem text-justify">
+                                        <ol>
+                                            <li>
+                                                Verhulst PF. Notice sur la loi que la population poursuit dans son accroissement. Correspondance mathématique et physique. 1838;10:113–21.
+                                            </li>
+                                            <li>
+                                                Pearl R, Reed L. On the Rate of Growth of the Population of the United States since 1790 and Its Mathematical Representation. Proceedings of the National Academy of Sciences. 1920;6(6):275-288.
-        <section id="Bibliography">
+                                            </li>
-            <div class="card card_igem wow fadeInUp" data-wow-duration="2s" data-wow-delay="0.1s">
+                                             <li>
-                <div class="card-title text-center">
+                                                Bailey J, Ollis D. Biochemical engineering fundamentals. 2nd ed. New York: McGraw-Hill; 1944.
-                    <h4>Bibliography</h4>
-                </div>
-                <div class="card-body text-justify">
-                    <div class="container">
-                        <div class="row">
-                            <div class="col-sm-10 mmmm mrfont">
-                                <p class="card-text card-text_igem text-justify">
-                                    <ol>
-                                        <li>
-                                             Verhulst PF. Notice sur la loi que la population poursuit dans son accroissement. Correspondance mathématique et physique. 1838;10:113–21.
-                                        </li>
-                                        <li>
-                                          Pearl R, Reed L. On the Rate of Growth of the Population of the United States since 1790 and Its Mathematical Representation. Proceedings of the National Academy of Sciences. 1920;6(6):275-288.
-                                        </li>
+                                            </li>
-                                        <li>
+                                            <li>
-                                          Bailey J, Ollis D. Biochemical engineering fundamentals. 2nd ed. New York: McGraw-Hill; 1944.
+                                                Part:pSB1C3 - parts.igem.org [Internet]. Parts.igem.org. 2008. Available from: http://parts.igem.org/Part:pSB1C3
+                                            </li>
-                                        </li>
+                                            <li>
-                                        <li>
+                                                Zettler J, Schütz V, Mootz HD. The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans-splicing reaction. FEBS Letters 2009;583:909–14.
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+                                                Al-Ali H, Ragan TJ, Gao X, Harris TK. Reconstitution of Modular PDK1 Functions on Trans-Splicing of the Regulatory PH and Catalytic Kinase Domains. Bioconjugate Chemistry 2007;18:1294–302.
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+                                            <li>
+                                                Brenzel S, Kurpiers T, Mootz HD. Engineering Artificially Split Inteins for Applications in Protein Chemistry:  Biochemical Characterization of the Split Ssp DnaB Intein and Comparison to the Split Sce VMA Intein. Biochemistry 2006;45:1571–8.
+                                            </li>
-                                        <li>
+                                             <li>
-                                             Zettler J, Schütz V, Mootz HD. The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans-splicing reaction. FEBS Letters 2009;583:909–14.
+                                                Kyte J, Doolittle RFA. simple method for displaying the hydropathic character of a protein. Journal of Molecular Biology. 1982;157(1):105–32.
-                                        </li>
+                                            </li>
-                                        <li>
+                                            <li>
-                                            Nichols NM, Benner JS, Martin DD, Evans TC. Zinc Ion Effects on Individual Ssp DnaE Intein Splicing Steps: Regulating Pathway Progression. Biochemistry 2003;42:5301–11.
+                                                ExPASy - ProtScale [Internet]. Web.expasy.org. Available from: https://web.expasy.org/protscale/
-                                        </li>
+                                            </li>
-                                        <li>
+                                            <li>
-                                            Al-Ali H, Ragan TJ, Gao X, Harris TK. Reconstitution of Modular PDK1 Functions on Trans-Splicing of the Regulatory PH and Catalytic Kinase Domains. Bioconjugate Chemistry 2007;18:1294–302.
+                                                Rim N, Roberts E, Ebrahimi D, Dinjaski N, Jacobsen M, Martín-Moldes Z, Buehler M, Kaplan D and Wong J. Predicting Silk Fiber Mechanical Properties through Multiscale Simulation and Protein Design. ACS Biomaterials Science & Engineering. 2017;3(8), pp.1542-1556.
-                                        </li>
+                                            </li>
-                                        <li>
+                                            <li>
-                                          Brenzel S, Kurpiers T, Mootz HD. Engineering Artificially Split Inteins for Applications in Protein Chemistry:  Biochemical Characterization of the Split Ssp DnaB Intein and Comparison to the Split Sce VMA Intein. Biochemistry 2006;45:1571–8.
+                                                Lin S, Ryu S, Tokareva O, Gronau G, Jacobsen M, Huang W, Rizzo D, Li D, Staii C, Pugno N, Wong J, Kaplan D and Buehler M. Predictive modelling-based design and experiments for synthesis and spinning of bioinspired silk fibres. Nature Communications. 2015;6(1).
-                                        </li>
+                                            </li>
+                                        </ol>
-                                        <li>
+                                 </div>
-                                            Kyte J, Doolittle RFA. simple method for displaying the hydropathic character of a protein. Journal of Molecular Biology. 1982;157(1):105–32.
-                                        </li>
-                                        <li>
-                                            ExPASy - ProtScale [Internet]. Web.expasy.org. Available from: https://web.expasy.org/protscale/
-                                        </li>
-                                        <li>
-                                            Rim N, Roberts E, Ebrahimi D, Dinjaski N, Jacobsen M, Martín-Moldes Z, Buehler M, Kaplan D and Wong J. Predicting Silk Fiber Mechanical Properties through Multiscale Simulation and Protein Design. ACS Biomaterials Science & Engineering. 2017;3(8), pp.1542-1556.
-                                        </li>
-                                        <li>
-                                          Lin S, Ryu S, Tokareva O, Gronau G, Jacobsen M, Huang W, Rizzo D, Li D, Staii C, Pugno N, Wong J, Kaplan D and Buehler M. Predictive modelling-based design and experiments for synthesis and spinning of bioinspired silk fibres. Nature Communications. 2015;6(1).
-                                        </li>
-                                    </ol>
-                                 </p>
                              </div>
                          </div>
                      </div>
                  </div>
-             </div>
+             </section>
-         </section>
+         </div>
      </div>
-</div>
 </div>
 <div class="footer">
@@ Line 1,030: / Line 978: @@
                      <div class="h2_footer_igem">Our Location</div>
                      University College London<br />
-  Gower St, Bloomsbury<br />London <br />WC1E 6BT
+                    Gower St, Bloomsbury<br />London <br />WC1E 6BT
                      <span></span>
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                  <center>
                      <div class="h2_footer_igem">Contact Us</div>
-                  ucl.igem@ucl.ac.uk
+                    ucl.igem@ucl.ac.uk
                      <a class="footer_img_igem" href="https://www.ucl.ac.uk/">
                          <img src="https://static.igem.org/mediawiki/2018/f/ff/T--UCL--UCLLogo.jpeg" height="100rem;" />