Team:Peking/Project/Luminesensor/Characterization

From 2012.igem.org

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  <h3 class="title1">Sensitivity</h3>
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  <h3 id="title1">Sensitivity</h3>
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  <p><b>Why high sensitivity & our luminesensor sensitivity</b><br/>We developed a light-switchable system that provides a robust and convenient way to control gene expression, and can be used to manipulate many biological processes in living systems with minimal perturbation on the basis of our innovative fusion protein.<br/><br/>Our light-switchable system has incomparable advantages and potentials not only because of the features we have discussed in the ‘motivation’ section of our project overview, but also because the system presents a higher level of sensitivity that researchers have never put into effect before. By taking advantage of this high level of sensitivity, we can easily accomplish the goal of controlling gene expression using light without the need for an extra energy source.<br/><br/>There are two critical reasons for why we endeavored ourselves into establishing such a highly sensitive Luminesensor. Firstly, sensitivity is one of the most critical evaluation criteria in optogenetics for the reason that the modularized systems can function under low intensity light, like natural light, as the sensor possess a high sensitivity with the purpose of minimizing the injury to the cells which possibly will be seriously affected by high intensity light exposure in the meantime maximizing the exploiting of natural light. According to information collected in the past decade, only about 25% of experimental results have achieved the goal of controlling gene expression by optogenetic approaches with the light intensity under 1W/m<sup>2</sup>, which is the exact equivalent of natural light, and is our greatest concern due to the availability and user-friendly characteristic of natural light (Figure 1:Optogenetic Approaches to Control Gene Expression under different light sensitivity):</p>
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  <p>
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<b>1. Set-up & Brief Procedure</b><br/>
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We tested the sensitivity of the <i>Luminesensor</i> by examining the light-dependent  transcriptional activity of a GFP-ssrA reporter. ssrA is a protein tag that induces fast degradation of protein, which in our case facilitated the observation of transcriptional activity. Based on the consideration of guaranteed accuracy and precision, our setup (Figure 1) is shown below:</p>
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   <img src="" alt="Figure 1" />
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   <img src="/wiki/images/a/a4/Peking2012_luminesensor_sen_div.jpg" alt="Figure 2" style="width:500px"/>
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   <p class="description">Figure 1. Optogenetic Approaches to Control Gene Expression under different light sensitivity.</p>
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   <p class="description">Figure 1. The setup we utilized to characterize the sensitivity of cells harboring the <i>Luminesensor</i></p>
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  <p>Secondly, most accessible methods used to trigger or block gene expression require an extremely complicated setup, sometimes even an invasive device, or a specialized laboratory technician to maneuver subjects to the rough Luminesensor modules. Although numerous efforts have been devoted to improving the experimental conditions, they still retain great handicaps. For example, the use of continuous laser irradiation is required in every measurement and cannot afford reversible monitoring.<br/><br/>Hence, based on the current situation talking above that the light sensors constructed previously always regrettably fail to achieve the goal, Peking iGEM 2012 spares no effort to assemble a high sensitivity light sensor and finally makes it come true. Incessant gene depression from our system using natural-light-intensity sensor optimized by amino acid sequence mutation has been considered a suitable option by means of the natural light intensity, which is very low in comparison with the laser intensity level and is easy to obtain without requiring toxic chemicals. The sensor we assembled solves the sensitivity problem in a new way: it is flexible and highly tunable, as well as modularized, and is constructed onto simple material with a user friendly setup.<br/><br/>To achieve such sensitivity while maintaining minor cell perturbation that is proper for experimental control, we had to verify our light-dependent impact on the transcriptional activity of a GFP-ssrA reporter which has the advantages of visualization and high-throughput driven by LexA binding sites fused with VVD after transformation in E.coli and incubator in our user-friendly setup. According to the literature we searched, we may safely draw a conclusion that our luminesensor possess a high sensitivity as a result of VVD itself owning a high sensitivity. Fully malleable and modularized components were realized utilizing tolerable intensity, natural-light-intensity irradiation to facilitate regulation of gene expression with diverse intentions. What’s more, with the intention of accelerating the degradation of GFP to observe the light-switchable phenomenon and enhance the stability of the light-induced fusion protein, we tagged our GFP reporter gene with an ssrA-tag that is known as a central feature of protein-quality control in all bacteria when the target protein is tagged with. Based on the consideration of guaranteeing accuracy and precision, our setup(Figure 2):</p>
