Team:Grenoble-EMSE-LSU/Project/Biology

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                <h3>A red fluorescent Killer</h3>
                <h3>A red fluorescent Killer</h3>
                <p>KillerRed is a red fluorescent protein <a href="#ref_bio_1">[1]</a>, meaning that by illuminating it with wavelengths from a certain portion of the visible spectrum, it re-emits light in another portion with longer (less energetic) wavelengths. Below is the absorption and emission spectra for the KillerRed protein:</p><br><br>
                <p>KillerRed is a red fluorescent protein <a href="#ref_bio_1">[1]</a>, meaning that by illuminating it with wavelengths from a certain portion of the visible spectrum, it re-emits light in another portion with longer (less energetic) wavelengths. Below is the absorption and emission spectra for the KillerRed protein:</p><br><br>
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                                         <p align="center"><img src="https://static.igem.org/mediawiki/2013/7/7f/KillerRed_spectra2.png" alt="Killer Red absorption-emission spectra" width="500px"></p>
                                         <p align="center"><img src="https://static.igem.org/mediawiki/2013/7/7f/KillerRed_spectra2.png" alt="Killer Red absorption-emission spectra" width="500px"></p>
                                         <p id="legend">The KillerRed protein absorption (left peak) and emission (right peak) spectra<br>
                                         <p id="legend">The KillerRed protein absorption (left peak) and emission (right peak) spectra<br>
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                                         Source:<a href="http://www.evrogen.com/products/KillerRed/KillerRed_Detailed_description.shtml">Detailed KillerRed description from Evrogen</a><br><br></p>
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                                         Source:<a href="http://www.evrogen.com/products/KillerRed/KillerRed_Detailed_description.shtml">Detailed KillerRed description from Evrogen</a><br><br></p>
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                <p>From the emission and absorption spectra, we can determine that the protein absorbs in the green portion of the spectrum with a peak at 585 nm and emits in the red portion of the spectrum with a peak at 610 nm, hence the name "KillerRed".<br>
                <p>From the emission and absorption spectra, we can determine that the protein absorbs in the green portion of the spectrum with a peak at 585 nm and emits in the red portion of the spectrum with a peak at 610 nm, hence the name "KillerRed".<br>
                Emitted light from bacteria is proportional to the amount of protein in the cells. This allows for measuring protein concentration in a cell culture.<br><br>
                Emitted light from bacteria is proportional to the amount of protein in the cells. This allows for measuring protein concentration in a cell culture.<br><br>
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                <h3>Structure</h3>                         
                <h3>Structure</h3>                         
                                         <p>In order to understand why KillerRed has its unique properties it is necessary to look at its structure. The protein is remarkably similar to other fluorescent proteins like GFP <em>(Aequorea victoria></em> and dsRed <em>(Discosoma striata)</em>, featuring a beta-barrel housing a central alpha helix with the fluorescent chromophore at its center<a href="#ref_bio_1">[2]</a>. Normally the chromophore is protected from the outside environment by the protein shell, but this isn't the case with KillerRed.<br><br></p>
                                         <p>In order to understand why KillerRed has its unique properties it is necessary to look at its structure. The protein is remarkably similar to other fluorescent proteins like GFP <em>(Aequorea victoria></em> and dsRed <em>(Discosoma striata)</em>, featuring a beta-barrel housing a central alpha helix with the fluorescent chromophore at its center<a href="#ref_bio_1">[2]</a>. Normally the chromophore is protected from the outside environment by the protein shell, but this isn't the case with KillerRed.<br><br></p>
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                <p align="center"><img src="https://static.igem.org/mediawiki/2013/8/8c/DsRed_alongside_KillerRed.png" alt="dsRed and KillerRed protein structures."></p>
                <p align="center"><img src="https://static.igem.org/mediawiki/2013/8/8c/DsRed_alongside_KillerRed.png" alt="dsRed and KillerRed protein structures."></p>
                                         <p id="legend">A comparison of the 3D structures of monomerix dsRed (left) and dimeric KillerRed (right)<br>
                                         <p id="legend">A comparison of the 3D structures of monomerix dsRed (left) and dimeric KillerRed (right)<br>
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                <p>KR was originally engineered from the anm2CP anthomedusa chromoprotein by individual amino acid mutations in order to obtain fluorescence and phototoxicity.<a href="#ref_bio_1">[1]</a></p>
