From c5c96e8529f052d71a021d2a6c2c0afa678d0fb4 Mon Sep 17 00:00:00 2001
From: Felisa Kowalewski <felisa.andrea.kowalewski@studium.uni-hamburg.de>
Date: Mon, 2 Dec 2024 12:25:13 +0000
Subject: [PATCH] Update description.html
---
wiki/pages/description.html | 54 ++++++++++++++++++-------------------
1 file changed, 27 insertions(+), 27 deletions(-)
diff --git a/wiki/pages/description.html b/wiki/pages/description.html
index 7dfba37..16d9577 100644
--- a/wiki/pages/description.html
+++ b/wiki/pages/description.html
@@ -674,7 +674,7 @@
<th>Saving Lives</th>
<td>By preventing infections through wound containment and reducing the need for surgery, reSkin
can significantly lower complications and risks associated with burn wounds. Because it is
- cheaper than other treatments, can be used for all skin tones and is easily applied and
+ cheaper than other treatments, can be used for all skin tones, and is easily applied and
removed, reSkin is usable in every clinic, including low-income countries, helping where it
is most needed.</td>
</tr>
@@ -692,15 +692,15 @@
</tr>
<tr>
<th>Pain reduction</th>
- <td>Minimized dressing changes by combining multiple treatment methods (wound containment, depth
- assessment, debridement, regeneration, wound-fluid absorption, infection prevention) in one
- step, reduces patient discomfort and stress. The non-invasive enzymatic debridement with a
- lower bromelain concentration cleans the wound less painfully.</td>
+ <td>Minimized amounts of dressing changes reduce patient discomfort and stress by combining multiple
+ treatment methods (wound containment, depth assessment, debridement, regeneration, wound-fluid
+ absorption, infection prevention) in one step. The non-invasive enzymatic debridement with a
+ lower bromelain concentration leads to a less painful wound cleaning.</td>
</tr>
<tr>
<th>Enhanced healing</th>
<td>Promotes faster wound healing by supporting collagen synthesis, reducing oxidative damage,
- and preserving healthy tissue, leading to reduced scarring and complications.</td>
+ and preserving healthy tissue, leading to reduced scarring and less complications.</td>
</tr>
<tr>
<th>Flexibility & Strength</th>
@@ -709,7 +709,7 @@
muscoskeletal system.</td>
</tr>
<tr>
- <th>Personalizable</th>
+ <th>Customizable</th>
<td>reSkin can be customized to include additional drugs tailored to individual patient needs,
expanding its versatility for different burn types and conditions.</td>
</tr>
@@ -748,7 +748,7 @@
</div>
<div class="col">
<p>
- The natural resilin consists of different protein regions. The interesting part is the highly repetitive Exon 1. Our Resilin polymer consists of 32+ repeats of this specific sequence. By improving the concatemerization technique [18] we developed our new repeat cloning method Repeatigo. Now we can assemble repeating sequences faster and have more control over the repeat length and the ligation into a vector. The Resilin polymer is then be expressed by Escherichia coli forming the best flexible basis for our reSkin hydrogel [16].
+ Natural resilin consists of different protein regions. The interesting part is the highly repetitive Exon 1. Our resilin polymer consists of 32+ repeats of this specific sequence. By improving the concatemerization technique [18] we developed our new repeat cloning method Repeatigo. Now we can assemble repeating sequences faster and have more control over the repeat length and the ligation into a vector. Then, the resilin polymer is be expressed by Escherichia coli, forming the best flexible basis for our reSkin hydrogel [16].
</p>
</div>
</div>
@@ -758,7 +758,7 @@
</div>
<div class="" style="padding: 10px; display: none;" id="desc-resilin-repeat">
<p>
- Our aim was to create super-long resilin filaments of 32-64 repeats to enable cross-linking. Since repetitive sequences of this extend can not be synthesized, we had to develop a new method - the Repeatigo method. This method enables us to generate repeats of the same gene sequence.
+ Our aim was to create super-long resilin filaments of 32 to 64 repeats to enable crosslinking. Since repetitive sequences of this extend cannot be synthesized, we had to develop a new method - Repeatigo. This enables us to generate repeats of the same gene sequence.
