Difference between revisions of "Team:Tel-Hai/Parts"

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<h3>Description</h3>
 
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<div class="description">
 
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<div class="image-holder"><img src="/wiki/images/8/86/T--Tel-Hai--des1.png" /></div>
 
  
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<h3>Cystic fibrosis (CF)</h3>
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<h3>Parts</h3>
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<p>CF is a genetic disorder that affects mostly the lungs, but also the pancreas, liver, kidneys, and intestine. Long-term issues include difficulty breathing and coughing up mucus as a result of frequent lung infections. Other signs and symptoms include sinus infections, poor growth, fatty stool, clubbing of the fingers and toes, and infertility in males, among others. </p>
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<p>CF is caused by a mutation in the gene cystic fibrosis transmembrane conductance regulator (CFTR). The most common mutation, ΔF508, is a deletion (Δ signifying deletion) of three nucleotides that results in a loss of the amino acid phenylalanine (F) at the 508th position on the protein. This mutation accounts for two-thirds (66–70%) of CF cases worldwide and 90% of cases in the United States; however, over 1500 other mutations can produce CF.</p>
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<div class="image-holder"><img src="/wiki/images/6/63/T--Tel-Hai--des2.png" /></div>
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<p>There is no known cure for Cystic Fibrosis. Lung infections are treated with antibiotics which may be given intravenously, inhaled, or orally. People with the disease take between 50-80 medications per day, and on a daily basis partake in physical therapy in order to facilitate the lungs, inhalation treatments to relieve respiratory distress, and are hospitalized every couple of months.</p>
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<p>Over the years, many drugs have been developed to help patients cope with symptoms and increase life expectancy. However, survival age is still only 40 years, as of 2015. In other words, these people are born with the knowledge that they will not have a long life.</p>
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<div class="image-holder"><img src="/wiki/images/7/79/T--Tel-Hai--des3.png" /></div>
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<p><strong>Our group has focused on solving the root cause i.e. correcting the CFTR mutation. Thus, we were looking for gene editing technology.</strong></p>
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<h3>CRISPR / Cas9</h3>
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<h4><a href="http://parts.igem.org/Part:BBa_k2061000">Part 1 - BBa_K2061000 tGFP ORF under EF1a promoter</a></h4>
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<p>The CRISPR/Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements and provides a form of acquired immunity. CRISPR associated proteins (Cas) use the CRISPR spacers to recognize and cut these exogenous genetic elements in a manner analogous to RNA interference in eukaryotic organisms.</p>
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<p>This part contains the GFP gene under the human Elongation Factor-1 alpha promoter, which is a constitutive promoter. This part enables the expression of fluorescent protein in mammalian cells. The part was cloned in pSB1C3 between EcoRI and XbaI sites. The reason we did not use the PstI site for cloning is because there are 2 PstI sites within the EF1a promoter sequence and 2 PstI sites inside the tGFP ORF.</p>
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<p>By delivering the Cas9 nuclease and appropriate guide RNAs into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added by homologous recombination resulting the correction of CFTR mutation.</p>
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<p>The idea is to expose lung epithelial cells to the cross-linked complex (the cholera toxin B subunit and this plasmid) and to look for GFP expression in the cells. This will indicate the insertion of the complex and expression from the plasmid.</p>
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<div class="image-holder"><img src="/wiki/images/7/79/T--Tel-Hai--des4.png" /></div>
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<p><strong>Next, our group though about a way to deliver CRISPR/Cas9 into the mutated cells.</strong></p>
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<h4><a href="http://parts.igem.org/Part:BBa_k2061001">PartI 2 -BBa_K2061001 CFTR gene exon 11 – donor part</a></h4>
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<p>This part contains the sequence of CFTR gene exon 11 with 400 bp of intron 10 and 340 bp of intron 11 on the flanking regions. The part was cloned in pSB1C3 between EcoRI and PstI sites. This part will serve as a donor sequence for gene editing of ΔF508 mutation in exon 11 of CFTR.</p>
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<h4><a href="http://parts.igem.org/Part:BBa_k2061002">Part 3 - BBa_K2061002 gRNA1 for editing F508 deletion</a></h4>
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<p>This part contains 20 bp of target sequence in CFTR exon 11, PAM sequence (NGG) and the gRNA scaffold under the control of U6 promoter and terminator. The part was cloned in pSB1C3 between EcoRI and PstI sites. This part will serve for generating the first gRNA leading the CAS9 to the CFTR exon 11 for gene editing.</p>
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<h4><a href="http://parts.igem.org/Part:BBa_k2061003">Part 4 - BBa_K2061003 gRNA2 for editing F508 deletion</a></h4>
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<p>This part contains 20 bp of target sequence in CFTR exon 11, PAM sequence (NGG) and the gRNA scaffold under the control of U6 promoter and terminator. The part was cloned in pSB1C3 between EcoRI and PstI sites. This part will serve for generating the second gRNA leading the CAS9 to the CFTR exon 11 for gene editing.</p>
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<h4><a href="http://parts.igem.org/Part:BBa_K2061005">Part 5 - BBa_K2061005</a></h4>
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<p>This part recombinant B-subunit of the E. coli Heat Labile Toxin (LTB)  fused via linker sequence to a short viral DNA binding domain (DBD). The part was cloned in pSB1C3 between EcoRI and XbaI sites. This part coded to a protein that can attached to a desired DNA cargo to be deliver to epithelial cell nucleus via endocytosis.</p>
  