                <p>KR was originally engineered from the anm2CP anthomedusa chromoprotein by individual amino acid mutations in order to obtain fluorescence and phototoxicity.<a href="#ref_bio_1">[1]</a></p>
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<h2>Construction of pLac-RBS-KR and pLac-RBS-mCherry</h2>
<h2>Construction of pLac-RBS-KR and pLac-RBS-mCherry</h2>
<p>The KillerRed gene that we obtained initially was in an eukaryotic plasmid. To express KR in <em>E. coli</em> and characterize its effects in response to light stimulations, we decided to clone KR into the commercial prokaryotic expression vector pQE30 (Qiagen). This plasmid contains a pLac promoter and a Shine-Dalgarno Ribosome Binding Site (RBS) that allow gene expression in response to the presence of Isopropyl β-D-1-thiogalactopyranoside (IPTG). The pLac-RBS-KR sequence in the pQE30 vector has been submitted as BBa_K1141001. Furthermore, the pLac-RBS-KR sequence was cloned into the pSB1C3 plasmid, to give the biobrick BBa_K1141002. Both BBa_K1141001 and BBa_K1141002 were sent to the standard registry part (Fig. 1).<br><br>
<p>The KillerRed gene that we obtained initially was in an eukaryotic plasmid. To express KR in <em>E. coli</em> and characterize its effects in response to light stimulations, we decided to clone KR into the commercial prokaryotic expression vector pQE30 (Qiagen). This plasmid contains a pLac promoter and a Shine-Dalgarno Ribosome Binding Site (RBS) that allow gene expression in response to the presence of Isopropyl β-D-1-thiogalactopyranoside (IPTG). The pLac-RBS-KR sequence in the pQE30 vector has been submitted as BBa_K1141001. Furthermore, the pLac-RBS-KR sequence was cloned into the pSB1C3 plasmid, to give the biobrick BBa_K1141002. Both BBa_K1141001 and BBa_K1141002 were sent to the standard registry part (Fig. 1).<br><br>
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                                         The choice of an inducible promoter is linked to the absence of literature about the effects of KR on cells in low light. Since KR could be cytotoxic and prevent bacteria from growing even at low doses of light, we wanted to be able to control its intracellular concentration. A negative control for KR characterization was also required. We decided to use the fluorescent protein mCherry, which displays the same excitation and emission spectra as KillerRed [1], and was shown not to be cytotoxic upon light illumination [2]. pSB1C3::pLac-RBS-mCherry (BBa_K1141000) was thus constructed from the existing biobricks BBa_R0010 and BBa_J06702 (Fig. 1).<br><br></p>
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                                         The choice of an inducible promoter is linked to the absence of literature about the effects of KR on cells in low light. Since KR could be cytotoxic and prevent bacteria from growing even at low doses of light, we wanted to be able to control its intracellular concentration. A negative control for KR characterization was also required. We decided to use the fluorescent protein mCherry, which displays the same excitation and emission spectra as KillerRed <a href="#ref_bio_1">[4]</a>, and was shown not to be cytotoxic upon light illumination <a href="#ref_bio_1">[5]</a>. pSB1C3::pLac-RBS-mCherry (BBa_K1141000) was thus constructed from the existing biobricks BBa_R0010 and BBa_J06702 (Fig. 1).<br><br></p>
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                                         <p align="center"><img src="https://static.igem.org/mediawiki/2013/0/00/Grenoble_Biobricks_KR_and_mCherry.png" alt="biobricks" width="750px"></p>
                                         <p align="center"><img src="https://static.igem.org/mediawiki/2013/0/00/Grenoble_Biobricks_KR_and_mCherry.png" alt="biobricks" width="750px"></p>
                                         <p id="legend">Figure 1.<br>Figure 1. Biobricks BBa_K1141002 <em>A</em> and BBa_K1141000 <em>A</em> used for characterizing KR. C. Picture of KR-expressing bacteria.</p>
                                         <p id="legend">Figure 1.<br>Figure 1. Biobricks BBa_K1141002 <em>A</em> and BBa_K1141000 <em>A</em> used for characterizing KR. C. Picture of KR-expressing bacteria.</p>
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                                         <p>We first decided to characterize KR in BW25113 bacteria, a wild-type (WT) strain derived from <em>E. coli</em> K12. Cells were successfully transformed with pQE30::KR and were shown to express the protein in response to IPTG induction. However, results of OD610 monitoring showed that BW25113 cells transformed with pQE30::KR grew really slowly (r = 0.08 h<sup>-1</sup>) as compared to WT cells (r = 0.77 h<sup>-1</sup>). One hypothesis was that repression of the pLac promoter by the endogeneous LacI repressor was not sufficient for preventing the expression of KR, a protein that could have affected cell growth even at low light levels.<br><br>
