Difference between revisions of "Template:BOKU-Vienna/Description"

(Created page with "<h1 id="project-description">Project Description</h1> <p>Heterogeneity of cell populations caused by quorum sensing leads to variability in gene expression that is hard to pre...")
 
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<h1 id="project-description">Project Description</h1>
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<h1 id="project-description-">Project Description:</h1>
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<p>Heterogeneity of cell populations caused by quorum sensing leads to variability in gene expression that is hard to predict. During biomanufacturing, elevating quorum-induced protein expression will lead to gain of profit. Decreasing this expression can also be beneficial in situations where undesirable biofilms may form on medical equipment or controlling virulence in bacteria. We will modify the existing bacterial quorum sensing system controlled by the Lsr operon by upregulating the synthesis and excretion of Autoinducer-2, a universal quorum molecule. This will increase population-scale AI-2 intake and phosphorylation after the initial AI-2 threshold concentrations have been reached to reduce variability in induced gene expression.</p>
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<h2 id="the-system-is-based-on-a-dcas9-toggle-switch-">The system is based on a dCas9 toggle switch.</h2>
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<p>A model is used to predict the impacts of manipulating the expression of quorum sensing genes and guide design and experimentation. DNA assembly of a Biobricks containing pLsr, T7 RNA Polymerase, LsrK, LsrACDB, LuxS, YdgG, and superfolding Green Fluorescent Protein (sfGFP) enables both the enhancement of natural quorum sensing and the quantification of protein activation among the bacteria in a colony. This device will improve the viability of autoinduction as an induction method in industries such as biomanufacturing by decreasing the variability of a cell phenotypes and increasaing expression within a single culture to reduce costs leading to an increase in profits. In addition, by insertion of T7 polymerase into a quorum-sensitive region of the genome of E. coli, we will create a chassis with specific, customizable quorum response for engineering and scientific applications.</p>
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<p>The ON and OFF gene classes comprise a strong constitutive promoter with an RNA polymerase II binding site and multiple copies of the same gRNA target site downstream next to it.
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All genes in a class are controlled by the same regulatory region, meaning their gene products are expressed simultaneously.
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At least one gene in each class codes for an RNA construct consisting of a gRNA sequence, flanked by two autonomously folding catalytic RNA sequences, which lead to the production of free gRNA when transcribed.
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Other genes in the ON genes class code for proteins which should be only expressed in the ON state of the system. Vice versa OFF genes encode for OFF state proteins.
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In theory there could be any number and combinations of genes which can define one of the states.
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However, we will only use one additional ON gene and no additional OFF gene.
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The ON gene contains coding sequence for a short lived GFP and will therefore be used as proof of concept.</p>
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<p>Our system has the ability to start or stop the expression of a gene of interest.
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By simply getting activated with the help of a signal molecule once, the state (ON or OFF) will sustain indefinitely, just like a lightbulb.
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This system is based on a so-called toggle-switch which consists of two NOR-gates that use an integrated dCas9 System, where specific guide RNAs bind to a DNA strand and thereby block the transcription.
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<em>toggle switch hier hin</em></p>
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<p>A NOR-gate is the result of the negation of the OR operator, which behaves according to the following truth table.</p>
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<table>
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<tr>
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<th colspan=2>Input</th>
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<th>Output</th>
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</tr>
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<tr>
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<td>A</td>
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<td>B</td>
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<td>A NOR B</td>
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</tr>
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<tr>
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<td>0</td>
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<td>0</td>
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<td>1</td>
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</tr>
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<tr>
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<td>1</td>
 +
<td>0</td>
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<td>0</td>
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</tr>
 +
<tr>
 +
<td>0</td>
 +
<td>1</td>
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<td>0</td>
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</tr>
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<tr>
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<td>1</td>
 +
<td>1</td>
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<td>0</td>
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</tr>
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</table>
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<p>As signals to control the toggle-switch we designed a copper, ethanol and estradiol pathway.</p>
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<p>However, neither of those would work well in agricultural applications.
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Choosing and designing these signal-molecules will be a very important step in future developments of this system, because they should not interfere with the ecosystem.
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As a reporter system for proof of concept in the laboratory we will use a short-lived ubiquitin
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GFP with a half-life of 7 minutes.</p>

Revision as of 09:45, 15 October 2018

Project Description:

The system is based on a dCas9 toggle switch.

The ON and OFF gene classes comprise a strong constitutive promoter with an RNA polymerase II binding site and multiple copies of the same gRNA target site downstream next to it. All genes in a class are controlled by the same regulatory region, meaning their gene products are expressed simultaneously. At least one gene in each class codes for an RNA construct consisting of a gRNA sequence, flanked by two autonomously folding catalytic RNA sequences, which lead to the production of free gRNA when transcribed. Other genes in the ON genes class code for proteins which should be only expressed in the ON state of the system. Vice versa OFF genes encode for OFF state proteins. In theory there could be any number and combinations of genes which can define one of the states. However, we will only use one additional ON gene and no additional OFF gene. The ON gene contains coding sequence for a short lived GFP and will therefore be used as proof of concept.

Our system has the ability to start or stop the expression of a gene of interest. By simply getting activated with the help of a signal molecule once, the state (ON or OFF) will sustain indefinitely, just like a lightbulb. This system is based on a so-called toggle-switch which consists of two NOR-gates that use an integrated dCas9 System, where specific guide RNAs bind to a DNA strand and thereby block the transcription. toggle switch hier hin

A NOR-gate is the result of the negation of the OR operator, which behaves according to the following truth table.

Input Output
A B A NOR B
0 0 1
1 0 0
0 1 0
1 1 0

As signals to control the toggle-switch we designed a copper, ethanol and estradiol pathway.

However, neither of those would work well in agricultural applications. Choosing and designing these signal-molecules will be a very important step in future developments of this system, because they should not interfere with the ecosystem. As a reporter system for proof of concept in the laboratory we will use a short-lived ubiquitin GFP with a half-life of 7 minutes.