diff --git a/wiki/footer.html b/wiki/footer.html
index 7347a63cb43cd1edc9f20d2532356ffff624cdc2..ec29e585454abffd38dc0cdda92607107e322740 100644
--- a/wiki/footer.html
+++ b/wiki/footer.html
@@ -12,23 +12,23 @@
</div>
<ul class="social-icon text-center row">
<li class="list-inline-item col-2">
- <a href="https://instagram.com">
+ <a href="https://www.instagram.com/igem.heidelberg/">
<img alt="Social Media Icon Instagram" src="https://static.igem.wiki/teams/5237/footer/instagram.svg">
</a>
</li>
<li class="list-inline-item col-2">
- <a href="https://linkedin.com">
+ <a href="https://www.instagram.com/igem.heidelberg/">
<img alt="Social Media Icon LinkedIn" src="https://static.igem.wiki/teams/5237/footer/linkedin-1.svg">
</a>
</li>
<li class="list-inline-item col-2">
- <a href="https://twitter.com">
+ <a href="https://igem-heidelberg.com/index.html">
<img alt="Social Media Icon Twitter" src="https://static.igem.wiki/teams/5237/footer/twitter.svg">
</a>
</li>
<li class="list-inline-item col-2">
- <a href="https://tiktok.com">
- <img alt="Social Media Icon Tiktok" src="https://static.igem.wiki/teams/5237/footer/tiktok.svg">
+ <a href="https://www.tiktok.com/@igem.heidelberg">
+ <img alt="Social Media Icon Tiktok" src="https://static.igem.wiki/teams/5237/model/igem-hd-logo-dark.svg-">
</a>
</li>
<li class="list-inline-item col-2">
diff --git a/wiki/pages/contribution.html b/wiki/pages/contribution.html
index a87c1aae39cbaefb6ff1414cfc3e65c0d112a94c..cef5fc3e93b9900c4b179e8a41b3e61dd1142f43 100644
--- a/wiki/pages/contribution.html
+++ b/wiki/pages/contribution.html
@@ -217,7 +217,7 @@
</ul>
</div>
- <h3 class="fs-h3 fw-semi-bold mt-4" id="elementary_school_workshop"> Elementary School Workshops</h3>
+
<p class="mt-3">
For our <b>primary school workshops</b>, we've created a full set of <b>teaching materials</b> designed to make running the workshops as easy and engaging as possible. This includes a detailed <b>lesson plan</b> that outlines each step of the workshop, <b>worksheets</b> for students to practice the concepts learned, and corresponding <b>answer keys</b> for teachers. Additionally, we’ve provided <b>certificates</b> to reward students for their participation, as well as important <b>background information</b> for educators, ensuring they feel well-prepared to teach the material and answer any questions.
diff --git a/wiki/pages/experiments.html b/wiki/pages/experiments.html
index e7c3fdb7ab9c655022016aaf4cbc4ec32a0870fa..a8339b4065aad8d5003b2ee4dd4a8b9a47b4c932 100644
--- a/wiki/pages/experiments.html
+++ b/wiki/pages/experiments.html
@@ -2352,7 +2352,7 @@
<div class="single-exp">
<h4 class="fs-h4 fw-bold">Plasmid Construction</h4>
<div class="text-img">
- <a href="{{ url_for('pages', page='results') }}" class="go-to-results-wrapper">
+ <a href="{{ url_for('pages', page='results') }}#cas_results_wetlab" class="go-to-results-wrapper">
<img class="go-to-results" src="https://static.igem.wiki/teams/5237/gotoresults.svg" alt="Go to results button">
</a>
<p>All plasmids created for the Cas staple project were constructed using Golden Gate assembly (GGA), with the exception of the fusion dCas plasmid being created by Gibson assembly . Inserted fragments were either annealed oligonucleotides or PCR amplified DNA fragments. PCRs were performed using Q5 2x Master Mix (New England Biolabs, USA).
@@ -2373,9 +2373,6 @@
<div class="single-exp">
<h4 class="fs-h4 fw-bold">Cell Culture Conditions</h4>
<div class="text-img">
- <a href="{{ url_for('pages', page='results') }}" class="go-to-results-wrapper">
- <img class="go-to-results" src="https://static.igem.wiki/teams/5237/gotoresults.svg" alt="Go to results button">
- </a>
<p>HEK293T cells were kept at 37°C and 5% CO<sub>2</sub> in 10 mL cell culture medium, containing Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific) with phenol red, supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and 10 units penicillin-streptomycin solution (Thermo Fisher Scientific).
