Difference between revisions of "Team:UNITN-Trento/Results"

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<p style="clear:both;">We have screened several parameters (media, temperature, time of induction) to discover  that the optimal expression conditions were in LB at 37C overnight in the presence of 10  &mu;M of all-trans retinal. Attempts to express the protein in the absence of retinal failed.  Proteorhodopsin is a membrane protein that needs the time to fold properly into the  membrane and requires retinal to bind the pocket and help the formation of the proper  folding.</p>
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<p style="clear:both;">We have screened several parameters (media, temperature, time of induction) to discover  that the optimal expression conditions were in LB at 37&deg;C    overnight in the presence of 10  &mu;M of all-trans retinal. Attempts to express the protein in the absence of retinal failed.  Proteorhodopsin is a membrane protein that needs the time to fold properly into the  membrane and requires retinal to bind the pocket and help the formation of the proper  folding.</p>
 
<p>The expected molecular size is 28 KDa. The SDS gel shows a band corresponding to around 37 KDa, as it was seen in other studies [4]. This is probably due to post-translational modifications.</p>
 
<p>The expected molecular size is 28 KDa. The SDS gel shows a band corresponding to around 37 KDa, as it was seen in other studies [4]. This is probably due to post-translational modifications.</p>
 
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<a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/1/12/Unitn_pics_results_prfalcons.jpg" title="Purification of Proteorhodopsin. "><img src="https://static.igem.org/mediawiki/2015/2/2c/Unitn_pics_results_prfalcons_thumb.jpg" alt="" style="width:100%; max-width:800px;"/></a>
 
<a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/1/12/Unitn_pics_results_prfalcons.jpg" title="Purification of Proteorhodopsin. "><img src="https://static.igem.org/mediawiki/2015/2/2c/Unitn_pics_results_prfalcons_thumb.jpg" alt="" style="width:100%; max-width:800px;"/></a>
<p class="image_caption"><span>Purification of Proteorhodopsin. </span>NEB10&beta; cells transformed with    BBa_K1604010 and BBa_K731201 were induced in LB at 37C in the presence of retinal.    The cell pellets were resuspended in 50 mM Tris-Cl pH 8 with 5 mM MgCl2 and sonicated.    The lysate was centrifuged at 10,000 rpm for 20 min at 4C. The supernatant was    ultracentrifuged for 100,000 g for 3 hours at 4C. The three tubes in front contain    proteorhodopsin purified fractions and the three tubes in the back are negative controls    treated in the same conditions</p>
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<p class="image_caption"><span>Purification of Proteorhodopsin. </span>NEB10&beta; cells transformed with    <a href="http://parts.igem.org/Part:BBa_K1604010" target="_blank">BBa_K1604010</a> and <a href="http://parts.igem.org/Part:BBa_K731201" target="_blank">BBa_K731201</a> were induced in LB at 37&deg;C    in the presence of retinal.    The cell pellets were resuspended in 50 mM Tris-Cl pH 8 with 5 mM MgCl2 and sonicated.    The lysate was centrifuged at 10,000 rpm for 20 min at 4C. The supernatant was    ultracentrifuged for 100,000 g for 3 hours at 4C. The three tubes in front contain    proteorhodopsin purified fractions and the three tubes in the back are negative controls    treated in the same conditions</p>
 
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<a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/2/26/Unitn_pics_results_prfalcons2.jpg" title="Proteorhodopsin expression in M9"><img src="https://static.igem.org/mediawiki/2015/0/01/Unitn_pics_results_prfalcons2_thumb.jpg" alt="" style="width:100%; max-width:600px;"/></a>
 
<a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/2/26/Unitn_pics_results_prfalcons2.jpg" title="Proteorhodopsin expression in M9"><img src="https://static.igem.org/mediawiki/2015/0/01/Unitn_pics_results_prfalcons2_thumb.jpg" alt="" style="width:100%; max-width:600px;"/></a>
<p class="image_caption" style="margin-top:1.1em;"><span>Proteorhodopsin expression in M9</span>Cells transformed with BBa_K1604010     and BBa_K731201 were grown in LB and transferred in M9 at an OD of 0.6 and induced    with arabinose with the presence of 10 &micro;M of retinal. After 6 hours of induction the cells    were centrifuged and the supernatant was discarded. From left to right: araC-pBAD    induced with retinal (A), proteorhodopsin induced with retinal (B), proteorhodopsin induced    (C) and not induced (D) both without retinal.</p>
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<p class="image_caption" style="margin-top:1.1em;"><span>Proteorhodopsin expression in M9</span>Cells transformed with <a href="http://parts.igem.org/Part:BBa_K1604010" target="_blank">BBa_K1604010</a> and <a href="http://parts.igem.org/Part:BBa_K731201" target="_blank">BBa_K731201</a> were grown in LB and transferred in M9 at an OD of 0.6 and induced    with arabinose with the presence of 10 &micro;M of retinal. After 6 hours of induction the cells    were centrifuged and the supernatant was discarded. From left to right: araC-pBAD    induced with retinal (A), proteorhodopsin induced with retinal (B), proteorhodopsin induced    (C) and not induced (D) both without retinal.</p>
 