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  <p>Our setup consisted of three central parts: light source, incubator, and 48-well plate. On account of high sensitivity, it is necessary to protect the system from the preventable light exposure with the purpose of acquiring the accurate results which is the true reflection of our sensitivity. In order to solve the problems, we focus on two foremost aspects: 1) utilizing attenuators to weaken the light intensity, and 2) using tin foil to avoid light leakage.  
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In our experiments, illumination with different light intensity conditions at 460nm peak light from blue LED arrays for 16 hours show marked light-depressed reporter gene transcription, which indicates that under different blue light exposure conditions, there was hardly any light-induced reporter gene transcriptional activity. But when in the dark environment (packaged with three layers of aluminum foil), our systems showed extremely high GFP expression (Figure 2).</p>
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   <img src="" alt="Figure 2" />
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   <img src="/wiki/images/f/fa/Peking2012_gfp_expression_2.jpg" alt="Figure 3" />
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   <p class="description">Figure 2. Setup</p>
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   <p class="description">Figure 2. GFP expression was repressed by Blue LED light and presented normally in the dark.</p>
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  <p>consists of three central parts: light source, incubator and 48-well plate.  On account of high sensitivity, protecting the system form the preventable light exposure with the purpose of acquiring the accurate results which is the true reflection of our sensitivity is necessary. In order to solve the problems, we focus on two foremost aspects: utilizing attenuators to weaken the light intensity and using tin foil to avoid light leakage. For more details about how we conducting the experiment, see experiment procedures. In our experiments, illumination with different light intensity conditions at 460nm peak light from blue LED arrays for 16 hours show marked light-depressed reporter gene transcription, which indicates that under different blue light exposure conditions, there was hardly any light-induced reporter gene transcriptional activity. But when in the dark environment (packaged with three layers of aluminum foil), our systems showed extremely high GFP expression (Figure 3)</p>
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  <p>There are a few plausible and conceivable reasons for why our system possesses such high sensitivity that can sense unbelievably weak light intensity. Firstly, it includes the unusually stable, photo-activated state of VVD, which causes the system to be extremely sensitive to light. Secondly, without relying on the addition of chromophores, which might impede the growth of cells, our system can directly regulate gene expression without trade-off.<br/><br/>
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<b>2. Quantitative Result</b><br/>
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Cells exposed to different light intensity expressing the <i>Luminesensor</i> showed manifest light-repressed reporter gene transcription. As shown in Figure 3, all of the cells with dissimilar attenuators showed incredible repression efficiency. <p>
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   <img src="" alt="Figure 3" />
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   <img src="/wiki/images/a/a8/Peking2012_Luminesensor_sensitivity_2.png" alt="Figure 4" style="width:500px"/>
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   <p class="description">Figure 3. Expression of GFP.</p>
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   <p class="description">Figure 3. luminance attenuation using different attenuators could also be sensed by the <i>Luminesensor</i>, which is much dimmer than natural light.</p>
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<p>There are a couple of plausible and conceivable reasons for why our system possesses such high sensitivity that can sense unbelievably weak light intensity. First, it includes the unusually stable, photo-activated state of VVD, which causes the system to be extremely sensitive to light. Secondly, without relying on the chromophores, our system can directly regulate gene expression without time delay.<br/><br/><b>Results and conclusions</b><br/>Our system exposed to different light intensity containing the LexA408 depression domain showed manifest light-depressed reporter gene transcription. As shown in the figure, all of the systems with dissimilar attenuators showed incredible depression efficiency. In the first four sets of data, which are completely exposed to blue light and covered by attenuators No. 2, 4, 7 respectively (see more details by clicking on the protocol, figure 4: luminance measurement of different attenuator, we choose the 48-well plates that covered with No. 2, 4, 7 attenuator as our experimental groups.)<p>
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  <p>It proves that once the cells are exposed to natural light, the transcription of reporter genes will be strongly repressed, although still present as a dose response. Besides, in the negative control group, which was entirely in the dark state, the expression of GFP ran up to a high degree of 50,000. As a matter of fact, as you can see later in "<a href="https://2012.igem.org/Team:Peking/Project/Communication/Results">Results of light communication between cells</a>", when we serially diluted light-emitting cells which express bacterial luciferase, the cells expressing the <i>Luminesensor</i> present significant dose response. Taking everything into account, our <i>Luminesensor</i> does possess high sensitivity across several orders of magnitude. <br/><br/>