                                         <p>We first decided to characterize KR in BW25113 bacteria, a wild-type (WT) strain derived from <em>E. coli</em> K12. Cells were successfully transformed with pQE30::KR and were shown to express the protein in response to IPTG induction. However, results of OD610 monitoring showed that BW25113 cells transformed with pQE30::KR grew really slowly (r = 0.08 h<sup>-1</sup>) as compared to WT cells (r = 0.77 h<sup>-1</sup>). One hypothesis was that repression of the pLac promoter by the endogeneous LacI repressor was not sufficient for preventing the expression of KR, a protein that could have affected cell growth even at low light levels.<br><br>
                                         We thus decided to switch to M15 cells (Qiagen), a commercial strain in which the lacI repressor is expressed at high levels from plasmid pREP4. M15 cells did express the KR protein in response to IPTG addition and displayed a faster growth rate than the BW25113 cells transformed with pQE30::KR (Fig. 2). For this reason, M15 cells were chosen to characterize KR.<br><br></p>
                                         We thus decided to switch to M15 cells (Qiagen), a commercial strain in which the lacI repressor is expressed at high levels from plasmid pREP4. M15 cells did express the KR protein in response to IPTG addition and displayed a faster growth rate than the BW25113 cells transformed with pQE30::KR (Fig. 2). For this reason, M15 cells were chosen to characterize KR.<br><br></p>
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                                         <p align="center"><img src="https://static.igem.org/mediawiki/2013/f/fa/Strain_choice.png" alt="strain choice" height="350px"></p>
                                         <p align="center"><img src="https://static.igem.org/mediawiki/2013/f/fa/Strain_choice.png" alt="strain choice" height="350px"></p>
                                         <p id="legend">Figure 2.<br>Comparison between the growth of pQE30::KR-containing BW25113 and M15 cells (without IPTG and in the dark). Cells were pre cultured ON in LB medium, supplemented with antibiotics. They were further re suspended in M9 medium, supplemented with antibiotics at OD610 = 0.1. OD610 was subsequently monitored in a 96-well plate for 600 min, using the Tristar LB941 microplate reader (Tristar, Bad Wildbad, Germany) available in the lab. Error bars represent the standard errors of 4 independent measurements.</p>
                                         <p id="legend">Figure 2.<br>Comparison between the growth of pQE30::KR-containing BW25113 and M15 cells (without IPTG and in the dark). Cells were pre cultured ON in LB medium, supplemented with antibiotics. They were further re suspended in M9 medium, supplemented with antibiotics at OD610 = 0.1. OD610 was subsequently monitored in a 96-well plate for 600 min, using the Tristar LB941 microplate reader (Tristar, Bad Wildbad, Germany) available in the lab. Error bars represent the standard errors of 4 independent measurements.</p>
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                                         First of all, KR fluorescence can be used as an indicator of the level of expression of the protein in our cell culture. Then, optical density (OD610) provides real-time information on the biomass of the system. However, it cannot be used to distinguish living and non-living cells. This is the reason why the number of colonies growing on agar plates was considered to be able to quantify live cells with an independent technique.<br><br>
                                         First of all, KR fluorescence can be used as an indicator of the level of expression of the protein in our cell culture. Then, optical density (OD610) provides real-time information on the biomass of the system. However, it cannot be used to distinguish living and non-living cells. This is the reason why the number of colonies growing on agar plates was considered to be able to quantify live cells with an independent technique.<br><br>
                                         Since the spectrophotometer available in the lab was not suitable for illuminating cell samples for extended periods of time, we decided to perform kinetics in 100 mL Erlenmeyer flasks, incubated at 37°C, 200 rpm. A LED light source, interfaced to a computer via a microcontroller, was placed into the incubator for illuminating cell samples. A customized software enabled us to tightly modulate the intensity of the light emitted by the source.<br><br></p>
                                         Since the spectrophotometer available in the lab was not suitable for illuminating cell samples for extended periods of time, we decided to perform kinetics in 100 mL Erlenmeyer flasks, incubated at 37°C, 200 rpm. A LED light source, interfaced to a computer via a microcontroller, was placed into the incubator for illuminating cell samples. A customized software enabled us to tightly modulate the intensity of the light emitted by the source.<br><br></p>
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                                         <p align="center"><img src="https://static.igem.org/mediawiki/2013/2/21/Grenoble_Incubateur_set_up.jpg" alt="" height="350px"></p>
                                         <p align="center"><img src="https://static.igem.org/mediawiki/2013/2/21/Grenoble_Incubateur_set_up.jpg" alt="" height="350px"></p>
                                         <p id="legend">Figure <br>Overview on the experimental set up used for KillerRed characterization.<br><br></p>
