diff --git a/wiki/pages/design.html b/wiki/pages/design.html
index 38d053b552ce614111f97850e61e93e5527f4d67..9a5f92a0e6c2f1a36edafacbcc166e183b5dbf31 100644
--- a/wiki/pages/design.html
+++ b/wiki/pages/design.html
@@ -78,18 +78,22 @@
</tbody>
</table>
</figure>
- <p>Table.1 two variables truth table</p>
- <p>There are two ways to present the 16 possible 2-input, 1-output logic gates: <strong>logic functions</strong>
- and
- <strong>Boolean algebra</strong>. For logic functions, each set of 4 numbers in the Logic functions column
- below
- represents f(1,1), f(1,0), f(0,1), and f(0,0). For example, 1001 represents f(1,1)=1, f(1,0)=0, f(0,1)=0,
- and
- f(0,0)=1. For Boolean algebra, A and B represent the inputs:
- </p>
+ <div style="color: #666666; padding: 2px;">Table.1 two variables truth table</div>
+ </center>
+ <p>There are two ways to present the 16 possible 2-input, 1-output logic gates: <strong>logic functions</strong>
+ and
+ <strong>Boolean algebra</strong>. For logic functions, each set of 4 numbers in the Logic functions column
+ below
+ represents f(1,1), f(1,0), f(0,1), and f(0,0). For example, 1001 represents f(1,1)=1, f(1,0)=0, f(0,1)=0,
+ and
+ f(0,0)=1. For Boolean algebra, A and B represent the inputs:
+ </p>
+ <center>
<p>A' and B' represent the complement of A and B (not-A and not-B)</p>
<p>A*B=A AND B</p>
<p>A+B=A OR B.</p>
+ </center>
+ <center>
<figure>
<table>
<thead>
@@ -213,7 +217,7 @@
</tbody>
</table>
</figure>
- <div tyle="color: #666666; padding: 2px;">Table.3 Intersection forms for 2-input, 1-output logic gates</div>
+ <div style="color: #666666; padding: 2px;">Table.3 Intersection forms for 2-input, 1-output logic gates</div>
</center>
<h3 id='22-implementation-of-a-and-not-a'>2.2 Implementation of A and not-A</h3>
<p>Next, how to implement A & not-A in genetic circuits? We decide to use inducible promoters and CRISPRi. As
@@ -223,12 +227,16 @@
<p>For example, taking IPTG as input A, GFP as output Z, a single inducible promoter equals an A logic gate. Then we
design a sgRNA that binds to a constant promoter and interferes with the binding of RNA polymerase to the
promoter. The sgRNA is attached to an IPTG-inducible promoter, thus achieving a not-A logic gate.</p>
- <p><img src="https://static.igem.wiki/teams/5276/wetlab/pictures/implementation1.webp" referrerpolicy="no-referrer"
- alt="a"> </p>
- <p>Figure 2: Implementation of A</p>
- <p><img src="https://static.igem.wiki/teams/5276/wetlab/pictures/implementation2.webp" referrerpolicy="no-referrer"
- alt="nota"> </p>
- <p>Figure 3: Implementation of not-A</p>
+ <center>
+ <img src="https://static.igem.wiki/teams/5276/wetlab/pictures/implementation1.webp" referrerpolicy="no-referrer"
+ alt="a" style="zoom: 60%;">
+ <div style="color: #666666; padding: 2px;">Figure 2: Implementation of A</div>
+ </center>
+ <center>
+ <img src="https://static.igem.wiki/teams/5276/wetlab/pictures/implementation2.webp" referrerpolicy="no-referrer"
+ alt="nota" style="zoom: 60%;">
+ <div style="color: #666666; padding: 2px;">Figure 3: Implementation of not-A</div>
+ </center>
<h2 id='3-circuit-design'>3. Circuit Design</h2>
<p>As stated in the Overview, our genetic circuit consists of 4 components. </p>
<h3 id='31-tangram'>3.1 Tangram</h3>
@@ -260,12 +268,12 @@
will be A.</p>
<center>
<img src="https://static.igem.wiki/teams/5276/wetlab/pictures/ra-new.png" alt="orthogonality-design-1.webp"
- style="zoom: 80%;">
+ style="zoom: 60%;">
<div style="color: #666666; padding: 2px;">Figure 5: How Register A transforms into different logic gates.