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  <img src="/wiki/images/a/a8/Peking2012_Luminesensor_sensitivity_2.png" alt="Figure 4" />
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</p>
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  <p class="description">Figure 4.  luminance measurement of different attenuator, we choose the 48-well plates that covered with No. 2, 4, 7 attenuator as our experimental groups.</p>
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  <p>It proves that once the system is exposed to natural light, reporter gene transcription will be absolutely depressed regardless of the attenuators we attached to it. In other words, all of the systems protected from equal to or lower than natural-light-intensity light blocked light-depressed gene expression as expected. Besides, in the fifth sets of data, which was entirely in the dark state, the expression of GFP ran up to a high degree of 50,000. Taking everything into account, our luminesensor does possess high sensitivity that across several orders of magnitude. Our next work plan is to improve our devices and attenuators in order to find out the threshold point in which the light-depressed gene expression will present linear results.<br/><br/>We are impartially proud of accomplishing the goal of controlling gene expression using light without excessive or unnecessary energy or substrates, and we have respectable and appropriate reasons to have complete faith in our system; that it has imposed specific innovative challenges of light-dependent process for the future researchers.</p>
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  <h3 class="title2">Orthogonal Test</h3>
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  <h3 id="title2">Orthogonality Test</h3>
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  <p><b>Abstract</b><br/>
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  <p>
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It is our biggest concern whether lexA408VVD works indepently with endogenous LexA. Two sections of testing expriments were carried out simultaneously. To our satisfication, the results of different support in vivo orthogonality.</b><br/>Amazingly sensitive as LexA-VVD system is, its application was limited for the wildspread of endogenous lexA repressor in cells. We have to maximum its biological orthogonality to get a really plug-and-play device. Therefore our part can work effciently in widely used host strains, e.g. DH5&alpha, BL21(DE3).<br/><br/>We had found from literature that LexA408, a lexA variant, works independently with wild-type lexA in E.coli; they recognize different sequences and have orthogonal DNA binding specfities.<br/><br/>We then designed the LexA408-VVD fusion protein and a series of its exclusive promoters fused to op408. These novel clones were achieved by point mutations from their wild-typel counterparts. To make things easier, we named the protein lexA408-VVD.<br/><br/>In our experiments, protein expression work was conducted in wild-type BL21(de3) strain. GFP was selected as a reporter and was fused downstream to op408. In this condition, GFP expression has a negative relation with repression activity of the protein. To be more specific, higher level of green fluorescent indicates weaker repression effect; lower expression of GFP stands for stronger repression.<br/><br/>
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  <b>Experimental Design</b><br/>To prove the orthogonality, facts that LexA408-VVD and endogenous LexA work totally independently are needed. Considering practical efficiency, three points of evidence are to collect:</p>
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Two sections of testing expriments were carried out. GFP was selected as a reporter and was fused downstream to two promoters (psulA408 and precA408) controlled by the <i>Luminesensor</i> with the 408 mutation and to two promoters (psulA and precA) controlled by the <i>Luminesensor</i> without the mutation. GFP expression is expected to have a negative relation with repression activity. To be more specific, higher level of green fluorescent indicates weaker repression effect; lower expression of GFP stands for stronger repression. <br/><br/>
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  <ul><li>1. LexA-responsive promoters are repressed in wild-type strains; LexA408-VVD-responsive promoters are not blocked in wild-type strains;
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  </li><li>2. LexA408-VVD efficiently represses its target in blue illumination, while it does not repress targets in total dark.
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  <b>1. Experimental Design(Figure 4)</b><br/>
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To prove the orthogonality, data and facts to prove that LexA408-VVD and the endogenous LexA work totally independently are needed. Considering the practical efficiency, two points of evidence were to be collected:</p>
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  <ul><li>1. Promoters psulA and precA are repressed in wild-type <i>Ecoli</i> strains while promoters psulA408 and precA408 are not blocked in wild-type strains;
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  </li><li>2. LexA408-VVD-<i>Luminesensor</i> efficiently represses its target under blue illumination, while it does not repress targets in total darkness.
  </li></ul>
  </li></ul>
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   <img src="" alt="Figure 5" />
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   <img src="/wiki/images/3/39/Peking2012_demonstration_of_orthogonal_result.png" alt="Figure 5" style="width:480px;"/>