</p>
<div class="row">
<!--<div class="col" style="text-align: center;">
@@ -767,7 +767,7 @@
</div>-->
<div class="col">
<p>
- The idea of this method is to synthesize single stranded oligos with a 5’-phosphorylization, varying from 23 to 45 bp, that will assemble while repetitively creating overhangs for further assembly. Finally, double-stranded DNA will be generated containing several consecutive repeats of the same sequence. The start and end oligos do not allow further assembly and stop the extension process. The start and end oligos incorporate a restriction recognition side. This allows specific restriction and ligation into a vector.
+ The idea of this method is to synthesize single stranded oligos with a 5’-phosphorylation, varying from 23 to 45 bp, that will assemble while repetitively creating overhangs for further assembly. Finally, double-stranded DNA will be generated containing several consecutive repeats of the same sequence. The start and end oligos do not allow for further assembly and stop the extension process. The start and end oligos incorporate a restriction recognition side: This allows specific restriction and ligation into a vector.
</p>
</div>
</div>
@@ -784,7 +784,7 @@
<ul>
<li>
<b>Middle Oligos:</b>
- The first resilin exon is the codon-optimized sequence for the expression in <i>E. coli</i>. This sequence is our forward oligo 1 (<a href="https://parts.igem.org/Part:BBa_K5077001">BBa_K5077001</a>). Oligo 2 repeat (<a href="https://parts.igem.org/Part:BBa_K5077002">BBa_K5077002</a>) is made up of the complementary bases of oligo 1 repeat shifted by half. This means that when these two oligos bind together, they only align partially and have overhangs on each side. This allows them to grow on both sides, building double-stranded resilin repeats in all different lengths.
+ The first resilin exon is the codon-optimized sequence for the expression in <i>E. coli</i>. This sequence is our forward oligo 1 (<a href="https://parts.igem.org/Part:BBa_K5077001">BBa_K5077001</a>). Oligo 2 repeat (<a href="https://parts.igem.org/Part:BBa_K5077002">BBa_K5077002</a>) is made up of the complementary bases of oligo 1 repeat shifted by half. This means, that when these two oligos bind together, they only align partially and have overhangs on each side. This allows them to grow on both sides, building double-stranded resilin repeats in all different lengths.
</li>
</ul>
</p>
@@ -800,7 +800,7 @@
<ul>
<li>
<b>Start Oligos: </b>
- The oligo NdeI 1 end (<a href="https://parts.igem.org/Part:BBa_K5077003">BBa_K5077003</a>) and oligo NdeI 2 end (<a href="https://parts.igem.org/Part:BBa_K5077004">BBa_K5077004</a>) contain the NdeI restriction site and random base pairs. The reverse oligo also binds to the first half of the oligo 1 repeat. This results in a fragment which ensures binding to the middle oligos and stops the extension.
+ The oligo NdeI 1 end (<a href="https://parts.igem.org/Part:BBa_K5077003">BBa_K5077003</a>) and oligo NdeI 2 end (<a href="https://parts.igem.org/Part:BBa_K5077004">BBa_K5077004</a>) contain the NdeI restriction site and random base pairs. The reverse oligo also binds to the first half of the oligo 1 repeat. This results in a fragment, which ensures binding to the middle oligos and stops the extension.
</li>
</ul>
</p>
@@ -823,9 +823,9 @@
</div>
</div>
<p>
- The complementary oligos are annealed separately and then ligated. To prevent false binding, the annealed start and middle oligos are first ligated, and then the annealed end oligos are added. The ligase can chain them together properly because of their 5-phoshorylization. To get the desired fragment length of 1.2 to 3 kilobases, it is necessary to run an agarose gel and cut and clean up the specific product sizes.
+ Complementary oligos are annealed separately and then ligated. To prevent false binding, the annealed start and middle oligos are first ligated, and then the annealed end oligos are added. The ligase can chain them together properly because of their 5'-phoshorylation. To get the desired fragment length of 1.2 to 3 kilobases, it is necessary to run an agarose gel and cut and clean up the specific product sizes.
<br>
- A polymerase-chain-reaction amplifies the cleaned ligation, which was again checked using an agarose gel. After a double digest of the resilin repeat ligation product and our pET28c(+) vector with NdeI and SacI, the restricted products can be ligated and transformed.
+ A polymerase-chain-reaction amplifies the cleaned ligation, which needs to be again checked using an agarose gel. After a double digest of the resilin repeat ligation product and our pET28c(+) vector with NdeI and SacI, the restricted products can be ligated and transformed.