- <p>Ara is the inducer of recombinase tp901, while ATc is the inducer of recombinase bxbI. Different sizes of
- triangles represent different pairs of recombinase-recognition sites. Once the inducers are added, the
- corresponding recombinases will bind to its specific recognition cites, catalyze inversion (when the
- sites are anti-aligned) or excision (when the sites are aligned). </p>
+ Ara is the inducer of recombinase tp901, while ATc is the inducer of recombinase bxbI. Different sizes of
+ triangles represent different pairs of recombinase-recognition sites. Once the inducers are added, the
+ corresponding recombinases will bind to its specific recognition cites, catalyze inversion (when the
+ sites are anti-aligned) or excision (when the sites are aligned).
</div>
</center>
<p>Register B operates similarly to Register A but uses different inducers and recombinases. While Ara is replaced
@@ -276,13 +284,12 @@
we have engineered two separate patches that are designed to express B' exclusively.</p>
<center>
<img src="https://static.igem.wiki/teams/5276/wetlab/pictures/rb-new.png" alt="orthogonality-design-1.webp"
- style="zoom: 80%;">
- <div style="color: #666666; padding: 2px;">Figure 6: How Register B transforms into different logic gates. <p>
- DAPG is the inducer of recombinase a118, while Xylose is the inducer of recombinase φC31. Different
- sizes of triangles represent different pairs of recombinase-recognition sites. Once the inducers are
- added, the corresponding recombinases will bind to its specific recognition cites, catalyze inversion
- (when the sites are anti-aligned) or excision (when the sites are aligned).
- </p>
+ style="zoom: 60%;">
+ <div style="color: #666666; padding: 2px;">Figure 6: How Register B transforms into different logic gates.
+ DAPG is the inducer of recombinase a118, while Xylose is the inducer of recombinase φC31. Different
+ sizes of triangles represent different pairs of recombinase-recognition sites. Once the inducers are
+ added, the corresponding recombinases will bind to its specific recognition cites, catalyze inversion
+ (when the sites are anti-aligned) or excision (when the sites are aligned).
</div>
</center>
<p>By combining the CRISPRi system, the recombinase expression system (Tangram section), and the designed sequences
@@ -290,22 +297,22 @@
orders (Figure 7-8).</p>
<center>
<img src="https://static.igem.wiki/teams/5276/wiki/modification/registera.webp"
- alt="orthogonality-design-1.webp" style="zoom: 80%;">
+ alt="orthogonality-design-1.webp" style="zoom: 60%;">
<div style="color: #666666; padding: 2px;">Figure 7: Register A</div>
</center>
<center>
<img src="https://static.igem.wiki/teams/5276/wiki/modification/registerb.webp"
- alt="orthogonality-design-1.webp" style="zoom: 80%;">
+ alt="orthogonality-design-1.webp" style="zoom: 60%;">
<div style="color: #666666; padding: 2px;">Figure 8: Register B</div>
</center>
<p> </p>
<h3 id='33-output'>3.3 Output</h3>
<p>The Output GFP is linked to a <em>lux</em> promoter, which turns on only when AHL & LuxR both exist, utilized
by the 2023 UCAS-China team. By using the <em>Lux</em> promoter, we can achieve the intersection of the Register
- A and Register B pathways <mark>(Figure 9)</mark>.</p>
+ A and Register B pathways (Figure 9).</p>
<center>
<img src="https://static.igem.wiki/teams/5276/wiki/modification/output.webp" alt="orthogonality-design-1.webp"
- style="zoom: 80%;">
+ style="zoom: 60%;">
<div style="color: #666666; padding: 2px;">Figure 9: Output</div>
</center>
<h3 id='34-patch'>3.4 Patch</h3>
@@ -315,13 +322,13 @@
Therefore, we additionally designed a patch system (Figure 10).</p>
<center>
<img src="https://static.igem.wiki/teams/5276/wiki/modification/patch.webp" alt="orthogonality-design-1.webp"
- style="zoom: 80%;">
+ style="zoom: 60%;">
<div style="color: #666666; padding: 2px;">Figure 10: A patch system using Cre/loxP system and fimE inversion
system</div>
</center>
<p> </p>
<p>In the original pathway, to achieve the output of A' + B', the dCas9 protein at B' inhibits the
- normal expression of A'. Therefore, we separately express B’ in the Patch system to realize A' + B'.