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   <p class="description">Figure 5. Shows observable color of plates where cell cultures growing under totally dark or light conditions. Different colors are used to indicate three levels of GFP expression.</p>
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   <p class="description">Figure 4. Shows observable color of plates where cell cultures growing under totally dark or light conditions. Different colors are used to indicate three levels of GFP expression.</p>
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  <p><b>Detailed Methods</b><br/>Experiments were carried out in parallel: taking photos of plates as visual evidence; measuring GFP to quatitify the effect. Detailed protocols are as follows:<p>
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  <p><b>2. Detailed Methods</b><br/>
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Experiments were carried out in parallel -- taking photos of the plates as visual evidence and measuring GFP to quantify the effect. Detailed protocols are as follows:<p>
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  <ul><li>1. Visible display:
  <ul><li>1. Visible display:
  </li><li>
  </li><li>
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<ul><li>Inoculate a single colony into 1.5ml centifuge tube, shaking cultivate cells at 250 rpm/min for 2-3 h at 30℃, then the cell culture are prepared for streaking;
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<ul><li>Inoculate a single colony into 1.5ml centifuge tube, shaking cultivate cells at 250 rpm/min for 2-3 h at 30℃, then the cell culture is ready for streaking;
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</li><li>Streak cell culture onto right plates;
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</li><li>Streak cell culture onto respective plates;
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</li><li>Ioncubate in totally dark and light state at 30℃ for 1 or 2 days;
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</li><li>Incubate in total darkness and under light state at 30℃ for 1 or 2 days;
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</li><li>Take photos of right plates;
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</li><li>Take photos of correct plates;
</li></ul>
</li></ul>
  </li><li>2. Data collection:  
  </li><li>2. Data collection:  
  </li><li>
  </li><li>
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<ul><li>Inoculate a proper colony in liquid media;
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<ul><li>Inoculate a proper single colony into liquid media;
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</li><li>Shaking cultivate cells until platform stage at 30 ℃;
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</li><li>Cultivate cells in a shaking state until platform stage at 30 ℃;
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</li><li>Dilute celll cultures with selective media in proportion of 1:500;
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</li><li>Dilute cell cultures with selective media at a ratio of 1:500;
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</li><li>Shaking cultivate cells until platform stage at 30 ℃ in fully dark or under blue light;
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</li><li>Cultivate cells in a shaking state until the stationary stage at 30 ℃ in complete darkness or under blue light;
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</li><li>Harvest cells by centrifugation, resuspend pellets in PBS;
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</li><li>Harvest cells by centrifugation, then re-suspend pellets in PBS;
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</li><li>Measuring GFP expression with 96-well-plate reader;
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</li><li>Measure GFP expression with 96-well-microplate reader;
</li></ul>
</li></ul>
  </li>
  </li>
  </ul>
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  <p><b>Results</b><br/>Below is a collage of our plates. The plates are placed in groups at 30℃ in either total dark or blue illumination. Visual results fit well with Figure1.Visual look of plates were positive evidence for us.</p>
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  <p><b>3. Results</b><br/>
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Below is a collage of our plates. The plates are placed in groups at 30℃ in either total darkness or under blue-light illumination. Visual results fit well with Figure 4. Visual appearance of the plates were positive evidence for us.</p>
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   <img src="" alt="Figure 6" />
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   <img src="/wiki/images/4/49/Peking2012_orthogonality_plate_group1_correction.png" alt="Figure 6(1)." style="width:500px;"/>
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   <p class="description">Figure 6. Plates display. Streaking certain strain onto selected media.</p>
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   <p class="description">Figure 5(1). Plate streaked with wild type <i>E.coli</i> transformed with wild type <i>Luminesensor</i> and GFP fused to LexA responsive promoters, and cultivated in the dark (A), or under light (B). Evidently there is no GFP expression, indicating that endogenous LexA proteins are capable of fully repress the expression of our wild type reporter gene, rendering our <i>Luminesensor</i> system no longer light-controllable. </p>
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<p>Apart from those photos, more accurate results were obtained with Fluorescent Microplate Reader. Below are histograms based on our experimmental data. They clarify how fluoresence changes with lighting condition in different strains.<p>
 
