Experiments | Paris_Bettencourt

Characterisation pOxyS - dpB.004

Abstract

Using elements from the native redox OxyRS transcription regulation, we designed a simple construct which is induced by the presence of electrochemically generated hydrogen peroxide (H2O2) (1). OxyR is a native Escherichia coli transcription factor that becomes oxidised by H2O2 and, in its oxidised form, will up-regulate the OxyS gene via its promoter pOxyS.

For our construct to act as an Input component, the coding sequence for the transcription factor CinR was added downstream of pOxyS, connecting it to the Processor component by pCinR. This will allow the Processor to be activated in the presence of hydrogen peroxide.

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Materials and methods

Cloning

We found the sequence of*pOxyS on NCBI and produced two Input constructs, which we ordered with IDT.

The first *pOxyS* Input construct has a downstream mScarlett fluorescent coding sequence to report on the activation of the pOxyS promoter in the presence of H2O2.
[dpB.004 - pB.006_pOxyS, U4m, C99m, T12m]

The second pOxyS Input construct has a downstream cinR coding sequence, which encodes the transcription factor for the downstream activation of the Processor construct. [dpB.005 - pB.006_pOxyS, U4m, C71m, T12m]

We assembled both these constructs into several vectors using Golden Gate cloning . First, we assembled the construct into the V35 vector plasmid (Bba_K4216046 ) . Then we assembled it into the three pDuet vector plasmids pCOLADuet (BBa_K4216045), pACYDuet(Bba_K4216043) and pCDFDuet (BBa_K4216044), so we could combine the Input, Processor, and Output components together. We transformed each of these plasmids into a DH5alpha strain of *E. coli*. We confirmed all transformations by sequencing.

dpB.004

Hydrogen peroxide dose response

We characterised the induction of pOxyS with H2O2 using the dpB.XXX construct (iGEM XXXX) in the V35 vector (iGEM XXXX) by performing a dose-response plate reader experiment and measuring fluorescence. 35% hydrogen peroxide was purchase from Sigma Aldrich and diluted in dionised water to create a stock solution of 1 mM. This stock solution was diluted at desired experimental concentrations in LB + Kanamycin.

Cells were grown overnight in LB with Kanamycin and diluted 1:100 and grown until an OD600 of 0.2 was reached. Triplicates of diluted cultures were inoculated in a 96-well plate containing LB media + Kanamycin with varying concentrations of H2O2 and grown for 12 hours in a Tecan Infinite® 200 PRO plate reader. The concentrations tested were 0, 1, 5, 10, 50, 100, 150, 200, and 250 uM. The final value of fluorescence was divided by OD600.

Results

After 12 hours, cells showed an increased fluorescence as the concentration of H2O2 increased. This shows that the pOxyS promoter is induced by H2O2. However, cells showed a decrease in growth at concentrations 100 uM and higher, likely due to oxidative stress hindering growth. This suggest a range of 50 to 100 uM for optimal induction of the pOxyS.

Future Work

Further experiments are needed to measure the potential of pOxyS to be electrochemically induced. The next step that we would have taken if we had more time is to generate different concentrations of H2O2 by applying a constant voltage potential to LB media to produce H2O2 from water. We would use varying periods of applied potential to generate varying concentrations of H2O2 and use the Pierce Quantitative Peroxide Assay Kit to measure the concentration precisely. We would test whether the applied voltage has an affect on the growth of the bacteria, and then measure the correlation of fluorescence to voltage potential.

References

Terrell, J.L.,Tschirhart, T., Jahnke, J.P. et al. Bioelectronic control of a microbial community using surface-assembled electrogenetic cells to route signals. Nat. Nanotechnol. 16, 688–697 (2021). https://doi.org/10.1038/s41565-021-00878-4