+ normal expression of A'. Therefore, we separately express B' in the Patch system to realize A' + B'.
</p>
<p>We introduced a cumate-inducible cymR promoter to express the Cre recombinase in the pathway; upon adding cumate,
the expressed Cre recombinase recognizes the <em>loxP</em> sequence, allowing the <em>LuxI</em> gene sequence to
@@ -341,19 +348,19 @@
continuous directed evolution system, and semi-rational design.</p>
<center>
<img src="https://static.igem.wiki/teams/5276/wiki/modification/workflow-of-the-experiment-verification.webp"
- alt="orthogonality-design-1.webp" style="zoom: 14%;">
+ alt="orthogonality-design-1.webp" style="zoom: 60%;">
<div style="color: #666666; padding: 2px;">Figure 11: Workflow of the experiment verification</div>
</center>
<p> </p>
<h3 id='42-orthogonality-matrix'>4.2 Orthogonality matrix</h3>
<center>
<img src="https://static.igem.wiki/teams/5276/wetlab/pictures/checkcheck.png" alt="orthogonality-design-1.webp"
- style="zoom: 80%;">
+ style="zoom: 50%;">
<div style="color: #666666; padding: 2px;">Figure 12: An orthogonality verification system</div>
</center>
<p> </p>
<p>To validate that the six recombinases selected for our project are mutually orthogonal, we designed an
- orthogonality verification experiment(Figure x). We created two plasmids: one plasmid expressing a specific
+ orthogonality verification experiment(Figure 12). We created two plasmids: one plasmid expressing a specific
recombinase under the <em>T7</em> promoter and the other plasmid expressing the inverted <em>gfp</em> using the
constitutive promoter <em>J23119</em>. Both the ribosome binding site (RBS) and the <em>gfp</em> have
recognition sites for a specific recombinase. We varied the recombinase genes and recognition site sequences in
@@ -366,20 +373,21 @@
LacI apart from promoters like Plac. Register 0 consists of four promoters (constant promoter J23111, trc
promoter, and inducible promoter lac operon, Rha promoter) with different directions, Three pairs of DNA
recognition sites(A118B-GG_A118P-GG, A118B-CC_A118P-CC, PhiC31B-AA_PhiC31P-AA), a Ribosome binding site, and
- msfGFP(Figure x).</p>
+ msfGFP(Figure 13).</p>
<center>
<img src="https://static.igem.wiki/teams/5276/wetlab/pictures/qs1.png" alt="orthogonality-design-2.webp"
- style="zoom: 35%;">
+ style="zoom: 50%;">
<div style="color: #666666; padding: 2px;">Figure 13: Design of Register 0</div>
</center>
- <h3 id='44-directed-evolution'>4.4 Directed evolution</h3>
+ <h3 id='44-directed-evolution'>4.4 Directed Evolution</h3>
<p>We use directed evolution to enhance recombinases efficiency for the under-expected performance of recombinases
in experiments.</p>
<h4 id='441-traditional-mutagenesis'>4.4.1 Traditional Mutagenesis</h4>
<p>In our first directed evolution system, we used error-prone PCR (epPCR) to generate a random mutant library of
the whole recombinases and selected the φX174 E phage lysis gene (Din et al., 2016) for selection and GFP gene
for fluorescence screening. We designed an expression module, which drives recombinase expression via IPTG
- induction (Fig. 12) and a selection module, which utilizes the φX174 E phage lysis gene and GFP gene (Fig. 13).
+ induction (Figure 14) and a selection module, which utilizes the φX174 E phage lysis gene and GFP gene (Figure
+ 15).