Cells were split twice a week using 5 mL Phosphate Buffer Saline (ThermoFisher Scientific, USA) and 1 mL Trypsin-EDTA (Thermo Fisher Scientific).
Cells were then diluted 1:10 into new cell culture medium.</p>
@@ -2384,10 +2381,6 @@
<div class="single-exp">
<h4 class="fs-h4 fw-bold">T7 Endonuclease I (T7EI) Assay</h4>
- <div class="text-img">
- <a href="{{ url_for('pages', page='results') }}" class="go-to-results-wrapper">
- <img class="go-to-results" src="https://static.igem.wiki/teams/5237/gotoresults.svg" alt="Go to results button">
- </a>
<p>24h after seeding 12,500 cells per well into a 96-well plate, cells were transfected by adding 200 ng DNA with Lipofectamine 2000 (ThermoFisher Scientific, USA) into each well.
After 72 h, cells were lysed by adding 70 µL H<sub>2</sub>O, 70 µL DirectPCR-Cell (VWR Chemicals), and 1.4 µL Proteinase K (Sigma-Aldrich, USA) into each well and incubating for 6 h at 55°C and 120 RPM.
Enzymes were then heat inactivated at 85°C for 45 min.
@@ -2401,9 +2394,6 @@
<div class="single-exp">
<h4 class="fs-h4 fw-bold">Dual Luciferase Assay</h4>
<div class="text-img">
- <a href="{{ url_for('pages', page='results') }}" class="go-to-results-wrapper">
- <img class="go-to-results" src="https://static.igem.wiki/teams/5237/gotoresults.svg" alt="Go to results button">
- </a>
<p>24h after seeding 12,500 cells per well into a 96-well plate, cells were transfected by adding 200 ng DNA with Lipofectamine 2000 (ThermoFisher Scientific, USA) into each well.
After 48 h, the medium was discarded and cells were lysed for 30 min in Passive Lysis 5X Buffer (Promega, USA) at room temperature, shaking at 500 RPM.
10 µL of each sample was transferred to white 96-well plates.
@@ -2419,7 +2409,7 @@
<div class="single-exp">
<h4 class="fs-h4 fw-bold">Molecular Cloning of the Cathepsin B-Cleavable Linker GFLG (p10)</h4>
<div class="text-img">
- <a href="{{ url_for('pages', page='results') }}" class="go-to-results-wrapper">
+ <a href="{{ url_for('pages', page='results') }}#results_cathepsinBliner" class="go-to-results-wrapper">
<img class="go-to-results" src="https://static.igem.wiki/teams/5237/gotoresults.svg" alt="Go to results button">
</a>
<p>
@@ -2431,9 +2421,6 @@
<div class="single-exp">
<h4 class="fs-h4 fw-bold">Molecular Cloning of the Cathepsin B Expression Cassette (p12)</h4>
<div class="text-img">
- <a href="{{ url_for('pages', page='results') }}" class="go-to-results-wrapper">
- <img class="go-to-results" src="https://static.igem.wiki/teams/5237/gotoresults.svg" alt="Go to results button">
- </a>
<p>
We amplified the gene block gBlock_Cathepsin_B with the primers fwd_gBlock_CatB and rev_gBlock_CatB. The gene block was composed of an upstream XhoI recognition sequence, followed by a Kozak sequence, the SV40 nuclear localization signal, a GGS linker, the human codon-optimized sequence for cathepsin B, and a downstream BamHI recognition sequence. We then inserted the amplicon into a pcDNA3.1 backbone via restriction cloning and transformed the ligated plasmid into <i class="italic">E. coli</i> Top10 cells.