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<h3>Cholera Toxin (CT)</h3>
 
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<p>CT is composed of two molecular subunits: Cholera toxin subunit A (CTA) and B (CTB). CTA is responsible for the toxic effect, whereas the non-toxic subunit B is responsible for the internalization and transport of the toxin into the cell. </p>
 
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<p>CTB is highly specific to epithelial cells (those are the mutated cells in CF). With its ability to bind the ganglioside receptor (GM1) in the epithelial cell membrane, we believe it will becomes a successful carrier for introducing the plasmids of the CRISPR in to the cells.</p>
 
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<div class="image-holder"><img src="/wiki/images/5/52/T--Tel-Hai--des5.png" /></div>
 
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<p><strong>In this project, we are combining these three elements together to develop a delivery system that can cure Cystic Fibrosis with a single treatment.</strong></p>
 
 
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Revision as of 11:30, 19 October 2016

iGEM Tel-Hai 2016

Parts

Part 1 - BBa_K2061000 tGFP ORF under EF1a promoter

This part contains the GFP gene under the human Elongation Factor-1 alpha promoter, which is a constitutive promoter. This part enables the expression of fluorescent protein in mammalian cells. The part was cloned in pSB1C3 between EcoRI and XbaI sites. The reason we did not use the PstI site for cloning is because there are 2 PstI sites within the EF1a promoter sequence and 2 PstI sites inside the tGFP ORF.

The idea is to expose lung epithelial cells to the cross-linked complex (the cholera toxin B subunit and this plasmid) and to look for GFP expression in the cells. This will indicate the insertion of the complex and expression from the plasmid.

PartI 2 -BBa_K2061001 CFTR gene exon 11 – donor part

This part contains the sequence of CFTR gene exon 11 with 400 bp of intron 10 and 340 bp of intron 11 on the flanking regions. The part was cloned in pSB1C3 between EcoRI and PstI sites. This part will serve as a donor sequence for gene editing of ΔF508 mutation in exon 11 of CFTR.

Part 3 - BBa_K2061002 gRNA1 for editing F508 deletion

This part contains 20 bp of target sequence in CFTR exon 11, PAM sequence (NGG) and the gRNA scaffold under the control of U6 promoter and terminator. The part was cloned in pSB1C3 between EcoRI and PstI sites. This part will serve for generating the first gRNA leading the CAS9 to the CFTR exon 11 for gene editing.

Part 4 - BBa_K2061003 gRNA2 for editing F508 deletion

This part contains 20 bp of target sequence in CFTR exon 11, PAM sequence (NGG) and the gRNA scaffold under the control of U6 promoter and terminator. The part was cloned in pSB1C3 between EcoRI and PstI sites. This part will serve for generating the second gRNA leading the CAS9 to the CFTR exon 11 for gene editing.

Part 5 - BBa_K2061005

This part recombinant B-subunit of the E. coli Heat Labile Toxin (LTB) fused via linker sequence to a short viral DNA binding domain (DBD). The part was cloned in pSB1C3 between EcoRI and XbaI sites. This part coded to a protein that can attached to a desired DNA cargo to be deliver to epithelial cell nucleus via endocytosis.