</p>
<p>We utilized epPCR to amplify the recombinase expression module to generate a random mutant library. The resulting
mutant expression plasmids were transformed with selection plasmids together and the efficient recombinase
@@ -399,7 +407,7 @@
<h4 id='442-epi-hypermutation-architectures-of-continuous-directed-evolution'>4.4.2 Epi-hypermutation Architectures
of continuous directed evolution</h4>
<p>Given the low efficiency of traditional mutagenesis, we developed an autonomous directed evolution system based
- on MutaT7, which was originally a fusion of cytosine deaminase and T7 RNA polymerase (fig. 14) (Park et al.,
+ on MutaT7, which was originally a fusion of cytosine deaminase and T7 RNA polymerase (Figure 16) (Park et al.,
2021). In the expression section, the gene for the recombinase is placed between the <em>T7</em> promoter and
<em>T7</em> terminator, allowing transcription by T7 polymerase while utilizing cytosine deaminase for
mutations.
@@ -426,9 +434,11 @@
<p>We transferred the above three modules into <em>E. coli</em>. We added IPTG and Ara to activate the
"Turnover" switch, inducing the expression of <em>eMutaT7</em> and mutating the <em>a118</em>
recombinase sequence. If the expressed A118 mutant retains its biological function as a recombinase, the
- <em>gfp</em> gene will be inverted and normally expressed. After sampling the strain, we removed IPTG and Ara and
+ <em>gfp</em> gene will be inverted and normally expressed. After sampling the strain, we removed IPTG and Ara
+ and
added ATc to activate the "Turn back" switch, relieving tetR repression on the <em>PLtetRO</em>
- promoter, which allows Gp44 protein expression to invert the <em>gfp</em> gene back to its original state. Mutant
+ promoter, which allows Gp44 protein expression to invert the <em>gfp</em> gene back to its original state.
+ Mutant
recombinases are screened after multiple “turn overâ€- “turn back†cycles.
</p>
<h4 id='443-semi-rational-design'>4.4.3 Semi-rational Design</h4>
@@ -443,29 +453,7 @@
<div style="color: #666666; padding: 2px;">Figure 17: Predicted structure of A118 and attP</div>
</center>
<p> </p>
- <h2 id='5-experiment-verification'>5. Experiment verification</h2>
- <h3 id='51-orthogonality-matrix'>5.1 Orthogonality matrix</h3>
- <center>
- <img src="https://static.igem.wiki/teams/5276/wetlab/pictures/checkcheck.png" alt="orthogonality-design-1.webp"
- style="zoom: 80%;">
- <div style="color: #666666; padding: 2px;">Figure 16: An orthogonality verification system</div>
- </center>
- <p>To validate that the six recombinases selected for our project are mutually orthogonal, we designed an
- orthogonality verification experiment(Figure 16). We created two plasmids: one plasmid expressing a specific
- recombinase under the <em>T7</em> promoter and the other plasmid expressing the inverted <em>gfp</em> using the
- constitutive promoter <em>J23119</em>. Both the ribosome binding site (RBS) and the <em>gfp</em> have
- recognition sites for a specific recombinase. We varied the recombinase genes and recognition site sequences in
- the two plasmids and co-transformed them into <em>E. coli</em>, then we have two methods to assess
- orthogonality. On the DNA level, we can use colony PCR to check whether the segments have been inverted. On the
- protein level, we can measure the intensity of green fluorescence.</p>
- <h3 id='52-length-between-the-promoter-and-the-start-codon'>5.2 Length between the promoter and the start codon</h3>
- <p>The optimal distance between a promoter and the start codon in a gene construct can vary depending on several
- factors, including the specific promoter used, the context of the gene expression system, and so on. As the
- distances between promoters and the start codon differ in our Register system, we hope to explore the
- relationship between the distances and transcription rates.</p>
- <p>We want to obtain some experimental data through experiments, construct a model with the modeling team members,
- and finally use the summarized data to optimize the design of the Register system.</p>
- <h2 id='6-references'>6. References</h2>
+ <h2 id='6-references'>5. References</h2>
<ol start=''>
<li>Din, M., Danino, T., Prindle, A. <em>et al.</em> Synchronized cycles of bacterial lysis for <em>in vivo</em>
delivery. <em>Nature</em> <strong>536</strong>, 81–85 (2016).</li>