</p>
@@ -2443,9 +2430,6 @@
<div class="single-exp">
<h4 class="fs-h4 fw-bold">Molecular Cloning of a Truncated and Mutated Form of Cathepsin B (p12.1)</h4>
<div class="text-img">
- <a href="{{ url_for('pages', page='results') }}" class="go-to-results-wrapper">
- <img class="go-to-results" src="https://static.igem.wiki/teams/5237/gotoresults.svg" alt="Go to results button">
- </a>
<p>To generate a truncated and mutated version of cathepsin B, we employed overlap extension PCR. We used the primers CatB_p12_frag1_fwd and CatB_p12_frag1_rev to remove the nucleotide sequence corresponding to the first twenty amino acids of wild-type cathepsin B using the gene block gBlock_Cathepsin_B as a template. We introduced three point mutations (D22A, H110A, and R116A) in the nucleotide sequence of cathepsin B using the primers CatB_p12_frag2_fwd, CatB_p12_frag2_rev, CatB_p12_frag3_fwd, CatB_p12_frag3_rev, CatB_p12_frag4_fwd, and CatB_p12_frag4_rev. After generating the amplicon (trunc_mut_CatB) with overlap extension PCR, we cloned it into the same pcDNA3.1 backbone used for the wild-type cathepsin B and transformed the ligated plasmid into <i class="italic">E. coli</i> Top10 cells.</p>
</div>
</div>
@@ -2453,9 +2437,6 @@
<div class="single-exp">
<h4 class="fs-h4 fw-bold">Cathepsin B Cleavage Fluorescence Readout Assay</h4>
<div class="text-img">
- <a href="{{ url_for('pages', page='results') }}" class="go-to-results-wrapper">
- <img class="go-to-results" src="https://static.igem.wiki/teams/5237/gotoresults.svg" alt="Go to results button">
- </a>
<p>To study cathepsin B-mediated cleavage of different linkers, we conducted a fluorescence readout assay. We seeded 10<sup>4</sup> HEK293T cells in each well of a 96-well plate with DMEM (10% FCS) and incubated the cells for 24 hours at 37 °C and 5% CO<sub>2</sub> before transfection. Plasmid solutions were prepared in OptiMEM, and transfections were performed using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham). For each linker, we included one negative control without the plasmid encoding cathepsin B, and two test samples with 30 ng and 60 ng of the plasmid. After transfection, we incubated the cells at 37 °C and 5% CO<sub>2</sub>. At 24 hours post-transfection, we added 500 nM doxorubicin to the cell supernatant. We measured eGFP and mCherry fluorescence intensities at 48 hours post-transfection using the Tecan Infinite F200 Pro plate reader (Tecan Group Ltd., Männedorf).</p>
</div>
</div>
@@ -2463,9 +2444,6 @@
<div class="single-exp">
<h4 class="fs-h4 fw-bold">Cell Lysis and Western Blotting</h4>
<div class="text-img">
- <a href="{{ url_for('pages', page='results') }}" class="go-to-results-wrapper">
- <img class="go-to-results" src="https://static.igem.wiki/teams/5237/gotoresults.svg" alt="Go to results button">
- </a>
<p>To confirm the overexpression of cathepsin B in HEK293T cells, we performed western blotting. We seeded 10<sup>5</sup> HEK293T cells per well in a 12-well plate with DMEM (10% FCS) and incubated for 24 hours at 37 °C and 5% CO<sub>2</sub> before transfecting cells separately with both wild-type and mutated cathepsin B constructs. Transfections were carried out in OptiMEM using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham), followed by incubation at 37 °C and 5% CO<sub>2</sub>. At 24 hours post-transfection, we supplemented the medium with 500 nM doxorubicin. 48 hours post-transfection, we washed the cells with DPBS. We then lysed the cells in RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% Sodium deoxycholate, 0.1% SDS, 1 mM dithiothreitol, 50 mM TRIS-HCl pH 7.6) supplemented with a protease inhibitor cocktail. Protein concentrations were quantified using the DC protein assay (Bio-Rad Laboratories GmbH, Feldkirchen) and measured with the Spark Multimode microplate reader (Tecan Group Ltd., Männedorf). For SDS PAGE, we used a Mini-PROTEAN TGX Stain-Free Precast gel (Bio-Rad Laboratories GmbH, Feldkirchen) and ran the gel in a Mini-PROTEAN Tetra cell system (Bio-Rad Laboratories GmbH, Feldkirchen) at 200 V for 30 minutes. Following SDS PAGE, we transferred proteins to a PVDF membrane via semi-dry western blotting using the Trans-Blot SD system (Bio-Rad Laboratories GmbH, Feldkirchen) at 10 V for 1 hour. The membrane was blocked with I-Block protein-based blocking reagent (Thermo Fisher Scientific, Waltham) and incubated overnight at 4 °C with a polyclonal anti-cathepsin B antibody and a monoclonal anti-beta-tubulin antibody (Proteintech, Rosemont) (1:1000 dilution). After washing thrice with TBS-T buffer, we incubated the membrane with anti-rabbit IgG DyLight 800 (Cell Signaling Technology, Inc., Danvers) and anti-mouse IgG Alexa Fluor 680 (Thermo Fisher Scientific, Waltham) (1:10000 dilution) for 1 hour at 4 °C. Afterwards, we washed the membrane three times with TBS-T buffer and imaged the membrane using the Odyssey XF Imager (LI-COR Biotechnology, Lincoln).</p>