                                         <p id="legend">Figure <br>Overview on the experimental set up used for KillerRed characterization.<br><br></p>
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                                         <p>During most of the kinetic experiments, 800 µL of medium were pipetted every 30-60 min. OD610 measurements were performed using a GENESYS 6 spectrophotometer (Thermo Scientific, Waltham, MA, USA) whereas fluorescence was measured with a Tristar LB941 microplate reader, equipped with a 540/630 nm filter set for excitation and emission. Bacterial cell plating on agar plates was also performed at each time point, using serial dilutions.<br><br></p>
                                         <p>During most of the kinetic experiments, 800 µL of medium were pipetted every 30-60 min. OD610 measurements were performed using a GENESYS 6 spectrophotometer (Thermo Scientific, Waltham, MA, USA) whereas fluorescence was measured with a Tristar LB941 microplate reader, equipped with a 540/630 nm filter set for excitation and emission. Bacterial cell plating on agar plates was also performed at each time point, using serial dilutions.<br><br></p>
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                                         <h4>Growth medium</h4>
                                         <h4>Growth medium</h4>
                                         <p>M9-glucose medium was privileged in our experiments. As a matter of fact, it displays very low auto fluorescence and contains a single carbon source - glucose – hence providing more repeatable results than Luria-Bertani (LB) medium. pRep4 and pQE30::KR are respectively kanamycin and ampicillin-resistant, and these antibiotics were used at 50 µg/µL and 200 µg/µL.<br><br></p>
                                         <p>M9-glucose medium was privileged in our experiments. As a matter of fact, it displays very low auto fluorescence and contains a single carbon source - glucose – hence providing more repeatable results than Luria-Bertani (LB) medium. pRep4 and pQE30::KR are respectively kanamycin and ampicillin-resistant, and these antibiotics were used at 50 µg/µL and 200 µg/µL.<br><br></p>
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                                         <h4>IPTG induction</h4>
                                         <h4>IPTG induction</h4>
                                         <p>One important point for our project was to reach a high level of KR expression, without slowing down cellular growth. As a matter of fact, to increase or decrease the amount of viable cells in our culture, we needed to make sure that the bacteria expressing KR could grow in the dark and be killed in response to light stimulations. Now, the more KR is present inside bacteria, the more ROS are produced upon illumination and the more likely the cells are to die. But is bacterial growth affected by high intracellular concentrations of KR? Is there an optimal IPTG concentration to reach high levels of KR without disturbing cell division?<br><br>
                                         <p>One important point for our project was to reach a high level of KR expression, without slowing down cellular growth. As a matter of fact, to increase or decrease the amount of viable cells in our culture, we needed to make sure that the bacteria expressing KR could grow in the dark and be killed in response to light stimulations. Now, the more KR is present inside bacteria, the more ROS are produced upon illumination and the more likely the cells are to die. But is bacterial growth affected by high intracellular concentrations of KR? Is there an optimal IPTG concentration to reach high levels of KR without disturbing cell division?<br><br>
                                         To answer these questions, we decided to induce KR expression with different concentrations of IPTG, while monitoring OD610 and fluorescence. M15 cells transformed with pSB1C3::pLac-RBS-mCherry (BBa_K1141000) were used as a negative control. To evaluate the amount of KR proteins per living cell, we normalized fluorescence by optical density. Results are shown in figure 3.<br><br></p>
                                         To answer these questions, we decided to induce KR expression with different concentrations of IPTG, while monitoring OD610 and fluorescence. M15 cells transformed with pSB1C3::pLac-RBS-mCherry (BBa_K1141000) were used as a negative control. To evaluate the amount of KR proteins per living cell, we normalized fluorescence by optical density. Results are shown in figure 3.<br><br></p>
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                                         <p align="center"><img src="https://static.igem.org/mediawiki/2013/4/41/Grenoble_Growth_mCherry_vs_KR.png" alt="mCherry vs KR" height="450px"></p>
                                         <p align="center"><img src="https://static.igem.org/mediawiki/2013/4/41/Grenoble_Growth_mCherry_vs_KR.png" alt="mCherry vs KR" height="450px"></p>
                                         <p id="legend">Figure 3.<br>OD610 (A and C) and Fluorescence/OD610 ratios (B and D) as a function of time for KillerRed (left panels) and mCherry (right panels)-expressing <em>E. coli</em>. The curves obtained with different concentrations of IPTG are represented in different colors as indicated on the legends at the right.<br><br></p>
