Team:LZU-China/wetlab2

From 2014.igem.org

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" " http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd"> LZU-China 2014

We constructed a pollutant substrate(PNP) bio-sensor coupling riboflavin synthetic gene cluster, the genetically modified E.coli can secrete riboflavin when added PNP in MFC anode medium.Riboflavin is a efficient redox mediator as well as a stimulator of MFC. We created a novel MFC devices and built a quantitative monitor system of PNP via measuring voltage increment.

GENETIC ENGINEERED BACTERIA

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.

Figure-1 The pathway of PNP sensor

Besides, we find many genes can catalyze the reduction of Cr(VI). They can be used in the cathode to reduce these heavy metal ions.

Figure-2 The pathway of the Cr ion reductase(yieF as an example)

ABOUT THE CONSTRUCTION OF PNP SENSOR

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-3 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-4 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-5 Gel picture of K1523101’s PCR

ABOUT THE REDUCING Cr IONS GENES

We found four genes to reduce the Cr(VI) to Cr(III). They are nahG, nahR, nahE, chrR and yieF. We didn’t do the deep research about them because of time limitation. They can be used in the general plasmids such as pBR322. We just made them to be the formation of Biobrick. More uses and functions need to be demonstrated and found.

nahG: We got the sequence from Pseudomonas putida

          (Primer:sense:5’---CCGGAATTCGCGGCCGCTTCTAGATGAAAAACAATAAACCTGGCTTGCGC---3’;
          anti-sense:5’---AAAATACTAGTAGCGGCCGCTGCAGTCACCCTTGACGTAGCACACC---3’)
nahE: We got the sequence from Pseudomonas putida
          (Primer:sense:5’---CCGGAATTCGCGGCCGCTTCTAGATGTCGAATAAAATTATGAAAACGTCGCG---3’;
          anti-sense:5’---AAAATACTAGTAGCGGCCGCTGCAGCTACTTCAATTCATTACTGTATTTAGCGTG---3’)
nahR: We got the sequence from Pseudomonas putida
          (Primer:sense:5’--- CCGGAATTCGCGGCCGCTTCTAGATGAAAAACAATAAACCTGGCTTGCGC---3’;
          anti-sense:5’---AAAATACTAGTAGCGGCCGCTGCAGTCACCCTTGACGTAGCACACC---3’)
chrR: We got the sequence from Escherichia coli str. K-12
          (Primer:sense:5’--- CCGGAATTCGCGGCCGCTTCTAGATGAAAAAAATAGTCCAGTCGGAAGG---3’;
          anti-sense:5’---AAAATACTAGTAGCGGCCGCTGCAGTCAGGCCTTTTTGTGCTGTTCAAC---3’)
yieF: We got the sequence from Escherichia coli str. K-12
          (Primer:sense:5’--- GAATTCGGATCCATGTCTGAAAAATTGCAGGTGG---3’;
          anti-sense:5’---GAATTCTCTAGATTAGATCTTAACTCGCTGAATAAA---3’)