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  <img src="" alt="Figure 7" />
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<img src="/wiki/images/8/88/Peking2012_orthogonality_plate_group2.png" alt="Figure 6(2)." style="width:500px;"/>
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  <p class="description">Figure 7. GFP expression under different promoters. The tests were carried out in strains capable of expressing LexA408-VVD. </p>
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<p class="description">Figure 5(2). Plate streaked with wild type <i>E.coli</i> transformed with GFP gene fused to the downstream of psulA408 (A) and precA408 (B). Evidently GFP is perfectly expressed, indicating endogenous LexA proteins are unable to bind to psulA408 and precA408, which suggested that the 408 promoter system is orthogonal to the endogenous SOS system.</p>
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<p>It repeartedly turned out that:</p>
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<ul><li>1. reporters under wild-type promoters hardly express in either illuminated or dark state; their colonies are yellow. In contrast, GFPs fused to 408 operators were not blocked in strains withour lexA408-VVD, thus they looks bright green.
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<img src="/wiki/images/e/e7/Peking2012_orthogonality_plate_group3.png" alt="Figure 6(3)." style="width:500px;"/>
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</li><li>2. BL21 (DE3) strains containing both lexA408-VVD and its reporters twist swiftly and sensitively from one states to the other, giving high definition output including lighting condition.
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<p class="description">Figure 5(3). Plate streaked with wild type <i>E.coli</i> transformed with mutated luminesensor and precA408-GFP or psulA408-GFP reporter gene and cultivated in the dark or under light.(A: luminesensor + precA408-GFP, cultivated in the dark. B: luminesensor + precA408-GFP, cultivated under light. C: luminesensor + psulA408-GFP, cultivated in the dark. D: luminesensor + psulA408-GFP, cultivated under light.) Just as we predicted, the reporter gene is expressed in the dark and supressed by our luminesensor under light. This clearly proved that our 408 system is orthogonal to bacteria SOS system while retaining light-controllability.</p>
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</li></ul>
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<p>To sum up, LexA408-VVD works independently with endogenous lexA. What’s more, thet are unrelated to most currently prevalent  ribosome-mRNA,  mRNA -rRNA orthogonal pairs, etc.
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Our devices are to help enrich logic gates system and enable the establishment of more complexed network in synthetic biology.  We feel proud thinking that more and more r resaerchers will benefit and like it, our lexA408-VVD.
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<h3 class="title3">Optimization</h3>
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<p>To test whether our designated mutations would optimize our luminesensor in respect to reversibility and the on/off ratio, we co-transformed the ColE-GFP (GFP driven by luminesensor repressible promoter ColE) plasmid with four versions of our luminesensor plasmid into BL21 (&Delta;LexA &Delta;SulA): the original LexA-VVD(WT), LexA-VVD(74), LexA-VVD(135), and LexA-VVD(74+135). The resulting four strains were designated LV-WT, LV-74, LV-135, LV-74-135, respectively. The overnight culture of the four strains were diluted 500 times and divided into two groups: one exposed to blue light and the completely wrapped with aluminum foil. After incubation for 16 hours, GFP expression levels were measured. As shown in figure X, LV-135 has an increased on/off ratio compared to the original LexA-VVD, while the LV-74 and LV-74-135 show reduced on/off ratios, which is in accordance with our model .</p>
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  <img src="/wiki/images/4/42/Peking2012_luminesensor_opt_1.png" alt="Figure 8" />
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  <p class="description">Figure 8. The on/off ratio of GFP expression level of the four strains.</p>
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  <p>In order to determine whether the LexA-VVD(135) has enhanced reversibility in comparison to the original LexA-VVD, the temporal change of GFP expression level of dilated overnight culture of the strains LV-WT and LV-135 under blue light were measured at 2 hour intervals for 26 hours. As shown in figure Y, the GFP expression level of both of the two strains began to rise after incubating at dark for about 10 hour. We speculated that the GFP expression level of the two strains is mainly determined by the equilibrium between GFP production and degradation and the dimerized LexA-VVD(WT) and LexA-VVD(135) dissociate in a shorter time scale compared to the time needed to establish the equilibrium of the GFP production and degradation.</p>
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  <p>Apart from those photos, more accurate results were obtained with Fluorescent Microplate Reader. Below are histograms based on our experimmental data. They clarify how fluoresence changes with lighting condition in different strains(Figure 6).</p>
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   <img src="" alt="Figure 9" />
