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

Revision as of 22:12, 5 September 2015

Results

Introduction to the Results

Proteorhodopsin

Proteorhodopsin (PR) is a light-powered proton pump that belongs to the rhodopsin family. It is a 7-transmembrane protein, which uses all-trans-retinal as the chromophore. It uses light energy to generate an outward proton flux. The increased proton motive force across the membrane can power cellular processes, such as ATP synthesis, chemiosmotic reactions and rotary flagellar motor [1]. Furthermore, it was demonstrated that light-activated proton pumping by proteorhodopsin can drive ATP synthesis as proton reenter the cell through the H+-ATP synthase complex[2].

Proposed mechanism of PR associated to the ATP-synthase complex Light-activated proteorhodopsin pumps protons outwardly, increasing the proton motive force. Protons can then reenter the cells through ATP-synthase complex, powering the ATP production.

The sequence of our part belongs to the uncultured marine Gammaproteobacteria of the SAR86 group. The original cluster is composed of 6 genes: four are involved in beta- carotene production; one is implied in beta carotene cleavage into two molecules of retinal, the other encodes for proteorhodopsin. From the analysis of our part sequence we found out that our protein belongs to the blue absorbing group. [3]

Schematic representation of the PR gene cluster identified in clone HF10_19P19Predicted transcription terminators are indicated in red. (Four genes are for beta-carotene synthesis, blh for retinal production, and PR itself.

PncB: nicotinic acid phosphorbosyl-transferase

Increasing the levels of NADH

Our goal was to boost electron production by increasing the concentration of electron carriers (i.e. NADH). To achieve this goal we decided to engineer E. coli with an enzyme that would provide more intracellular NAD, and thus NADH.

PncB catalyzes one of the rate-limiting step in the NAD synthesis pathway. This gene is naturally found in E. coli and encodes for the enzyme NAPRTase (nicotinic acid phosphorbosyl- transferase) that catalyzes the formation of nicotinate mono-nucleotide, a direct precursor of NAD, from NA (nicotinic acid).

Our device is controlled by an inducible arabinose promoter built by the Unitn iGEM team in 2012. PncB was extracted by E. coli genome, the illegal site PstI was removed, and it was placed in pSB1C3 (BBa_K1604030). Subsequently it was placed under the araC-pBAD promoter (BBa_K1604030).

PncB is not toxic if overexpressed in E.coli

NEB10β transformed with BBa_K1604030 (araC-pBAD-pncB) or BBa_K731201 (i.e. araC-pBAD) were grown up to an OD of 0.6, splitted in two tubes of 23 mL each and induced with 5 mM of arabinose. Negative controls were not induced.
The OD (600 nm) was measured every 45 minutes for 5 hours. All measurements were done for 3 different biological samples and 3 technical measures.

Although the growth rate is slightly decreased, due to the cell stress when expressing pncB, the data indicate that this enzyme does not have toxicity effect on the cells.

Growth rate of BBa_K1604031 (aracpbad-pncb) and BBa_K731201 (i.e. aracpBad). Cells were grown up to an OD of 0.6 and splitted before induction with arabinose. BBa_K1604031 (Orange line) and BBa_K731201 (green line) induced with 5 mM arabinose. BBa_K1604031 (yellow line) and BBa_K731201 (blue line) not induced.

PncB enhances NAD production by ~2.5 fold

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

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

__Title of the Figure__ NAD and NADH levels were quantified with Sigma NAD /NADH quantification kit (MAK037) following the instructions described in the technical bulletin. Panel A: Standard curve (0, 20, 40, 60, 80, 100 pmole/well of NADH)). Panel B: NAD/NADH levels for three biological samples of BBa_K1604030 (green) and one negative control BBa_K731201 (blue). The cells were grown as described previously.

__Title of the Figure__ Lane B samples 2-7 calibration curve (0, 20, 40, 60, 80, 100 pmole/well of NADH). Lane C samples 2-9 NAD total levels; Lane D samples 2-9 NAD total repeated with a 2 fold concentrated sample; Lane E NADH only; Lane F NADH only, repeated with a 2 fold concentrated sample. In lanes C-F the order of the samples is: 2 technical replicates of the negative control, and 2 technical replicates of each of the 3 biological samples of BBa_K1604031. The plate was read with a Tecan Infinite M-200 pro instrument at 450 nm. The measurements were taken after 0.5, 1, 2, 3, 4 hours to allow color development. The data shown are representative of the best measurement at 2 hours.

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.

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 										<p>Proteorhodopsin (PR) is a light-powered proton pump that belongs to the rhodopsin family. It is a 7-transmembrane protein, which uses all-trans-retinal as the chromophore. It uses <span class="i_enph">light energy</span> to generate an <span class="i_enph">outward proton flux</span>. The increased proton motive force across the membrane can power cellular processes, such as ATP synthesis, chemiosmotic reactions and rotary flagellar motor [1]. Furthermore, it was demonstrated that light-activated proton pumping by proteorhodopsin can drive ATP synthesis as proton reenter the cell through the H+-ATP synthase complex[2].</p>
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-										<a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/5/50/Unitn_pics_results_prscheme.jpg" title="Proposed mechanism of PR associated to the ATP-synthase complex"><img src="https://2015.igem.org/File:Unitn_pics_results_prscheme_thumb.jpg" alt="" style="width:100%; max-width:700px;"/></a>
+										<a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/5/50/Unitn_pics_results_prscheme.jpg" title="Proposed mechanism of PR associated to the ATP-synthase complex"><img src="https://static.igem.org/mediawiki/2015/2/2b/Unitn_pics_results_prscheme_thumb.jpg" alt="" style="width:100%; max-width:700px;"/></a>
 										<p class="image_caption"><span>Proposed mechanism of PR associated to the ATP-synthase complex</span> Light-activated proteorhodopsin pumps protons outwardly, increasing the proton motive force. Protons can then reenter the cells through ATP-synthase complex, powering the ATP production.</p>
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 										<a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/1/1b/Unitn_pics_project_cluster_pr.png" title="Schematic representation of the PR gene cluster identified in clone HF10_19P19"><img src="https://static.igem.org/mediawiki/2015/d/db/Unitn_pics_project_cluster_pr_thumb.png" alt="" style="width:100%; max-width:700px;"/></a>
 										<p class="image_caption"><span>Schematic representation of the PR gene cluster identified in clone HF10_19P19</span>Predicted transcription terminators are indicated in red. (Four genes are for beta-carotene synthesis, blh for retinal production, and PR itself.</p>