Difference between revisions of "Team:Vilnius-Lithuania/Description"
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− | <p style="border-left: 5px solid rgb(236,151,31); padding-left: 3px; border-bottom: none"> | + | <p style="border-left: 5px solid rgb(236,151,31); padding-left: 3px; border-bottom: none"><strong>Figure 1.</strong> The CRISPR-Cas system. Cascade protein complex use crRNA to recognize target DNA sequence. Cas3 protein binds to the single stranded DNA and cleaves it.</p> |
<p class="text-justify">We use this bacterial protection CRISPR-Cas system to create a method by which we could programme the life of genetically modified bacteria. One of the main goals of our project is regulating a specific number of life cycles that genetically altered bacteria could go through, when it is released into natural wildlife conditions. This is Coliclock – an automatic timer, which starts when you set the bacteria free to do its function.</p> | <p class="text-justify">We use this bacterial protection CRISPR-Cas system to create a method by which we could programme the life of genetically modified bacteria. One of the main goals of our project is regulating a specific number of life cycles that genetically altered bacteria could go through, when it is released into natural wildlife conditions. This is Coliclock – an automatic timer, which starts when you set the bacteria free to do its function.</p> | ||
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− | <p style="border-left: 5px solid rgb(236,151,31); padding-left: 3px; border-bottom: none"> | + | <p style="border-left: 5px solid rgb(236,151,31); padding-left: 3px; border-bottom: none"><strong>Figure 2.</strong> System function differences, when bacteria are in the lab (above) and when in the environment (below). In the lab repressor cI is synthesized, because of presence of IPTG, and pLux/cI promoter is repressed - Cd and Cas3 are not transcribed. In the environment IPTG does not occur, so repressor cI is not synthesized - Cd and Cas are transcribed.</p> |
<p class="text-justify">This is the point at which our system switches from laboratory mode into wildlife mode. Once the concentration of cI repressor reaches a minor treshold, the transcription of LuxR and LuxI begins. At first it is not very indicative, but through the positive loop expression increases dramatically. </p> | <p class="text-justify">This is the point at which our system switches from laboratory mode into wildlife mode. Once the concentration of cI repressor reaches a minor treshold, the transcription of LuxR and LuxI begins. At first it is not very indicative, but through the positive loop expression increases dramatically. </p> | ||
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− | <p style="border-left: 5px solid rgb(236,151,31); padding-left: 3px; border-bottom: none"> | + | <p style="border-left: 5px solid rgb(236,151,31); padding-left: 3px; border-bottom: none"><strong>Figure 3.</strong> Positive loop. Positive loop occurs, when LuxR and LuxI interacts with each other and increase transcription of itselves.</p> |
<p class="text-justify">The product of LuxI sequence is HL (homoserine lactone, which, together with the LuxR protein, acts as an activation complex for pLux/cl promoters. This creates a positive loop of expression – the first transcribed proteins join into a complex, which activates the further transcription of the same proteins. The genes within these promoters are intensively transcribed. We call this whole construct the "switch-on" of the functional Coliclock system subunit.</p> | <p class="text-justify">The product of LuxI sequence is HL (homoserine lactone, which, together with the LuxR protein, acts as an activation complex for pLux/cl promoters. This creates a positive loop of expression – the first transcribed proteins join into a complex, which activates the further transcription of the same proteins. The genes within these promoters are intensively transcribed. We call this whole construct the "switch-on" of the functional Coliclock system subunit.</p> | ||
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<img src="https://static.igem.org/mediawiki/2015/1/11/Vilnius15_plazmides1.png" style="width: 500px; " /> | <img src="https://static.igem.org/mediawiki/2015/1/11/Vilnius15_plazmides1.png" style="width: 500px; " /> | ||
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− | <p style="border-left: 5px solid rgb(236,151,31); padding-left: 3px; border-bottom: none"> | + | <p style="border-left: 5px solid rgb(236,151,31); padding-left: 3px; border-bottom: none"><strong>Figure 4.</strong> Regulatory unit. This unit consists RBS, pLux/cI and pLac promoter, LuxR, LuxI and cI. This unit is essential for system regulation.</p> |
<p class="text-justify">Once the functional subunit is switched on and the transcription of functional subunit genes is present, Cascade, together with the crRNA molecules, forms a big ribonucleoprotein complex, which targets DNA polymerase III and RNA polymerase genes. They are essential for the survival of bacteria. Those genes are later cleaved by the cas3 nuclease. </p> | <p class="text-justify">Once the functional subunit is switched on and the transcription of functional subunit genes is present, Cascade, together with the crRNA molecules, forms a big ribonucleoprotein complex, which targets DNA polymerase III and RNA polymerase genes. They are essential for the survival of bacteria. Those genes are later cleaved by the cas3 nuclease. </p> | ||
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− | <p style="border-left: 5px solid rgb(236,151,31); padding-left: 3px; border-bottom: none"> | + | <p style="border-left: 5px solid rgb(236,151,31); padding-left: 3px; border-bottom: none"><strong>Figure 5.</strong> Functional unit. Three types of functional unit are different from each other, because of RBS (weak, medium, strong). Also Cascade (Cd), Cas3 and crRNA are in this regulatory unit. </p> |
<p class="text-justify">In our project, we aimed to regulate the amount of cell divisions that a cell can make, in other words, we wanted to control the start of a functional system unit. To achieve this, we cloned different strength RBS (ribosome binding site) sequences in front of each CRISPR-Cas gene. According to these RBS sequences, the expression of CRISPR-Cas genes either is stronger or weaker, which reflects on bacterial lifetime: the stronger RBS sequence, the bigger CRISPR gene expression, the shorter bacterial lifetime. Regulatory and functional units were cloned in parallel. Regulatory unit was cloned from Standart Assembly Biobricks, sent from iGEM Headquarters, while functional units were cloned from <i>A. actinomycetemcomitans</i> by amplifying the desired genes by using PCR.</p> | <p class="text-justify">In our project, we aimed to regulate the amount of cell divisions that a cell can make, in other words, we wanted to control the start of a functional system unit. To achieve this, we cloned different strength RBS (ribosome binding site) sequences in front of each CRISPR-Cas gene. According to these RBS sequences, the expression of CRISPR-Cas genes either is stronger or weaker, which reflects on bacterial lifetime: the stronger RBS sequence, the bigger CRISPR gene expression, the shorter bacterial lifetime. Regulatory and functional units were cloned in parallel. Regulatory unit was cloned from Standart Assembly Biobricks, sent from iGEM Headquarters, while functional units were cloned from <i>A. actinomycetemcomitans</i> by amplifying the desired genes by using PCR.</p> |
Latest revision as of 11:58, 18 September 2015