Difference between revisions of "Part:BBa K1604030"

Latest revision as of 22:40, 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. The cells were grown for additional 20 hours. NAD<sup+</sup> and NADH levels were quantified with a colorimetric assay as described in Figure 2. 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.

Susana J. Berrıos-Rivera, K.-Y. San, and G. N. Bennett. (2002) Metabolic Engineering 4, 238–247
Marcel G. Wubbolts, Peter Terpstra, Jan B. van Beilen, Jaap Kingma, H. A. Rene Meesters, and Bernard Witholt. (1990) JBC, 265 (29), 17665-72
Ka-Yiu San, George N. Bennett, Susana J. Berríos-Rivera, Ravi V. Vadali, Yea-Tyng Yang, Emily Horton, Fred B. Rudolph, Berna Sariyar, and Kimathi Blackwood (2002) Metabolic Engineering, 4, 182–192

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 1106
1000
COMPATIBLE WITH RFC[1000]

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 <div style="text-align:center"><html><img src="https://static.igem.org/mediawiki/parts/e/ed/Unitn_pics_2015_NADpathway.png" style="width:75%;"></img></div></html>
 <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. This is considered the limiting step of the synthesis of NAD+. </p>
+text-align:justify "><b>FIGURE 1. Biochemical pathway of NAD<sup>+</sup> synthesis.</b> 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+. </p>
 <div style="text-align:center"><html><img src="https://static.igem.org/mediawiki/parts/b/b3/Unitn_pics_2015_WellRatioNAD-NADH.png" style="width:80%;"></img></div></html>
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 <div style="text-align:center"><html><img src="https://static.igem.org/mediawiki/parts/6/63/Unitn_pics_2015_NADAnaerobic.png" style="width:50%;"></img></div></html>
 <p style="width:600px; margin-left:150px; margin-bottom:60px;
-text-align:justify "><b>FIGURE 3. PncB enhances NAD production by 13 fold in anaerobic condition in LB broth.</b> 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. </p>
+text-align:justify "><b>FIGURE 3. PncB enhances NAD production by 13 fold in anaerobic condition in LB broth.</b> 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. The cells were grown for additional 20 hours. NAD<sup+</sup> and NADH levels were quantified with a colorimetric assay as described in Figure 2. 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. </p>