                                         <p id="legend">Figure 3.<br>OD610 (A and C) and Fluorescence/OD610 ratios (B and D) as a function of time for KillerRed (left panels) and mCherry (right panels)-expressing <em>E. coli</em>. The curves obtained with different concentrations of IPTG are represented in different colors as indicated on the legends at the right.<br><br></p>
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                                         <p><strong>This new experiment confirms that the expression level of KillerRed has an effect on cell growth that isn't observed for a control red fluorescent protein, mCherry. This effect is threshold-based, meaning that if we go over a certain concentration of IPTG and thus a certain protein expression level, then the cells start growing much more slowly. We observe that at an IPTG concentration of 0.05 mM, there is no effect on cell growth compared to control, while protein expression at that concentration is the best out of all the curves. This experiment allows us to define the IPTG concentration used thereafter in KillerRed characterization: 0.05 mM. </strong></p>
                                         <p><strong>This new experiment confirms that the expression level of KillerRed has an effect on cell growth that isn't observed for a control red fluorescent protein, mCherry. This effect is threshold-based, meaning that if we go over a certain concentration of IPTG and thus a certain protein expression level, then the cells start growing much more slowly. We observe that at an IPTG concentration of 0.05 mM, there is no effect on cell growth compared to control, while protein expression at that concentration is the best out of all the curves. This experiment allows us to define the IPTG concentration used thereafter in KillerRed characterization: 0.05 mM. </strong></p>
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<h3>Comparison with mCherry: Cellular Death is ROS-mediated</h3>
<h3>Comparison with mCherry: Cellular Death is ROS-mediated</h3>
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<p>We demonstrated in the previous section that KillerRed-expressing bacteria could be killed upon white light illumination. However, exposure to white light and incubation outside of the normal temperature range were shown to affect bacterial growth [1]. Therefore, we decided to perform additional kinetics, using mCherry-expressing bacteria as a negative control. Results of these experiments demonstrated that KillerRed is responsible for cell death in response to white light stimulations.<br><br></p>
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<p>We demonstrated in the previous section that KillerRed-expressing bacteria could be killed upon white light illumination. However, exposure to white light and incubation outside of the normal temperature range were shown to affect bacterial growth <a href="#ref_bio_1">[6]</a>. Therefore, we decided to perform additional kinetics, using mCherry-expressing bacteria as a negative control. Results of these experiments demonstrated that KillerRed is responsible for cell death in response to white light stimulations.<br><br></p>
<h4>Kinetics</h4>
<h4>Kinetics</h4>
                                         <p> mCherry and KillerRed-expressing M15 bacteria were inoculated at OD610 = 0.015 in LB medium, supplemented with antibiotics and 0.05 mM IPTG. Cell samples were subsequently incubated at 37°C, 200 rpm. Fluorescence (540/630 nm) and OD610 measurements were performed every 20-100 min for 535 min. Erlenmeyers were kept in the dark for the first 180 min, and were then illuminated (P = 0.03 µW/cm<sup>2</sup>) for the rest of the experiment.<br><br></p>
                                         <p> mCherry and KillerRed-expressing M15 bacteria were inoculated at OD610 = 0.015 in LB medium, supplemented with antibiotics and 0.05 mM IPTG. Cell samples were subsequently incubated at 37°C, 200 rpm. Fluorescence (540/630 nm) and OD610 measurements were performed every 20-100 min for 535 min. Erlenmeyers were kept in the dark for the first 180 min, and were then illuminated (P = 0.03 µW/cm<sup>2</sup>) for the rest of the experiment.<br><br></p>
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<p align="center"><img src="https://static.igem.org/mediawiki/2013/a/a9/Grenoble_mCherry_vs_KR.png" alt="mCherry vs KR" width="750px"></p>
<p align="center"><img src="https://static.igem.org/mediawiki/2013/a/a9/Grenoble_mCherry_vs_KR.png" alt="mCherry vs KR" width="750px"></p>
<p id="legend">Figure 1.<br>OD610 <strong>A</strong> and fluorescence <strong>B</strong> as a function of time of mCherry and KillerRed expressing M15 bacteria. Constant light illumination at maximum intensity was applied from 180 min to 535 min. Temperature was measured in each Erlenmeyer during illumination and was shown to stay constant and equal to 37°C. The error bars represent the standard errors of 2 independent measurements.<br><br></p>