</p>
<div style="text-align: center;">
<img src="https://static.igem.wiki/teams/5077/description/fig2.png" alt="" style="width: 80%;">
@@ -837,7 +837,7 @@
</div>
<div class="" style="padding: 10px; display: none;" id="desc-resilin-exp">
<p>
- Our pET28c(+)_Resilin_Re32 needs to be treated with care. Most E. coli strains have naturally occurring recombinases like recA, which tend to delete repeating sequences. We decided to use the E. coli strain JM109, which is recA-deficient and, therefore, more unlikely to destroy our plasmid. pET28c(+) has a lac operon, so resilin production can be easily started by adding IPTG [19].
+ Our pET28c(+)_Resilin_Re32 needs to be treated with care. Most E. coli strains have naturally occurring recombinases like recA, which tend to delete repeating sequences. We decided to use the E. coli strain JM109, which is recA-deficient and, therefore, less likely to destroy our plasmid. pET28c(+) has a lac operon, so resilin production can be easily started by adding IPTG [19].
</p>
</div>
<div class="heading" style="margin-top: 10px; cursor: pointer;"
@@ -849,7 +849,7 @@
Since our resilin is expressed with a C-terminal His-tag comprising six histidines, we purify it by affinity chromatography using a magnetic beads kit with nickel ions bound to nitrilotriacetic acid [20].
<br>
When adequately purified and detected via SDS-PAGE, the resilin polymer can be used for crosslinking with hyaluronic acid.
- The lysate is then added to an equilibrated magnetic beads solution, incubated, and washed according to the kit protocol. The final elution holds the purified resilin, which can be detected by running an SDS PAGE. Depending on the number of repeats, the molecular weight will vary.
+ The lysate is then added to an equilibrated magnetic beads solution, incubated, and washed according to the kit protocol. The final elution holds the purified resilin, which can be detected by running an SDS-PAGE. Depending on the number of repeats, the molecular weight will vary.
</p>
</div>
</div>
@@ -862,7 +862,7 @@
<div class="row">
<div class="col">
<p>
- The other key element of our hydrogel is the integration of hyaluronic acid. It is a natural negatively charged polymer which has a very high water capacity. It can hold up to 1000 times its volume in water [21] which is perfect for the hydration of the burn wound and the absorption of the wound fluid [22].
+ The other key element of our hydrogel is the integration of hyaluronic acid. It is a naturally negatively charged polymer, which has a very high water capacity. It can hold up to 1000 times its volume in water [21], which is perfect for the hydration of the burn wound and the absorption of wound fluid [22].
</p>
</div>
<div class="col" style="text-align: center;">
@@ -883,7 +883,7 @@
<p>Fig. 4: Hyaluronic acid pathway. [12]</p>
</div>
<p>
- Some specific strains of E. coli are predestined to use for the synthesis of hyaluronic acid, as they already have an incomplete pathway for the synthesis. By using metabolically engineered E. coli we can synthesize hyaluronic acid naturally without depending on chemical synthesis or the extraction from animals. The E. coli strand Top 10 already owns most of the genes for this synthesis. By transforming the hasA-gene, which codes the hyaluronic acid synthetase, we can complete the pathway and synthesize hyaluronic acid in the bacteria [25].
+ Some specific strains of E. coli are predestined to use for the synthesis of hyaluronic acid, as they already have an incomplete pathway for the synthesis. By using metabolically engineered E. coli, we can synthesize hyaluronic acid naturally without depending on chemical synthesis or the extraction from animals. The E. coli strain Top10 already owns most of the genes for this synthesis. By transforming the HasA gene, which codes for hyaluronic acid synthetase, we can complete the pathway and synthesize hyaluronic acid in the bacteria [25].
</p>
</div>
@@ -894,7 +894,7 @@
<p>
To determine how well the HasA expression in our <i>E. coli</i> is working, we added the sequence for enhanced green fluorescent protein (eGFP): If the expression is successful, UV light will induce fluorescence [26].
<br>
- To do this, we first insert the sequence for eGFP and a linker behind the HasA gene in our puc_HasA plasmid, which codes for hyaluronic acid synthetase. For this, both vector and eGFP have to be linearized by conducting a PCR. Afterwards, they can be merged by a SLiCE homologous recombination [27]. Our plasmid is now ready to express the synthetase marked by eGFP and is transformed in the competent Top10Â E. coli. The cells can be multiplied by incubation (overnight culture) at 37 degrees Celsius, the optimum temperature for our bacteria [28].