</div>
</div>
@@ -2473,9 +2451,6 @@
<div class="single-exp">
<h4 class="fs-h4 fw-bold">Molecular Cloning of an Intein-dCas9 Fusion Protein (p13)</h4>
<div class="text-img">
- <a href="{{ url_for('pages', page='results') }}" class="go-to-results-wrapper">
- <img class="go-to-results" src="https://static.igem.wiki/teams/5237/gotoresults.svg" alt="Go to results button">
- </a>
<p>To enable functionalization of Cas staples <i class="italic">in vivo</i>, we cloned a dCas9-intein fusion protein using HiFi DNA assembly. We PCR-amplified the inteins NpuC and NpuN from the gene block gBlock_dCas9_Inteins using the primers SV40_NpuC_Cage_fwd, SV40_NpuC_rev, NpuN_SV40_fwd, and NpuN_Cage_SV40_rev. We amplified the backbone (p70) and dCas9 (p505) with the primers p70_BB_SV40_fwd, p70_BB_SV40_rev and dCas9_fwd, dCas9_rev creating the amplicons p70_backbone and dCas9, respectively. Finally, we assembled the fragments using the NEBuilder HiFi DNA assembly reaction mix (New England Biolabs, Ipswich).</p>
</div>
</div>
@@ -2489,7 +2464,7 @@
<div class="single-exp">
<h4 class="fs-h4 fw-bold">Constructs for FRET Assay</h4>
<div class="text-img">
- <a href="{{ url_for('pages', page='results') }}" class="go-to-results-wrapper">
+ <a href="{{ url_for('pages', page='results') }}#results_fret_measurment_wetlab" class="go-to-results-wrapper">
<img class="go-to-results" src="https://static.igem.wiki/teams/5237/gotoresults.svg" alt="Go to results button">
</a>
<p>
@@ -2514,9 +2489,6 @@
<div class="single-exp">
<h4 class="fs-h4 fw-bold">Constructs for Protein Expression and Purification</h4>
<div class="text-img">
- <a href="{{ url_for('pages', page='results') }}" class="go-to-results-wrapper">
- <img class="go-to-results" src="https://static.igem.wiki/teams/5237/gotoresults.svg" alt="Go to results button">
- </a>
<p>Expression plasmids for simple staple constructs were based on p3. Plasmid p3.6 (Oct1-mNeonGreen) was generated by
inverse PCR with BS-9, BS-10. Plasmid p3.7 (tetR-mScarlet-I) was constructed by digesting p3 with NdeI and XhoI
and assembling the PCR-amplified fragments (p3 template with primer BS-9 + BS-10 and BS-11 + BS-12) using NEBuilder
@@ -2539,9 +2511,6 @@
<h5 class="fw-semi-bold">Protein Expression and Purification</h5>
<div class="text-img">
- <a href="{{ url_for('pages', page='results') }}" class="go-to-results-wrapper">
- <img class="go-to-results" src="https://static.igem.wiki/teams/5237/gotoresults.svg" alt="Go to results button">
- </a>
<p>Proteins were expressed in <i>E. coli</i> BL21 DE3 (NEB, Ipswich, MA) carrying the necessary constructs under a T7
promoter. A 500 mL culture in DYT medium was inoculated 1:100 with an overnight culture, grown to OD<sub>600</sub>
of 0.4-0.6, and induced with 0.1 mM IPTG. Cells were harvested 16-18 hours post-induction at 32 °C and centrifuged
@@ -2557,9 +2526,6 @@
</div>
<h5 class="fw-semi-bold">SDS-PAGE Analysis</h5>
<div class="text-img">
- <a href="{{ url_for('pages', page='results') }}" class="go-to-results-wrapper">
- <img class="go-to-results" src="https://static.igem.wiki/teams/5237/gotoresults.svg" alt="Go to results button">
- </a>
<p>The general protocol was followed. Shortly, samples were mixed with 4x Laemmli buffer (Thermo Fisher Scientific) and
heated to 95 °C before loading on 4-15%
precast TGX stain-free gels (Bio-Rad Laboratories). For Coomassie staining, gels were incubated in staining solution
@@ -2568,9 +2534,6 @@
</div>
<h5 class="fw-semi-bold">Electrophoretic Mobility Shift Assay (EMSA)</h5>
<div class="text-img">
- <a href="{{ url_for('pages', page='results') }}" class="go-to-results-wrapper">
- <img class="go-to-results" src="https://static.igem.wiki/teams/5237/gotoresults.svg" alt="Go to results button">
- </a>
<p>Purified proteins were incubated with annealed DNA oligos and loaded onto 10% TGX stain-free gels pre-equilibrated
with TBE (Bio-Rad Laboratories). Usually 0.5 µM DNA were loaded with protein ranging from 50 - 0.01 µM in a 10 µL
reaction.