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<p>Proteorhodpsin is a light activated proton pump that exploits the conformational change of  all trans-retinal to all cis-retinal. The different absorption properties are due to a single  amino acid, at position 105 in the retinal binding pocket. The presence of a highly  conserved Gln at position 105 in BBa_K1604010 indicates that it belongs to the blue  absorbing family. [3]</p>
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<p>Proteorhodpsin is a light activated proton pump that exploits the conformational change of  all trans-retinal to all cis-retinal. The different absorption properties are due to a single  amino acid, at position 105 in the retinal binding pocket. The presence of a highly  conserved Gln at position 105 in <a class="i_linker" href="http://parts.igem.org/Part:BBa_K1604010" target="_blank">BBa_K1604010</a> indicates that it belongs to the blue  absorbing family. [3]</p>
 
 
 
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Anaerobic chamber.</p>
 
Anaerobic chamber.</p>
 
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<p>We tested if light activation with a white light bulb (160W) containing the blue wavelength, activates proteorhodopsin, thus making the bacteria survive better anaerobically.</p>
 
<p>We tested if light activation with a white light bulb (160W) containing the blue wavelength, activates proteorhodopsin, thus making the bacteria survive better anaerobically.</p>
 
<p>Anaerobiosis was achieved using sealed glass bottles with a rubber septum. We got from the local pharmacy 12 sterile bottles of physiological solution. After removing the liquid, washing them and autoclaving them, the bottles were ready to host our bacteria!</p>   
 
<p>Anaerobiosis was achieved using sealed glass bottles with a rubber septum. We got from the local pharmacy 12 sterile bottles of physiological solution. After removing the liquid, washing them and autoclaving them, the bottles were ready to host our bacteria!</p>   
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<p class="image_caption" style="margin-top:0;"><span>Anaerobioc growth of BBa_K1600410</span> E. coli transformed with BBa_K1604010 (blue line) and BBa_K731201 (green line) were grown in LB at 37C untilan OD of 0.6 and induced in M9 minimal medium with 5 mM arabinose and 10 uM retinal in the dark. After 5 hours of induction the culture were transferred in sealed bottles in the anaerobic chamber and placed again in the thermoshaker. Sample in the dark were kept in aluminum foil. Light exposed samples were excited with a 160W halogen light bulb placed outside the incubator. The blue line (proteorhodopsin) is the result of the average of 6 different samples (3 in the dark and 3 in the light) while the green line (araC-pBAD) is the average of 1 sample in the dark and 1 in the light.</p>
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<p class="image_caption" style="margin-top:0;"><span>Anaerobioc growth of BBa_K1600410</span> E. coli transformed with <a href="http://parts.igem.org/Part:BBa_K1604010" target="_blank">BBa_K1604010</a> (blue line) and <a class="i_linker" href="http://parts.igem.org/Part:BBa_K731201" target="_blank">BBa_K731201</a> (green line) were grown in LB at 37&deg;C    untilan OD of 0.6 and induced in M9 minimal medium with 5 mM arabinose and 10 uM retinal in the dark. After 5 hours of induction the culture were transferred in sealed bottles in the anaerobic chamber and placed again in the thermoshaker. Sample in the dark were kept in aluminum foil. Light exposed samples were excited with a 160W halogen light bulb placed outside the incubator. The blue line (proteorhodopsin) is the result of the average of 6 different samples (3 in the dark and 3 in the light) while the green line (araC-pBAD) is the average of 1 sample in the dark and 1 in the light.</p>
 
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<p>After five hours of induction in the dark (i.e. the samples were wrapped in aluminum foils)  the cultures were split in the anaerobic chamber in light and dark conditions. The cultures  were placed in the thermoshaker that was illuminated from the outside. Half of the cultures  were kept in the dark and the other half were exposed to the light. <br /> The OD<sub>600</sub> was constantly monitored because E. coli’s growth is slowed down in stressful conditions such as the lack of oxygen.</p>
 