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   <img src="/wiki/images/4/44/Peking2012_precA408-GFP_with_axis_correction1.png" alt="Figure 7" style="width:600px;" />
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   <p class="description">Figure 9. The temporal change of GFP expression of the LV-WT and LV-135</p>
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   <p class="description">Figure 6(1). GFP expression level under control of precA408 promoter. Column 1 shows expression of precA-GFP reporter gene when transformed into wild-type <i>E.coli</i> without luminesensor. The high expression level indicates that the 408 reporter system is not interfered by endogenous LexA protein. The column 2 and column 3 are expression level of precA-GFP reporter gene co-transformed with our 408 form luminesensor into wild-type <i>E.coli</i>, and cultivated under light (column 2), or under light (column 3). The low expression level under light and hign expression level in the dark indicate that our 408 luminesensor system is totally light-controllable, and possessing a high dynamic range.</p>
  </div>
  </div>
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<div class="floatC">
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<img src="/wiki/images/9/95/Peking2012_psulA408-GFP_fluorescence_with_axis1.png" alt="Figure 7" style="width:600px;" />
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<p class="description">Figure 6(2). GFP expression level under control of psulA408 promoter. Every column shows data acquired under conditions same as its conterpart in Figure 7(1). The only difference is that the precA408 promoter has been replaced by psulA408 promoter.</p>
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</div>
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<p>It repeatedly turned out that:
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<br /><br />
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1.  reporters under wild-type promoters (psulA and precA) hardly expressed in either illuminated or dark state; their colonies are yellow. In contrast, GFPs fused to promoters psulA408 and precA408 were not blocked in strains, thus they looks bright green.
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<br /><br />
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2. BL21 (DE3) strains expressing both the <i>Luminesensor</i> and its reporters shifted swiftly and sensitively from one states to the other, giving high definition output including lighting condition.
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<br /><br />
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To sum up, LexA408-VVD-<i>Luminesensor</i> works independently of host genetic context.
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</p>
</div>
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  <h3 class="title4">Conclusion</h3>
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<h3 id="title5">Reference</h3>
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  <p>In the end, we have finally constructed a LexA-VVD fusion protein that will function as a transcription repressor in response to blue light. And since we have introduced mutations into the LexA DNA binding domain, the <i>Luminesensor</i> proved orthogonal to endogenous bacteria SOS system. Futher introduced mutations in VVD photosensitive domain significantly improved the dynamic range of our optogenetic module. Equipped with this optimized LexA408-VVD74 <i>Luminesensor</i>, we are now able to apply it in real bacteria system to achieve amazing accomplishments.</p>
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<p></p>
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<ul class="refer"><li id="ref1">
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<div class="PKU_context floatR">
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1. Wang, X., Chen, X. & Yang, Y.(2012). spatiotemporal control of gene expression
by a light-switchable transgene system. <i>Nat. Methods</i>, 9: 266: 269
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<h3 class="title3">Reference</h3>
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  </li><li id = "ref2">
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  <p>reference here </p>
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2. Farrell, C.M., Grossman, A.D., and Sauer., R.T.(2005). Cytoplasmic degradation of ssrA-tagged proteins. <i>Mol. Microbiol.</i>, 57: 1750: 61
 +
  </li><li id = "ref3">
 +
3. Roche, E.D., and Sauer., R.T.(2001). Identification of Endogenous SsrA-tagged Proteins Reveals Tagging at Positions Corresponding to Stop Codons. <i>J. Biol. Chem.</i>, 276: 28509: 28515
 +
  </li><li id = "ref3">
 +
4. Dimitrova,D., <i>et al.</i>(1997). A new LexA-based genetic system for monitoring and analyzing protein heterodimerization in <i>Escherichia coli</i>.<i>Mol. Gen. Genet.</i>, 257: 205: 212
 +
  </li><li id = "ref3">
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5. Voigt., C.A.(2006). Genetic parts to program bacteria. <i>Curr. Opin. Biotechnol.</i>, 17: 548: 557
 +
  </li><li id = "ref3">
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6. Thliveris, A.T., Mount., D.W.(1992). Genetic identification of the DNA binding domain of <i>Escherichia coli</i> LexA protein. <i>Proc. Natl. Acad. Sci. USA</i>, 89 (10): 4500: 4504
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  </li></ul>
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Latest revision as of 13:20, 26 October 2012