@@ -2585,9 +2548,6 @@
<div class="single-exp">
<h4 class="fs-h4 fw-bold">Cell Growth and Fluorescence Assays</h4>
<div class="text-img">
- <a href="{{ url_for('pages', page='results') }}" class="go-to-results-wrapper">
- <img class="go-to-results" src="https://static.igem.wiki/teams/5237/gotoresults.svg" alt="Go to results button">
- </a>
<p>Cells were grown overnight to stationary phase and inoculated 1:100 into fresh medium, grown to OD<sub>600</sub>
0.1. In a 96-well clear flat-bottom plate (200 µL final volume), cells were incubated at 37 °C with shaking at 200
rpm. Measurements were taken every 30 minutes using a Tecan Spark plate reader (Männedorf, Switzerland) for
@@ -2600,9 +2560,6 @@
<div class="single-exp">
<h4 class="fs-h4 fw-bold">Data Analysis</h4>
<div class="text-img">
- <a href="{{ url_for('pages', page='results') }}" class="go-to-results-wrapper">
- <img class="go-to-results" src="https://static.igem.wiki/teams/5237/gotoresults.svg" alt="Go to results button">
- </a>
<p>Data were managed and cleaned using Excel, and final graphs were generated with GraphPad Prism 10.</p>
</div>
</div>
@@ -2616,7 +2573,7 @@
<div class="single-exp">
<h4 class="fs-h4 fw-bold">Cloning of pHelper_RP4 (Helper Plasmid Encoding the RP4 Conjugation Machinery)</h4>
<div class="text-img">
- <a href="{{ url_for('pages', page='results') }}" class="go-to-results-wrapper">
+ <a href="{{ url_for('pages', page='results') }}#results_delivery_system" class="go-to-results-wrapper">
<img class="go-to-results" src="https://static.igem.wiki/teams/5237/gotoresults.svg" alt="Go to results button">
</a>
<p>
@@ -2628,9 +2585,6 @@
<div class="single-exp">
<h4 class="fs-h4 fw-bold">Cloning of pmob_b (Mobilizable Plasmid for Conjugation Between Bacteria)</h4>
<div class="text-img">
- <a href="{{ url_for('pages', page='results') }}" class="go-to-results-wrapper">
- <img class="go-to-results" src="https://static.igem.wiki/teams/5237/gotoresults.svg" alt="Go to results button">
- </a>
<p>The pSC101 ori of 2374_pSC101 was swapped with p15A ori from pRep_p15A to generate pmob_b. The backbone for pmob_b was amplified via PCR from 2374_pSC101 with the primers DS3 and DS4. The p15A origin of replication (ori) was amplified via PCR from pRep_p15A with the primers DS5 and DS6. In both cases, the primers were designed to produce the necessary homology regions for subsequent utilization in Gibson Assembly.</p>
</div>
</div>
@@ -2638,9 +2592,6 @@
<div class="single-exp">
<h4 class="fs-h4 fw-bold">Cloning of pmob_m_CMV (Mobilizable Plasmid for Conjugation between Bacteria and Mammalian Cells)</h4>
<div class="text-img">
- <a href="{{ url_for('pages', page='results') }}" class="go-to-results-wrapper">
- <img class="go-to-results" src="https://static.igem.wiki/teams/5237/gotoresults.svg" alt="Go to results button">
- </a>
<p>pmob_m_CMV was cloned using four-fragment Golden Gate Assembly. The two parts of the backbone were amplified via PCR from pcDNA3.1 respectively with the primer pairs DS7, DS8 and DS11, DS12. The p15A ori was amplified via PCR from pRep_p15A with the primers DS9 and DS10. The origin of transfer (<i class="italic">oriT</i>) sequence was amplified via PCR from 2374_pSC101 with the primers DS13 and DS14. All resultant PCR amplicons possessed 5’ and 3’ extensions with BsmBI recognition sites and were designed to be assembled directionally after cleavage by BsmBI. Following gel extraction, the four fragments were then assembled via Golden Gate Assembly using BsmBI.</p>
</div>
</div>
@@ -2648,9 +2599,6 @@
<div class="single-exp">
<h4 class="fs-h4 fw-bold">Cloning of pmob_m_CEA (Mobilizable Plasmid for Conjugation Between Bacteria and Mammalian Cells)</h4>
<div class="text-img">
- <a href="{{ url_for('pages', page='results') }}" class="go-to-results-wrapper">
- <img class="go-to-results" src="https://static.igem.wiki/teams/5237/gotoresults.svg" alt="Go to results button">
- </a>