<p id="legend">Figure 1.<br>OD610 <strong>A</strong> and fluorescence <strong>B</strong> as a function of time of mCherry and KillerRed expressing M15 bacteria. Constant light illumination at maximum intensity was applied from 180 min to 535 min. Temperature was measured in each Erlenmeyer during illumination and was shown to stay constant and equal to 37°C. The error bars represent the standard errors of 2 independent measurements.<br><br></p>
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<p>Both cell strains display similar growth dynamics in the dark, with growth rates of 1.39h<sup>-1</sup> and 0.57 h<sup>-1</sup> in early (0-120 min) and late (120-180 min) exponential phase, respectively. At t = 255 min occurs a strong decrease in the growth rate of KR-expressing cells as compared to mCherry-expressing cells. This phenomenon, described in the previous section, is due to the killing of bacteria in response to light stimulations. Since the viability of mCherry-expressing cells is not affected, we conclude that KR is responsible for the decrease in the number of living bacteria when illuminating the sample with white light. Cell death is coupled to a decrease in the amount of fluorescing KR proteins. This phenomenon, known as photobleaching, was shown to be a good indicator of the amount of ROS produced by KR upon light illumination [3]. Free radicals such as H<sup>2</sup>O<sup>2</sup> are highly reactive, and cause damage to endogenous proteins and DNA, ultimately leading to cell death. <em>E. coli</em> defense mechanisms against oxidative stress, including the superoxide dismutase and catalase enzymes [4], seem insufficient in preventing significant and irreversible ROS-mediated damages inside bacteria.</p>
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<p>Both cell strains display similar growth dynamics in the dark, with growth rates of 1.39h<sup>-1</sup> and 0.57 h<sup>-1</sup> in early (0-120 min) and late (120-180 min) exponential phase, respectively. At t = 255 min occurs a strong decrease in the growth rate of KR-expressing cells as compared to mCherry-expressing cells. This phenomenon, described in the previous section, is due to the killing of bacteria in response to light stimulations. Since the viability of mCherry-expressing cells is not affected, we conclude that KR is responsible for the decrease in the number of living bacteria when illuminating the sample with white light. Cell death is coupled to a decrease in the amount of fluorescing KR proteins. This phenomenon, known as photobleaching, was shown to be a good indicator of the amount of ROS produced by KR upon light illumination <a href="#ref_bio_1">[7]</a>. Free radicals such as H<sup>2</sup>O<sup>2</sup> are highly reactive, and cause damage to endogenous proteins and DNA, ultimately leading to cell death. <em>E. coli</em> defense mechanisms against oxidative stress, including the superoxide dismutase and catalase enzymes <a href="#ref_bio_1">[8]</a>, seem insufficient in preventing significant and irreversible ROS-mediated damages inside bacteria.</p>
<h3>Cell Growth Recovery after Stopping Illumination</h3>
<h3>Cell Growth Recovery after Stopping Illumination</h3>
<p>We showed that we could either increase or decrease the amount of living cells within our sample, by modulating the amount of light reaching the culture. Indeed, KR-expressing cells were shown to be able to divide in the dark whereas they were killed upon appropriate illumination. But can a culture, initially illuminated, recover and grow again ? In other words: what is the viability status of cells that survive oxidative stress ?<br><br>
<p>We showed that we could either increase or decrease the amount of living cells within our sample, by modulating the amount of light reaching the culture. Indeed, KR-expressing cells were shown to be able to divide in the dark whereas they were killed upon appropriate illumination. But can a culture, initially illuminated, recover and grow again ? In other words: what is the viability status of cells that survive oxidative stress ?<br><br>
To answer this question, we decided to perform a kinetic experiment, in which a square light function (120 min, P = 0.03 µW/cm<sup>2</sup>) was applied. Cells were inoculated at OD610 = 0.02 in 25 mL LB medium, supplemented with antibiotics and 0.05 mM IPTG. The first measurement was performed 30 min after IPTG induction.<br><br></p>
To answer this question, we decided to perform a kinetic experiment, in which a square light function (120 min, P = 0.03 µW/cm<sup>2</sup>) was applied. Cells were inoculated at OD610 = 0.02 in 25 mL LB medium, supplemented with antibiotics and 0.05 mM IPTG. The first measurement was performed 30 min after IPTG induction.<br><br></p>
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<h4>Results</h4>
<h4>Results</h4>
<p align="center"><img src="https://static.igem.org/mediawiki/2013/2/26/Grenoble_recovery_graph.png" alt="results" width="750px"></p>
<p align="center"><img src="https://static.igem.org/mediawiki/2013/2/26/Grenoble_recovery_graph.png" alt="results" width="750px"></p>
<p id="legend">Figure2.<br>OD610 <strong>A</strong> and Fluorescence <strong>B</strong> responses of a culture exposed to a 120 min constant light illumination (P = 0.03 µW/cm2). The illuminated sample is represented in red, the dark sample in blue. Error bars represent the standard errors of duplicates.<br><br></p>