Sensitivity

1. Set-up & Brief Procedure
We tested the sensitivity of the Luminesensor by examining the light-dependent transcriptional activity of a GFP-ssrA reporter. ssrA is a protein tag that induces fast degradation of protein, which in our case facilitated the observation of transcriptional activity. Based on the consideration of guaranteed accuracy and precision, our setup (Figure 1) is shown below:

Figure 2

Figure 1. The setup we utilized to characterize the sensitivity of cells harboring the Luminesensor

Our setup consisted of three central parts: light source, incubator, and 48-well plate. On account of high sensitivity, it is necessary to protect the system from the preventable light exposure with the purpose of acquiring the accurate results which is the true reflection of our sensitivity. In order to solve the problems, we focus on two foremost aspects: 1) utilizing attenuators to weaken the light intensity, and 2) using tin foil to avoid light leakage. In our experiments, illumination with different light intensity conditions at 460nm peak light from blue LED arrays for 16 hours show marked light-depressed reporter gene transcription, which indicates that under different blue light exposure conditions, there was hardly any light-induced reporter gene transcriptional activity. But when in the dark environment (packaged with three layers of aluminum foil), our systems showed extremely high GFP expression (Figure 2).

Figure 3

Figure 2. GFP expression was repressed by Blue LED light and presented normally in the dark.

There are a few plausible and conceivable reasons for why our system possesses such high sensitivity that can sense unbelievably weak light intensity. Firstly, it includes the unusually stable, photo-activated state of VVD, which causes the system to be extremely sensitive to light. Secondly, without relying on the addition of chromophores, which might impede the growth of cells, our system can directly regulate gene expression without trade-off.

2. Quantitative Result
Cells exposed to different light intensity expressing the Luminesensor showed manifest light-repressed reporter gene transcription. As shown in Figure 3, all of the cells with dissimilar attenuators showed incredible repression efficiency.

Figure 4

Figure 3. luminance attenuation using different attenuators could also be sensed by the Luminesensor, which is much dimmer than natural light.

It proves that once the cells are exposed to natural light, the transcription of reporter genes will be strongly repressed, although still present as a dose response. Besides, in the negative control group, which was entirely in the dark state, the expression of GFP ran up to a high degree of 50,000. As a matter of fact, as you can see later in "Results of light communication between cells", when we serially diluted light-emitting cells which express bacterial luciferase, the cells expressing the Luminesensor present significant dose response. Taking everything into account, our Luminesensor does possess high sensitivity across several orders of magnitude.

Orthogonality Test

Two sections of testing expriments were carried out. GFP was selected as a reporter and was fused downstream to two promoters (psulA408 and precA408) controlled by the Luminesensor with the 408 mutation and to two promoters (psulA and precA) controlled by the Luminesensor without the mutation. GFP expression is expected to have a negative relation with repression activity. To be more specific, higher level of green fluorescent indicates weaker repression effect; lower expression of GFP stands for stronger repression.

1. Experimental Design(Figure 4)
To prove the orthogonality, data and facts to prove that LexA408-VVD and the endogenous LexA work totally independently are needed. Considering the practical efficiency, two points of evidence were to be collected:

  • 1. Promoters psulA and precA are repressed in wild-type Ecoli strains while promoters psulA408 and precA408 are not blocked in wild-type strains;
  • 2. LexA408-VVD-Luminesensor efficiently represses its target under blue illumination, while it does not repress targets in total darkness.
Figure 5

Figure 4. Shows observable color of plates where cell cultures growing under totally dark or light conditions. Different colors are used to indicate three levels of GFP expression.