<p>pmob_m_CEA was cloned from pmob_m_CMV by exchanging the CMV-promoter with the CEA-promoter. The backbone was amplified from pmob_m_CMV via PCR with the primers DS15 and DS16. The CEA-promoter was ordered as a gene block (DSg2) and amplified via PCR with the primers DS17 and DS18. Both resultant PCR amplicons carried 5’ and 3’ extensions with BsmBI recognition sites and were designed to be assembled directionally after cleavage by BsmBI. PCR product clean-up was performed for the amplified CEA promoter and the PCR product of the pmob_m_CMV backbone was gel purified. The promoter and the backbone were then assembled via Golden Gate Assembly using BsmBI.</p>
</div>
</div>
@@ -2658,9 +2606,6 @@
<div class="single-exp">
<h4 class="fs-h4 fw-bold">Cloning of pNeae2_7D12 (for Surface Expression of Synthetic Adhesin Against EGFR on <i class="italic">E. coli</i> 10-beta)</h4>
<div class="text-img">
- <a href="{{ url_for('pages', page='results') }}" class="go-to-results-wrapper">
- <img class="go-to-results" src="https://static.igem.wiki/teams/5237/gotoresults.svg" alt="Go to results button">
- </a>
<p>The cloning of pNeae2_7D12 will be done by two-fragment Gibson Assembly using pNeae2 (addgene #168300) as the backbone. The backbone will be amplified using the primers - DS19 and DS20. The gene block encoding wt 7D12 adhesin (DSg3) will be amplified using the primers - DS21 and DS22. The PCR amplicons are designed to possess the necessary homology regions for Gibson Assembly. Following clean-up, the PCR amplicons will then be assembled via Gibson Assembly.</p>
</div>
</div>
@@ -2668,9 +2613,6 @@
<div class="single-exp">
<h4 class="fs-h4 fw-bold">Solid Media Bacterial Conjugation Assay</h4>
<div class="text-img">
- <a href="{{ url_for('pages', page='results') }}" class="go-to-results-wrapper">
- <img class="go-to-results" src="https://static.igem.wiki/teams/5237/gotoresults.svg" alt="Go to results button">
- </a>
<p>In preparation for the conjugation assay, the corresponding plasmids were transformed into respective chemically competent E. coli strains and plated on appropriate antibiotic-containing LB agar plates to create the necessary donor and recipient cells. Single colonies were picked and 5 ml overnight cultures were set up in LB medium supplemented with the appropriate antibiotics.</p>
<p>The experimental design involved a positive control, two negative controls, and a test group, as outlined in Table 4 below. In all cases, the recipients were made to carry a plasmid conferring resistance against ampicillin (pAmpR) in order to enable selection against donors. </p>
@@ -2715,9 +2657,6 @@
<div class="single-exp">
<h4 class="fs-h4 fw-bold">Liquid Media Bacterial Conjugation Assay</h4>
<div class="text-img">
- <a href="{{ url_for('pages', page='results') }}" class="go-to-results-wrapper">
- <img class="go-to-results" src="https://static.igem.wiki/teams/5237/gotoresults.svg" alt="Go to results button">
- </a>
<p>Bacteria from the same clones used for conjugation on solid media were used for conjugation on liquid media. The same steps as for solid media conjugation were followed until the first resuspension step. The first resuspension was performed in 1 ml of antibiotic-free LB medium, followed by OD<sub>600</sub> adjustment (to 10). For each experiment group, 120 µl of the corresponding OD-adjusted liquid cultures were mixed together and then centrifuged for 1 min at 11,000 rpm. After resuspension of the pellets in 1.2 ml of LB medium each, 1 ml of cell suspension from each experiment group was pipetted into a well of a 12-well plate and incubated at 37 °C for 18 hours without shaking. After incubation, the bacterial suspensions were removed from the wells and their OD<sub>600</sub> was adjusted to 2.4 using the diluent. Serial dilutions were prepared and plated on selective agar plates (same as for solid media conjugation). Three technical replicates were used for the test group. The plates were then incubated overnight at 37 °C.</p>