<p id="legend">Figure2.<br>OD610 <strong>A</strong> and Fluorescence <strong>B</strong> responses of a culture exposed to a 120 min constant light illumination (P = 0.03 µW/cm2). The illuminated sample is represented in red, the dark sample in blue. Error bars represent the standard errors of duplicates.<br><br></p>
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<p>As mentioned before, photobleaching of KR is a good indicator of the cytotoxicity induced by this protein upon light stimulation. This phenomenon occurs right after the beginning of the illumination (t = 210 min), the moment at which ROS start being produced and accumulate inside bacteria (Fig 2.B). Fluorescence of the illuminated cell sample still increases during illumination, probably because KR is still being produced by <em>E. coli</em>. There is thus a progressive accumulation of intracellular damages caused by oxidative stress during light illumination. A duration of 120 min of illumination seems long enough for the cell population to accumulate sufficient ROS damage. Indeed, at this illumination threshold, a significant decrease in the amount of living cells is measurable, ultimately leading to the stabilization of the OD610 between 365 and 510 min (Fig 2.A). The cells that have survived the light-induced oxidative stress divide again after time point 510 min.<br><br>
<p>As mentioned before, photobleaching of KR is a good indicator of the cytotoxicity induced by this protein upon light stimulation. This phenomenon occurs right after the beginning of the illumination (t = 210 min), the moment at which ROS start being produced and accumulate inside bacteria (Fig 2.B). Fluorescence of the illuminated cell sample still increases during illumination, probably because KR is still being produced by <em>E. coli</em>. There is thus a progressive accumulation of intracellular damages caused by oxidative stress during light illumination. A duration of 120 min of illumination seems long enough for the cell population to accumulate sufficient ROS damage. Indeed, at this illumination threshold, a significant decrease in the amount of living cells is measurable, ultimately leading to the stabilization of the OD610 between 365 and 510 min (Fig 2.A). The cells that have survived the light-induced oxidative stress divide again after time point 510 min.<br><br>
Thus, it seems possible for an illuminated culture to recover a growth phase with comparable dynamics as for the culture that was kept in the dark (Fig 1.A).
Thus, it seems possible for an illuminated culture to recover a growth phase with comparable dynamics as for the culture that was kept in the dark (Fig 1.A).
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<p>We demonstrated that illumination of a culture of KR-expressing bacteria at maximal intensity (corresponding power density : P = 0.03 µW/cm<sup>2</sup>) could trigger an important decrease in the number of viable cells. How about being able to stabilize the number of living bacteria around a steady value? We thus decided to see whether or not we could change the rate at which cells were killed, by modulating the intensity of the illumination.<br><br>
<p>We demonstrated that illumination of a culture of KR-expressing bacteria at maximal intensity (corresponding power density : P = 0.03 µW/cm<sup>2</sup>) could trigger an important decrease in the number of viable cells. How about being able to stabilize the number of living bacteria around a steady value? We thus decided to see whether or not we could change the rate at which cells were killed, by modulating the intensity of the illumination.<br><br>
                                         In these experiments we simply put an additional light source inside the incubator in order to illuminate two cultures at once, at 100% and 50% light intensity respectively. The light sources were switched on 195 minutes after inoculation, until the end of the kinetic experiment (600 min). Another sample of KR-expressing M15 bacteria was kept in the dark, as a negative control. Results of OD610 and fluorescence measurements are shown in Fig. 2.<br><br></p>
                                         In these experiments we simply put an additional light source inside the incubator in order to illuminate two cultures at once, at 100% and 50% light intensity respectively. The light sources were switched on 195 minutes after inoculation, until the end of the kinetic experiment (600 min). Another sample of KR-expressing M15 bacteria was kept in the dark, as a negative control. Results of OD610 and fluorescence measurements are shown in Fig. 2.<br><br></p>
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                                         <p align="center"><img src="https://static.igem.org/mediawiki/2013/9/9d/Grenoble_Intensity_Graph_%282%29.png" alt="" width="750px"></p>
                                         <p align="center"><img src="https://static.igem.org/mediawiki/2013/9/9d/Grenoble_Intensity_Graph_%282%29.png" alt="" width="750px"></p>
                                         <p id="legend">Figure 2.<br>OD610 (A) and fluorescence (630 nm) (B) as a function of time for 3 different bacterial cell samples, under different light conditions. The sample kept in the dark is represented in blue, the ones illuminated at 50 and 100% of the maximal intensity in red and green, respectively. The light sources were switched on 195 min after inoculation, until the end of the experiment. Error bars represent standard errors of duplicates.<br><br></p>