+ To do this, we first insert the sequence for eGFP and a linker behind the HasA gene in our pUC_HasA plasmid. For this, both vector and eGFP have to be linearized by conducting a PCR. Afterwards, they can be merged by a SLiCE homologous recombination [27]. Our plasmid is now ready to express the synthetase marked by eGFP and is transformed in the competent Top10Â E. coli. The cells can be multiplied by incubation (overnight culture) at 37 degrees Celsius, the optimum temperature for our bacteria [28].
</p>
<div style="text-align: center;">
<img src="https://static.igem.wiki/teams/5077/description/fig5.png" alt="" style="width: 80%;">
@@ -903,7 +903,7 @@
<p>
The expression of hyaluronic acid synthetase can then be induced by adding arabinose, as our vector has an arabinose promotor.
<br>
- All the steps have to be continuously checked by running gel electrophoreses. For a successful insertion of eGFP into the pucHasA plasmid we expect to see bands at 5982 base pairs. The expression of the synthetase can be shown by testing fluorescence, but also by running an SDS PAGE.
+ All the steps have to be continuously checked by running gel electrophoreses. For a successful insertion of eGFP into the pUC_HasA plasmid, we expect to see bands at 5982 base pairs. The expression of the synthetase can be shown by testing fluorescence, but also by running an SDS-PAGE.
</p>
</div>
<div class="heading" style="margin-top: 10px; cursor: pointer;"
@@ -912,7 +912,7 @@
</div>
<div class="" style="padding: 10px; display: none;" id="desc-acid-extract">
<p>
- To extract Hyaluronic Acid, the sample must be prepared beforehand. For this, a fermentation broth sample that contains HA-producing cells is diluted with an equal volume of 0.1% w/v SDS and incubated for 10 minutes at room temperature. The SDS helps to dissolve the cell membrane and releases the HA. To precipitate the HA and to purify it, 1.5-2 volumes of ethanol are added to the sample and incubated at 4°C for 1 hour. After the incubation, the sample is centrifuged at 3000 rpm for 20 minutes at room temperature. HA will form a pellet at the bottom of the tube, and the supernatant will be carefully discarded. The HA pellet must be resuspended with the same volume of 0.1 M NaCl used for the sample. The pellet is incubated in NaCl for 10 minutes to fully dissolve the HA [25].
+ To extract hyaluronic acid (HA), the sample must be prepared in advance. For this, a fermentation broth sample that contains HA-producing cells is diluted with an equal volume of 0.1% w/v SDS and incubated for 10 minutes at room temperature. The SDS helps to dissolve the cell membrane and releases the HA. To precipitate the HA and to purify it, 1.5 to 2 volumes of ethanol are added to the sample and incubated at 4 degrees Celsius for 1 hour. After the incubation, the sample is centrifuged at 3000 rpm for 20 minutes at room temperature. HA will form a pellet at the bottom of the tube, and the supernatant will be carefully discarded. The HA pellet must be resuspended with the same volume of 0.1 M NaCl used for the sample. The pellet is incubated in NaCl for 10 minutes to fully dissolve the HA [25].
</p>
</div>
<div class="heading" style="margin-top: 10px; cursor: pointer;" onclick="toggleCollapsible('desc-acid-verify')">
@@ -920,9 +920,9 @@
</div>
<div class="" style="padding: 10px; display: none;" id="desc-acid-verify">
<p>
- The modified carbazole assay is used to measure hyaluronic acid. In this assay, one of the critical components, glucuronic acid, is measured to quantify HA. For this, 0.25 ml of our resuspended sample is mixed with 1.5 ml chilled sulphuric acid reagent containing sodium borate decahydrate. This will hydrolyze the HA into glucuronic acid and N-acetylglucosamine. The mixture is placed in a boiling water bath for 20 minutes to break down the HA into measurable components. After 20 minutes, immediately cool the sample, as it prevents further reactions and stabilizes the glucoronic acid.
+ A modified carbazole assay is used to measure hyaluronic acid: In this assay, one of the critical components, glucuronic acid, is measured to quantify HA. For this, 0.25 ml of our resuspended sample are mixed with 1.5 ml chilled sulphuric acid reagent containing sodium borate decahydrate. This will hydrolyze the HA into glucuronic acid and N-acetylglucosamine. The mixture is placed in a boiling water bath for 20 minutes to break down the HA into measurable components. After 20 minutes the sample has to be cooled immedeatly, as this prevents further reactions and stabilizes the glucoronic acid.
