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

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<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>
 
<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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<h4 class="header4 wow fadeInDown delay05"> <span>Light or no light that is the question.</span> <i class="faabig flaticon-shield114"></i></h4>
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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>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>
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  <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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<a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/3/3a/Unitn_pics_results_pr7.jpg" title="Apparatus for anaerobiosis growth"><img src="https://static.igem.org/mediawiki/2015/7/75/Unitn_pics_results_pr7_thumb.jpg" alt="" style="width:100%; max-width:700px;"/></a>
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<p class="image_caption"><span>Apparatus for anaerobiosis growth</span>Panel A) sealed sterile bottles. Panel B)
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Anaerobic chamber.</p>
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<p class="image_caption"><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>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>
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    <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>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 10:31, 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]

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!

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

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.