<p>In both solid and liquid media conjugation assays, following the last overnight incubation step, the conjugation efficiency was calculated by counting colonies at a certain dilution on the ampicillin+kanamycin plate (selecting for transconjugants) and dividing them by the number of colonies on the corresponding ampicillin plate (selecting for total number of recipients).</p>
</div>
diff --git a/wiki/pages/results.html b/wiki/pages/results.html
index 6e1c6050b343f928f82613a81a50faa5228c22f3..f58df5e7155a299aabf603e2ad614e32d316e872 100644
--- a/wiki/pages/results.html
+++ b/wiki/pages/results.html
@@ -277,7 +277,7 @@
DNA loci into proximity. </p>
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- <h3 class="fs-h3 fw-semi-bold mt-5">Results</h3>
+ <h3 class="fs-h3 fw-semi-bold mt-5" id="cas_results_wetlab">Results</h3>
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With these approaches, we aim to induce structural and functional changes in protein-stapled DNA selectively in cancerous tissue.</p>
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- <h3 class="fs-h3 fw-semi-bold mt-5">Results</h3>
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</p>
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- <h3 class="fs-h3 fw-semi-bold mt-5">Results</h3>
+ <h3 class="fs-h3 fw-semi-bold mt-5" id="results_fret_measurment_wetlab">Results</h3>
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- <h2 class="fs-h2 fw-semi-bold">Delivery System</h2>
+ <h2 class="fs-h2 fw-semi-bold" id="results_delivery_system">Delivery System</h2>
<p class="mt-3 mb-3">As part of the PICasSO toolbox, we propose <b>bacterial conjugation</b> as a <b>cheap, simple and scalable alternative</b> to conventional <b>DNA delivery methods</b>, particularly suited for large plasmid constructs <b>encoding protein-based staples for controlled modulation of mammalian 3D genome organization</b>.
Bacterial conjugation is one of the key mechanisms for <b>horizontal gene transfer</b> between bacteria on solid media. We validated the proficiency of the <b>RP4 conjugative machinery</b> in mediating conjugation between bacteria on solid media. Future experiments will expand the <b>DNA delivery capabilites</b> of the <b>Type IV secretion system</b> to mammalian cells by employing <b>synthetic adhesins</b> to enhance <b>cell-cell contact.</b> </p>
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diff --git a/wiki/pages/safety.html b/wiki/pages/safety.html
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<h3 class="fs-h3 fw-semi-bold mt-4">The PICasSO System: Identifying Possible Risks</h3>
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- <p class="mt-3 mb-3"> After consultations with experts, we came to the conclusion that our project poses <b>minimal risk</b>. However, bringing two DNA sequences into close proximity <i class="italic">in vivo</i>, especially if one of them is an enhancer, poses potential risks, such as carcinogenesis and cell dysfunction. Additionally, off-target effects and the incomplete understanding of 3D genome organization make it challenging to fully assess all possible outcomes.
+ <p class="mt-3 mb-3"> After consultations with experts, we came to the conclusion that our project poses <b>minimal risk</b>. However, bringing two DNA sequences into close proximity <i class="italic">in vivo</i>, especially if one of them is an enhancer, poses potential risks, such as carcinogenesis and cell dysfunction. Additionally, off-target effects and the incomplete understanding of 3D genome organization make it challenging to fully assess all possible outcomes. Of note, these risks would only apply to applications in animal models or even patients and are of no danger in context of cell and tissue culture experiments.
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<h3 class="fs-h3 fw-semi-bold mt-4">The PICasSO System: How We Eliminate These Risks</h3>
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