<p>After five hours of induction in the dark (i.e. the samples were wrapped in aluminum foils)  the cultures were split in the anaerobic chamber in light and dark conditions. The cultures  were placed in the thermoshaker that was illuminated from the outside. Half of the cultures  were kept in the dark and the other half were exposed to the light. <br /> The OD<sub>600</sub> was constantly monitored because E. coli’s growth is slowed down in stressful conditions such as the lack of oxygen.</p>
     <p>The bacteria expressing proteorhodopsin have an increased lifetime when compared to a negative control with araC-pBAD (BBa_K731201). However we did not observe significant changes between light and dark with this test. The explanations could be several. Most likely we were not exciting properly the system. However it seems that there is a basal functionality even in the absence of light, probably due to activation of the proton pump independently from light exposure.</p>
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     <p>The bacteria expressing proteorhodopsin have an increased lifetime when compared to a negative control with araC-pBAD (<a class="i_linker" href="http://parts.igem.org/Part:BBa_K731201" target="_blank">BBa_K731201</a>). However we did not observe significant changes between light and dark with this test. The explanations could be several. Most likely we were not exciting properly the system. However it seems that there is a basal functionality even in the absence of light, probably due to activation of the proton pump independently from light exposure.</p>
 
<p>While we decided to explore different light sources, we built a solar mimicking apparatus, that would allow us to directly illuminate the samples without the glass of the thermoshaker.</p>  
 
<p>While we decided to explore different light sources, we built a solar mimicking apparatus, that would allow us to directly illuminate the samples without the glass of the thermoshaker.</p>  
 
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Revision as of 10:40, 7 September 2015

Results

  • Proteorhodopsin

  • PncB NAD Booster

Introduction to the Results

Proteorhodopsin

Proteorhodpsin is a light activated proton pump that exploits the conformational change of all trans-retinal to all cis-retinal. The different absorption properties are due to a single amino acid, at position 105 in the retinal binding pocket. The presence of a highly conserved Gln at position 105 in BBa_K1604010 indicates that it belongs to the blue absorbing family. [3]

Apparatus for anaerobiosis growthPanel A) sealed sterile bottles. Panel B) Anaerobic chamber.

We tested if light activation with a white light bulb (160W) containing the blue wavelength, activates proteorhodopsin, thus making the bacteria survive better anaerobically.

Anaerobiosis was achieved using sealed glass bottles with a rubber septum. We got from the local pharmacy 12 sterile bottles of physiological solution. After removing the liquid, washing them and autoclaving them, the bottles were ready to host our bacteria!

After five hours of induction in the dark (i.e. the samples were wrapped in aluminum foils) the cultures were split in the anaerobic chamber in light and dark conditions. The cultures were placed in the thermoshaker that was illuminated from the outside. Half of the cultures were kept in the dark and the other half were exposed to the light.
The OD600 was constantly monitored because E. coli’s growth is slowed down in stressful conditions such as the lack of oxygen.

The bacteria expressing proteorhodopsin have an increased lifetime when compared to a negative control with araC-pBAD (BBa_K731201). However we did not observe significant changes between light and dark with this test. The explanations could be several. Most likely we were not exciting properly the system. However it seems that there is a basal functionality even in the absence of light, probably due to activation of the proton pump independently from light exposure.

While we decided to explore different light sources, we built a solar mimicking apparatus, that would allow us to directly illuminate the samples without the glass of the thermoshaker.

PncB: nicotinic acid phosphorbosyl-transferase

Our goal was to demonstrate that pncB increased intracellular levels of NAD and thus NADH. We quantified the levels of NAD by a colorimetric test that measures the levels of NAD indirectly by quantifying the concentration of NAD total (NAD + NADH) and NADH only. To make precise quantitation a standard curve with NADH was built. The test provides the ratio of NAD/NADH

NADtotal = Amount of total NAD (NAD+NADH) in unknown sample (pmole) from standard curve.
NADH = Amount of NADH in unknown sample (pmole) from standard curve.

BBa_K1604031 does increase NAD levels by 126% (2.5 fold) and NADH levels by 44% (1.4 fold) when expressed in NEB10β. Although we did see an enhancement in NAD levels, this did not correlate to a proportional boost in NADH levels. We plan in the future to add a NAD reducing enzyme and to give a medium able to enhance the cell metabolism to further increase NADH intracellular levels.