<br>
- Next, 50 µl of carbazole reagent is added to the cooled sample. Carbazole reacts with glucuronic acid to form a purple-colored complex. Place the sample back into a boiling water bath for 15 minutes to enhance the color development of the purple complex. Afterward, cool the sample again to stop the reaction. Finally, the absorbance can be measured using a spectrophotometer at the appropriate wavelength (530 nm). The HA titer can be calculated as 2.05 times the glucuronic acid concentration. A standard curve of known glucuronic acid concentration can be used to calculate the amount of glucuronic acid in the sample [25].
+ Next, 50 µl of carbazole reagent are added to the cooled sample. Carbazole reacts with glucuronic acid to form a purple-colored complex. Placed back in boiling water for 15 minutes, the sample will develop the purple complex with an enhanced color. Afterwards, it needs cooling again to stop the reaction. Finally, the absorbance can be measured using a spectrophotometer at the appropriate wavelength (530 nm). The HA titer can be calculated as 2.05 times the glucuronic acid concentration. A standard curve of known glucuronic acid concentration can be used to calculate the amount of glucuronic acid in the sample [25].
<br>
Since our hyaluronic acid needs to be modified to form crosslinks, we have to add a functional group. We chose methacrylic anhydride (MA) for that purpose. Our HA is incubated for 24 hours at 5 degrees Celsius in a pH 8 solution with an excess of MA to produce MA-HA [29].
</p>
@@ -935,7 +935,7 @@
</div>
<div class="content" style="padding: 10px; display: none;" id="desc-cross">
<p>
- After the production of our specially synthesized resilin (RE) and methacrylated hyaluronic acid (HA) the next step on our way on creating a hydrogel is the photo-crosslinking of both components. It is necessary to figure out the correct proportions of all the ingredients as well as the right irradiation time for our purposes on the go: every little change has an effect on the degree of crosslinking and therefore on the properties of the product [30]. Our aim is to induce crosslinking between resilin-molecules via dityrosine bonds on the phenyl rings, between molecules of hyaluronic acid via the methacrylic groups, and also between both resilin and hyaluronic acid.
+ After the production of our specially synthesized resilin (RE) and methacrylated hyaluronic acid, the next step on our way to creating a hydrogel is the photo-crosslinking of both components. It is necessary to figure out the correct proportions of all the ingredients as well as the right irradiation time for our purposes on the go: Every little change has an effect on the degree of crosslinking and therefore on the properties of the product [30]. Our aim is to induce crosslinking between resilin-molecules via dityrosine bonds on the phenyl rings, between molecules of hyaluronic acid via the methacrylic groups, and also between both resilin and hyaluronic acid.
</p>
<div style="text-align: center;">
<img src="https://static.igem.wiki/teams/5077/crosslinking-home-seite.webp" alt="" style="width: 80%;">
@@ -962,7 +962,7 @@
</div>
<div class="" style="padding: 10px; display: none;" id="desc-cross-buffer">
<p>
- We chose phosphate-buffered saline (PBS) as a buffer because, like RFP, it produces no cytotoxicity and is already a proven component in crosslinking reactions [33]. PBS matches the osmolarity and ion concentrations of the human body and keeps the pH level at 7.4.
+ We chose phosphate-buffered saline (PBS) as a buffer, because, like RFP, it produces no cytotoxicity and is already a proven component in crosslinking reactions [33]. PBS matches the osmolarity and ion concentrations of the human body and keeps the pH level at 7.4.
</p>
</div>
<div class="heading" id="desc-cross-head" style="margin-top: 20px; cursor: pointer;"
@@ -980,9 +980,9 @@
</div>
<div class="" style="padding: 10px; display: none;" id="desc-cross-stacon">
<p>
- We dissolve HA (2.5% w/v) and RE (20 % w/v) in PBS and mix them together in differing ratios. Then, we dissolve RF in PBS as well and add it to our solutions according to the RF concentration to be tested.
+ We dissolve MA-HA (2.5% w/v) and RE (20 % w/v) in PBS and mix them together in differing ratios. Then, we dissolve RFP in PBS as well and add it to our solutions according to the RFP concentration to be tested.
<br>
- Mixing ratios for HA/RE assays:
+ Mixing ratios for MA-HA/RE assays:
<ol>
<li>
1 : 1
--
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