Part:BBa_K1523101

PyeaR->Riboflavin synthase

It can sense p-nitrophenol by Pyear.When PNP exits,it can produce electry in MFC.

Abstract

Microbial Fuel Cells (MFCs) which can convert contaminants in wastewaters into energy is an ideal approach to solve both pollution problem and energy crisis. However, MFCs is hard to determine the contaminants concentrations which is a major drawback for MFCs applications. We found that the electricity produced by MFCs was somewhat related to the substrates concentrations such as P-nitrophenol (PNP) in anode or Chromium (VI) in cathode. Then a novel MFC system was designed. For anode strain, we’ve cloned a PNP sensor sequence and a riboflavin into Escherichia coli. The recombinants is able to detect PNP and produce riboflavin to boost electrical generation when co-cultured with Shewanella oneidensis.

Construction

We found that the part BBa_K381001 by iGEM10_BCCS-Bristol is a sensor which can detect the appearance of nitrate and nitrite. So we perfomed an experience by this part to see if this part can detect PNP(p-Nitrophenol). We got a good result.You can see the PNP can also induce the green fluorescence.

Figure-1 Fluorescence of different system. a.bacterial liquid with 10mM PNP;b.bacterial liquid with ddH2O;c.bacterial liquid with 10mM KNO3;d.bacterial liquid with 10mM KCl.

Firstly we got the plasmid with K1172303 from Registry. We cut the plasmid by EcoRI and XbaI, then put the Pnsr(K216005+B0030 this sequence was synthesized by company) into the gap.

Figure-2 Construction of K1523101

The size of the whole part is about 3700bp(without plasmid), we test the assembling result by PCR(sense:5’---TTCCCATCTATAATCCTCCCTGATTCTTCG---3’;anti-sense:5’---GAATTCTCTAGATTACAACTGTTGTTCAAGCTGTT---3’). From the gel picture we can see the size is right.

Figure-3 Gel picture of K1523101’s PCR

Pathway

Figure-4 The pathway of PNP sensor

We designed this pathway for our purpose. As you can see, the NsrR is constitutive expressed in almost all E. coli orgnisms and it can repress the promoter PyeaR. In our project we found that this sensitive system is also adjusted to the PNP(p-nitrophenol). So we constructed this sensor pathway.

The sensor will cause the expression of riboflavin, a kind of mediator which can induce the generation of current in MFCs, to show the appearance of PNP. We can estimate the concentration of PNP.

Application

Figure-5 The riboflavin producing value on the increasing of PNP.

Discussion:

As you can see, the concentration of riboflavin is increasing with the increasing of PNP under 0.5mM. Then it decreased. A reason is that the PNP can kill the strain so they cannot produce riboflavin. While the strain always can live in the 20mM PNP so we should determine the real reason in the future.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1243
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]