View source for Part:BBa K1604031

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<partinfo>BBa_K1604031 short</partinfo>

pncB encodes for the enzyme NAPRTase (nicotinic acid phosphorbosyl-transferase). It catalyzes the formation of nicotinate mono-nucleotide, a direct precursor of NAD, from NA (nicotinic acid). This device is controlled by an inducible arabinose promoter.


===Usage and Biology===
PncB catalyzes one of the rate-limiting step in the NAD synthesis pathway. It has been shown that the overexpression of pncB in <i>E.coli</i> increases the intracellular level of NAD(1). 



<div style="text-align:center">[[Image:201_3b.jpg]]</div>
<p style="width:600px; margin-left:150px; margin-bottom:60px;
text-align:justify "><b>FIGURE 1. Biochemical pathway of NAD synthesis.</b> PncB gene encodes the transcription of the NARPTase which catalyzes the formation of nicotinate mono-nucleotide from nicotinic acid.</p>



<div style="text-align:center">[[Image:201_3b.jpg]]</div>
<p style="width:600px; margin-left:150px; margin-bottom:60px;
text-align:justify "><b>FIGURE 2. pncB does not affect cells growth rate.</b> Neb10&beta; cells transformed BBa_K1604031 and grown overnight in LB. The day after cells were diluted 1:100 and grown until they reached an optical density (OD600) of 0.5. The cells were splitted in two different aliquotes of 23 mL and induced with 5mM of arabinose. OD was measured every 45 minutes for 5 hours. Negative control were  cells transformed with an empty plasmid bearing the arabinose promoter from UniTN Trento 2012 (BBa_K   ). All measurements were done for 3 different biological samples and 3 technical measures.</p>



<div style="text-align:center">[[Image:201_3b.jpg]]</div>
<p style="width:600px; margin-left:150px; margin-bottom:60px;
text-align:justify "><b>FIGURE 3. pncB enhances NAD production by ~2.5 fold.</b> Cells expressing pncB (BBa_) and the negative control (BBa_) were grown as described before (figure 2) for a total of 5 hours. After 5 hours the cells the OD was measured again for normalization. 1 mL of cells corresponding to 10<8 were centrifuged and the supernatant was discarded. The NAD and NADH levels were calculated with a colorimetric assay using the Sigma  NAD /NADH quantification kit (MAK037) following the instructions of the technical bulletin. Quantification was based on a standard curve with 0, 20, 40, 60, 80 pmole/well of NADH standard. The kit provides the measures of NAD levels indirectly from total levels of NAD + NADH and NADH only. All samples were normalized by removing the blank.</p>



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<span class='h3bb'>Sequence and Features</span>
<partinfo>BBa_K1604031 SequenceAndFeatures</partinfo>


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===Functional Parameters===
<partinfo>BBa_K1604031 parameters</partinfo>
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