Team:LZU-China/wetlab3

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<!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.

PNP PROMOTE THE RIBOFLAVIN'S EXPRESS

Genetically modified E.coli was able to produce quantitative riboflavin dependent on substrate’s concentration.

Using a pollutant substrate bio-sensor coupling riboflavin synthetic gene cluster, the genetically modified E.coli with plasmid pET28A can secrete riboflavin when we add PNP in medium.

The E.coli was incubate with LB broth overnight at 37℃,200rpm and feed with quantitative PNP and incubate for another 12 hours fermentation. Supernatant was collected by centrifuge at 4000×g and adjusted pH to 3~4 in order to eliminate the PNP’s absorb of blue light, which may disorder the measurement of riboflavin. Supernatant was transferred to spectrophotometer and measured absorb value at 444nm. E.coli strain DH5αfermented LB broth was treated by same method above as the control group set at 0 absorption value. A riboflavin standard curve was established for quantitatively measurement (figure 6).

Figure-6. Riboflavin OD value standard curve at 444nm.

E.coli SUPER SEVEN!
We test many single colonies of genetically modified E.coli and screen their riboflavin production at same condition, then we get the most efficiency bacteria strain—Super Seven—as our seed bacteria. As shown in figure6, when treated with concentration gradient of PNP, Super Seven was able to secrete a stabilized and quantitatively amount of riboflavin. PNP, a toxic industrial pollutant could influence the microorganism’s metabolism and growth, so that curve goes down when PNP addition is high.

Figure-7. Concentration of riboflavin secreted by Super Seven treated with different amount of PNP.

We used Gauss function to fit that curve, it’s a good mathematics model so far (figure 8).

Figure-8. A Gauss function was built to fit curve in figure 7, the formula of this curve is
y=0.4186 + (0.96352/(0.369*SQRT(3.1416/2)))*EXP(-2*((x-0.44771)/0.369)^2)

and the adjusted R-square value is 0.929.

Riboflavin can stimulate the MFC power output.

When E.coli Super Seven secrete riboflavin to the anode medium or add riboflavin manually, the MFC system will have a better performance. The riboflavin is a kind of redox mediator; it could enhance the MFC power output in a great scale in just a few minute (figure 8).

Figure-9. Gradient concentrations of riboflavin could raise the MFC voltage, measured with 200Ω external resistance，peak voltages were recorded within 5 hours after riboflavin added.

We found that the most important dependent variable was not the power density or peak voltage as we think previously, instead, voltage increment value reflect riboflavin’s concentration best. Another fact was that the voltage increments were often dependent on former power output.

Measure PNP concentrations with MFC system

Measure PNP concentrations with a PNP bio-sensor coupling riboflavin MFC stimulate system.

We tried to make E.coli Super Seven co-exist with Shewanella in anode medium at the same time. When PNP pollutant was added, Super Seven could secrete riboflavin; as a result, MFC voltage would rise and reflect the PNP concentrations. Though riboflavin’s expression quantity was relative low as the iGEM authority plasmid pSB1C3 is not the best expression vector, we still found the interrelationship between PNP addition and voltage increment.

Figure-10. Using MFC devices measuring concentrations of PNP obtained a fitting curve.

We introduced(ΔE) as dependent variable, PNP concentration is independent variable and the previous stable voltage of MFC without pollutant treatment, E1, is an important parameter in this interrelationship. We also created a constant value, which dependent on our novel MFC devices and experimental data.

We conclude an empirical formula for our devices:

In our experiments with MFC devices designed by ourselves, the MFC Characteristic Constant is a certain value, C=1.4034.

We can measure the voltage value,ΔE, and deduce the PNP pollutant concentration, vice versa.