2. Detailed Methods
Experiments were carried out in parallel -- taking photos of the plates as visual evidence and measuring GFP to quantify the effect. Detailed protocols are as follows:

  • 1. Visible display:
    • Inoculate a single colony into 1.5ml centifuge tube, shaking cultivate cells at 250 rpm/min for 2-3 h at 30℃, then the cell culture is ready for streaking;
    • Streak cell culture onto respective plates;
    • Incubate in total darkness and under light state at 30℃ for 1 or 2 days;
    • Take photos of correct plates;
  • 2. Data collection:
    • Inoculate a proper single colony into liquid media;
    • Cultivate cells in a shaking state until platform stage at 30 ℃;
    • Dilute cell cultures with selective media at a ratio of 1:500;
    • Cultivate cells in a shaking state until the stationary stage at 30 ℃ in complete darkness or under blue light;
    • Harvest cells by centrifugation, then re-suspend pellets in PBS;
    • Measure GFP expression with 96-well-microplate reader;

3. Results
Below is a collage of our plates. The plates are placed in groups at 30℃ in either total darkness or under blue-light illumination. Visual results fit well with Figure 4. Visual appearance of the plates were positive evidence for us.

Figure 6(1).

Figure 5(1). Plate streaked with wild type E.coli transformed with wild type Luminesensor and GFP fused to LexA responsive promoters, and cultivated in the dark (A), or under light (B). Evidently there is no GFP expression, indicating that endogenous LexA proteins are capable of fully repress the expression of our wild type reporter gene, rendering our Luminesensor system no longer light-controllable.

Figure 6(2).

Figure 5(2). Plate streaked with wild type E.coli transformed with GFP gene fused to the downstream of psulA408 (A) and precA408 (B). Evidently GFP is perfectly expressed, indicating endogenous LexA proteins are unable to bind to psulA408 and precA408, which suggested that the 408 promoter system is orthogonal to the endogenous SOS system.

Figure 6(3).

Figure 5(3). Plate streaked with wild type E.coli transformed with mutated luminesensor and precA408-GFP or psulA408-GFP reporter gene and cultivated in the dark or under light.(A: luminesensor + precA408-GFP, cultivated in the dark. B: luminesensor + precA408-GFP, cultivated under light. C: luminesensor + psulA408-GFP, cultivated in the dark. D: luminesensor + psulA408-GFP, cultivated under light.) Just as we predicted, the reporter gene is expressed in the dark and supressed by our luminesensor under light. This clearly proved that our 408 system is orthogonal to bacteria SOS system while retaining light-controllability.

Apart from those photos, more accurate results were obtained with Fluorescent Microplate Reader. Below are histograms based on our experimmental data. They clarify how fluoresence changes with lighting condition in different strains(Figure 6).

Figure 7

Figure 6(1). GFP expression level under control of precA408 promoter. Column 1 shows expression of precA-GFP reporter gene when transformed into wild-type E.coli without luminesensor. The high expression level indicates that the 408 reporter system is not interfered by endogenous LexA protein. The column 2 and column 3 are expression level of precA-GFP reporter gene co-transformed with our 408 form luminesensor into wild-type E.coli, and cultivated under light (column 2), or under light (column 3). The low expression level under light and hign expression level in the dark indicate that our 408 luminesensor system is totally light-controllable, and possessing a high dynamic range.

Figure 7

Figure 6(2). GFP expression level under control of psulA408 promoter. Every column shows data acquired under conditions same as its conterpart in Figure 7(1). The only difference is that the precA408 promoter has been replaced by psulA408 promoter.

It repeatedly turned out that:

1. reporters under wild-type promoters (psulA and precA) hardly expressed in either illuminated or dark state; their colonies are yellow. In contrast, GFPs fused to promoters psulA408 and precA408 were not blocked in strains, thus they looks bright green.

2. BL21 (DE3) strains expressing both the Luminesensor and its reporters shifted swiftly and sensitively from one states to the other, giving high definition output including lighting condition.

To sum up, LexA408-VVD-Luminesensor works independently of host genetic context.

Reference

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by a light-switchable transgene system. Nat. Methods, 9: 266: 269
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  • 3. Roche, E.D., and Sauer., R.T.(2001). Identification of Endogenous SsrA-tagged Proteins Reveals Tagging at Positions Corresponding to Stop Codons. J. Biol. Chem., 276: 28509: 28515
  • 4. Dimitrova,D., et al.(1997). A new LexA-based genetic system for monitoring and analyzing protein heterodimerization in Escherichia coli.Mol. Gen. Genet., 257: 205: 212
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