                                         <p id="legend">Figure 2.<br>OD610 (A) and fluorescence (630 nm) (B) as a function of time for 3 different bacterial cell samples, under different light conditions. The sample kept in the dark is represented in blue, the ones illuminated at 50 and 100% of the maximal intensity in red and green, respectively. The light sources were switched on 195 min after inoculation, until the end of the experiment. Error bars represent standard errors of duplicates.<br><br></p>
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                                         <p>Optical density values of the 3 bacterial cell samples start differing 105 min after the light sources are switched on. ROS-mediated intracellular damages start accumulating inside the bacteria after t = 195 min, leading to a significant change in the number of living cells after t = 300 min (Fig 2.A). For all cultures, OD610 increases after time point 300 min, but at different rates that depend on the intensity of illumination.<br><br>
                                         <p>Optical density values of the 3 bacterial cell samples start differing 105 min after the light sources are switched on. ROS-mediated intracellular damages start accumulating inside the bacteria after t = 195 min, leading to a significant change in the number of living cells after t = 300 min (Fig 2.A). For all cultures, OD610 increases after time point 300 min, but at different rates that depend on the intensity of illumination.<br><br>
                                         According to our expectations, the sample in which the biomass increases the most is the one that was kept in the dark, the condition in which no ROS is produced by KR.<br>
                                         According to our expectations, the sample in which the biomass increases the most is the one that was kept in the dark, the condition in which no ROS is produced by KR.<br>
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                                         <h2 id="ref_bio_1">References</h2>
                                         <h2 id="ref_bio_1">References</h2>
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                                         <p>[1] M.E. Bulina et al., A genetically encoded photosensitizer, <em>Nature Biotechnology</em>, January 2006.<br>
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                                         <p><strong>[1]</strong> M.E. Bulina et al., A genetically encoded photosensitizer, <em>Nature Biotechnology</em>, January 2006.<br>
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                                           [2] Sergei Pletnev et al., Structural Basis for Phototoxicity of the Genetically Encoded Photosensitizer KillerRed, <em>The Journal of Biological Chemistry</em>, November 2009.<br>
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                                           <strong>[2]</strong> Sergei Pletnev et al., Structural Basis for Phototoxicity of the Genetically Encoded Photosensitizer KillerRed, <em>The Journal of Biological Chemistry</em>, November 2009.<br>
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                                           [3] Russell B. Vegh et al., Reactive oxygen species in photochemistry of the red fluorescent protein ‘‘Killer Red’’, <em>Chem. Commun.</em>, 2011.</p>
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                                           <strong>[3]</strong> Russell B. Vegh et al., Reactive oxygen species in photochemistry of the red fluorescent protein ‘‘Killer Red’’, <em>Chem. Commun.</em>, 2011.<br>
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                                          <strong>[4]</strong> 1] N C Shaner et al., Improved monomeric red, orange and yellow fluorescent proteins derived from <em>Discosomasp.</em> red fluorescent protein, <em>Nature Biotechnology</em>, 2004.<br>
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                                          <strong>[5]</strong> [2] W Waldeck et al., ROS-mediated killing efficiency with visible light of bacteria carrying different red fluorochrome proteins, <em>Journal of Photochemistry and Photobiology</em>, 2012.<br>
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                                          <strong>[6]</strong> J D Oliver, Recent findings on the viable but nonculturable state in pathogenic bacteria, <em>FEMS Microbiol. Rev.</em>, 2010.<br>
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                                          <strong>[7]</strong> M E Bulina et al., Chromophore-assisted light inactivation (CALI) using the phototoxic fluorescent protein KillerRed, <em>Nature Protocol</em>, 2006.<br>
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                                          <strong>[8]</strong> E Cabiscol et al. Oxidative stress in bacteria and protein damage by reactive oxygen species, <em>International Microbiology</em>, 2000.<br></p>
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