|
|
Line 117: |
Line 117: |
| </li> | | </li> |
| </ul> | | </ul> |
− | <p>*Next to the rbGlob polyA (upstream) there is a sequence coding for a synthetic miRNA, targeting a region of firefly luciferase [<a href='#ref11_pd'>11</a>]. Named FF4, it is a <strong>posttranscriptional repressor</strong>.</p> | + | <p>*Next to the rbGlob polyA (upstream) there is a sequence coding for a synthetic miRNA, targeting a region of firefly luciferase [<a href='#ref11_pd'>11</a>]. It is a <strong>post-transcriptional repressor</strong>, named FF4.</p> |
| <p>As a result, using these <a href='https://2017.igem.org/Team:Greece/Basic_Part'>Basic Parts</a>, a large number of circuit topologies can be envisioned.</p> | | <p>As a result, using these <a href='https://2017.igem.org/Team:Greece/Basic_Part'>Basic Parts</a>, a large number of circuit topologies can be envisioned.</p> |
| </div> | | </div> |
Line 162: |
Line 162: |
| <p>Analytically, <a href='http://parts.igem.org/Part:BBa_K515105'>BBa_K515105</a> consists of superfolder GFP (sfGFP), a very brightly fluorescent protein under the control of the bacterial constitutive promoter J23100 and is used as a reporter to simplify the validation process during cloning. </p> | | <p>Analytically, <a href='http://parts.igem.org/Part:BBa_K515105'>BBa_K515105</a> consists of superfolder GFP (sfGFP), a very brightly fluorescent protein under the control of the bacterial constitutive promoter J23100 and is used as a reporter to simplify the validation process during cloning. </p> |
| <div style='text-align:center'><img class='sub_images' style='width: 65%; height: 700px' src='https://static.igem.org/mediawiki/2017/4/4d/Greekom_Design_Petri40.jpeg' /></div> | | <div style='text-align:center'><img class='sub_images' style='width: 65%; height: 700px' src='https://static.igem.org/mediawiki/2017/4/4d/Greekom_Design_Petri40.jpeg' /></div> |
− | <p>It is flanked by two recognition sites for BbsI, a type IIS restriction enzyme and two annealing sites for a universal M13 forward & reverse primer. As type IIS restriction enzymes recognize asymmetric DNA sequences and cleave outside of their recognition sequence, they are central to our approach for fusing miRNA target sequences into the 3’-untranslated region, as described in the next section. Examples of these constructs include:</p> | + | <p>BBa_K515105 is flanked by two recognition sites for BbsI, a type IIS restriction enzyme and two annealing sites for a universal M13 forward & reverse primer. As type IIS restriction enzymes recognize asymmetric DNA sequences and cleave outside of their recognition sequence, they are central to our approach for fusing miRNA target sequences into the 3’-untranslated region, as described in the next section. Examples of these constructs include:</p> |
| <table style='text-align:center'> | | <table style='text-align:center'> |
| <tr><td><img class='sub_images' src='https://static.igem.org/mediawiki/2017/5/58/Greekom_Design_Composite1.png' /></td></tr> | | <tr><td><img class='sub_images' src='https://static.igem.org/mediawiki/2017/5/58/Greekom_Design_Composite1.png' /></td></tr> |
Line 212: |
Line 212: |
| <section class='sub_sections'> | | <section class='sub_sections'> |
| <div style='text-align: justify;'> | | <div style='text-align: justify;'> |
− | <p>Type I pilli, surface rod-shaped organelles 7nm in diameter and 1μm in length, are the best studied system of bacterial adhesion [<a href='#ref15_pd'>15</a>].They are heteropolymers of four proteins with FimA being the main structural protein of the pilli, which polymerizes approximately 1000 times to form a right-handed helix that constitutes the main axis of the structure and includes smaller concentrations of FimG, FimF and FimH [<a href='#ref16_pd'>16-18</a>]. FimH is the functional component of the structure as it alone confers the ability to bind to a-D-mannose of various eukaryotic cells and is located at the tip and the shafts of the pilus, whereas FimF and FimG seem to be responsible for docking FimH to FimA [<a href='#ref19_pd'>19-20</a>]. To achieve selective adhesion to colorectal cancer cells using type I pilli we need to disrupt their natural ability to bind to a-D-mannose and introduce a mechanism to facilitate adhesion to CRC cells by a mannose-independent mechanism. A mutation in the 49th amino acid of FimH has been demonstrated to completely abolish mannose binding [<a href='#ref21_pd'>21</a>]. In addition, a small peptide called RPMrel has been identified through phage display assays due to its ability to bind to five different colorectal cancer cell lines as well as cancerous tissues obtained by biopsies and not to other kinds of cancer [<a href='#ref22_pd'>22</a>]. Taken together these two modifications perform both the functions specified for CRC selective binding and have been successfully used by previous iGEM teams, iGEM Harvard 2015 and iGEM Ankara 2016 to that end. We employed the same part <a href=’http://parts.igem.org/Part:BBa_K1850011’>BBa_K1850011</a> that was submitted by iGEM Harvard 2015, in a fimH KO strain. Having achieved selective adhesion to colorectal cancer cells we move on to the second half of our device, internalization and transference of genetic material. Strains of the bacteria E. coli can be modified so that they will express two key proteins : invasin and listeriolysin O [<a href='#ref23_pd'>23-24</a>]. Invasin gives the bacteria, the ability to enter epithelial and other non phagocytic cells [<a href='#ref25_pd'>25-26</a>]. Listeriolysin O, on the other hand, has to do with what happens to the bacteria after they enter the target-cell. This particular protein allows the bacteria to free themselves of the vesicle that was used for their phagocytosis, without damaging the plasmatic/cell membrane of the target-cell. This happens due to the low pH of the vesicle (~5.9-6) that is also the optimal pH range for the protein listeriolysin [<a href='#ref27_pd'>27-28</a>]. The modified strains of <i>E. coli</i>, through expressing these proteins are able to not only enter non phagocytic target-cells that express b1-integrins but also to transfer their load to them, through escaping the phagolysosome [<a href='#ref23_pd'>23</a>]. Finally, we aim to put invasin and listeriolysin O under quorum control, through the use of the <i>lux</i> genetic circuit of <i>Vibrio fischeri</i>, as this operon has been utilized to achieve cell-density dependent invasion. During our communication with the safety committee, we were extremely glad to hear that we submitted a thorough and analytical Check In form and the Committee Members advised us to focus on the safe implementation of our classifier module and then consider a transfer method. As a result, we switched our focus and put great effort to characterize the components of our classifier to demonstrate the while integrating feedback from the scientific community about the health risks of our conceptual anticancer agent, formulating what we term, a <strong>5-STAR security system</strong>, which represents the culmination of our proposed modifications:</p> | + | <p>Type I pilli, surface rod-shaped organelles of 7nm in diameter and 1μm in length, are the best studied system of bacterial adhesion [<a href='#ref15_pd'>15</a>].They are heteropolymers of four proteins with FimA being the main structural protein of the pilli, which polymerizes approximately 1000 times to form a right-handed helix that constitutes the main axis of the structure and includes smaller concentrations of FimG, FimF and FimH [<a href='#ref16_pd'>16-18</a>]. FimH is the functional component of the structure as it alone confers the ability to bind to a-D-mannose of various eukaryotic cells and is located at the tip and the shafts of the pilus, whereas FimF and FimG seem to be responsible for docking FimH to FimA [<a href='#ref19_pd'>19-20</a>]. To achieve selective adhesion to colorectal cancer cells using type I pilli we need to disrupt their natural ability to bind to a-D-mannose and introduce a mechanism to facilitate adhesion to CRC cells by a mannose-independent mechanism. A mutation in the 49th amino acid of FimH has been demonstrated to completely abolish mannose binding [<a href='#ref21_pd'>21</a>]. In addition, a small peptide called RPMrel has been identified through phage display assays due to its ability to bind to five different colorectal cancer cell lines as well as cancerous tissues obtained by biopsies and not to other kinds of cancer [<a href='#ref22_pd'>22</a>]. Taken together these two modifications perform both the functions specified for CRC selective binding and have been successfully used by previous iGEM teams, iGEM Harvard 2015 and iGEM Ankara 2016 to that end. We employed the same part <a href=’http://parts.igem.org/Part:BBa_K1850011’>BBa_K1850011</a> that was submitted by iGEM Harvard 2015, in a fimH KO strain. Having achieved selective adhesion to colorectal cancer cells we move on to the second half of our device, internalization and transference of genetic material. Strains of the bacteria E. coli can be modified so that they will express two key proteins : invasin and listeriolysin O [<a href='#ref23_pd'>23-24</a>]. Invasin gives the bacteria, the ability to enter epithelial and other non phagocytic cells [<a href='#ref25_pd'>25-26</a>]. Listeriolysin O, on the other hand, has to do with what happens to the bacteria after they enter the target-cell. This particular protein allows the bacteria to free themselves of the vesicle that was used for their phagocytosis, without damaging the plasmatic/cell membrane of the target-cell. This happens due to the low pH of the vesicle (~5.9-6) that is also the optimal pH range for the protein listeriolysin [<a href='#ref27_pd'>27-28</a>]. The modified strains of <i>E. coli</i>, through expressing these proteins are able to not only enter non phagocytic target-cells that express b1-integrins but also to transfer their load to them, through escaping the phagolysosome [<a href='#ref23_pd'>23</a>]. Finally, we aim to put invasin and listeriolysin O under quorum control, through the use of the <i>lux</i> genetic circuit of <i>Vibrio fischeri</i>, as this operon has been utilized to achieve cell-density dependent invasion. During our communication with the safety committee, we were extremely glad to hear that we submitted a thorough and analytical Check In form and the Committee Members advised us to focus on the safe implementation of our classifier module and then consider a transfer method. As a result, we switched our focus and put great effort to characterize the components of our classifier to demonstrate the while integrating feedback from the scientific community about the health risks of our conceptual anticancer agent, formulating what we term, a <strong>5-STAR security system</strong>, which represents the culmination of our proposed modifications:</p> |
| <strong> | | <strong> |
| Level 1:</br> | | Level 1:</br> |