Difference between revisions of "Part:BBa K1604030"

Revision as of 22:36, 17 September 2015

pncB

pncB encodes for the enzyme NAPRTase (nicotinic acid phosphoribosyl-transferase). It catalyzes the formation of nicotinate mono-nucleotide, a direct precursor of NAD, from NA.[1] [2] [3].

Usage and Biology

FIGURE 1. Biochemical pathway of NAD⁺ synthesis. PncB gene encodes the transcription of the NARPTase which catalyzes the formation of nicotinate mono-nucleotide from nicotinic acid. This is considered the limiting step of the synthesis of NAD+.

FIGURE 2. PncB increases NAD+ levels by 2.5 fold and NADH levels by 1.4 fold when expressed in NEB10β. NAD+ and NADH levels were calculated with a colorimetric assay using the Sigma NAD/NADH quantification kit (MAK037) following the instructions described in the technical bulletin. The kit provides the measures of NAD+ levels indirectly from total levels of NAD + NADH and NADH only. Panel A. Colorimetric assay for NAD/NADH quantification. Panel B. NAD/NADH levels for three biological samples expressing BBa_K1604031 and one negative control expressing BBa_K731201.

FIGURE 3. PncB enhances NAD production by 13 fold in anaerobic condition in LB broth. NAD/NADH ratio between negative control and cell expressing BBa_K1604031, both samples were growth in LB medium. Both samples after 6 hours of induction in Thermoshaker (37°C, 190 rpm) were transferred in sealed glass bottles with a rubber septum under anaerobic work station to keep samples without oxygen. PncB does increase NAD+ levels by ~ 13 fold. In this graphic there are no bars of standard deviation because test was made for an only biological sample for negative control araC-pBAD and two samples for pncB because we have limiting glass to put under anaerobic work station.

For more information about the pncB (BBa_K1604030) characterization go on BBa_K1604031 page, or on Unitn iGEM 2015 project checking out our Wiki: Unitn iGEM wiki 2015.