Experiments | Paris_Bettencourt

pSoxS - dpB.002

Abstract

Using redox responsive elements from the native E. coli SoxRS regulon, we designed constructs that are induced by the presence of the redox molecule pyocyanin, in it’s oxidised form. We contacted Joshua Lawrence who was part of the 2018 Imperial College iGEM team and who authored a paper that demonstrated that a redesigned version of the native pSoxRS could be used as an electrochemically induced promoter via electronically modulating the redox state of pyocyanin (1).
We reproduced a dose response pyocyanin induction with their redesigned pSoxR for two constructs in our electrogenetic system We also tested two of the constructs in the Lawrence et al. paper, one meant to activate a fluorescent reporter gene in the presence of oxidised pyocyanin (Act106), and one meant to repress expression (Inv106). The sequences of the Act106 and Inv106 constructs can be found on Joshua Lawrence’s GitHub page (https://github.com/JLawrence96/ElectrogeneticsToolset). When pyocyanin is in its reduced form, it does not activate the pSoxS promoter.
The Act106 construct functioned as intended, but we found contradictory results for the Inv106 construct.

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Pyocyanin activates pSoxS in its oxydised form and not in its reduced form. To notice a change in expression using this promoter in the presence of pyocyanin, we reduced pyocyanin by applying a potential using our open-source potentiostat hardware. In order to have a construct that functions as an electrically activated promoter, such that with the electrically reduced form of pyocyanin induces the expression of a gene, we also constructed an Input construct that incorporates a Not Gate.
Our bioelectronic setup showed a different in expression after applying a voltage potential of -0.5 V for several hours. Further experiments need to be performed here to characterise this induction properly.

Materials and methods

Cloning

We designed two constructs with pSoxS, one with a downstream mScarlet fluorescent coding sequence to report on the activity of the promoter (dpB.002 -Bba_K4216027) and one with a downstream CinR transcription factor coding sequence (dpB.003 -Bba_K4216028) to act as an Input in our multiple plasmid system. We ordered the constructs from IDT and used Golden Gate cloning to insert them into the V35 vector plasmid (iGEM XXX) and the pDuet vector plasmids pCOLADuet (Bba_K4216045), pACYDuet(Bba_K4216043) and pCDFDuet (Bba_K4216044). These were transformed in a DH5alpha strain of E. coli and confirmed by sequencing.

We received the Act106 and Inv106 shipped to us by Joshua Lawrence and transformed these into DH5alpha E. coli and confirmed them by sequencing.

We used the Act106 and Inv106 constructs to extract the phiF and pPHIF parts by PCR amplification and assembled them with our parts to construct our own Inverter dpB.063. We used Gibson assembly to assemble the parts into the pDuets vectors because the pPHIF promoter used to inhibit the expression of fluorescence containes a BsaI restriction site, which made it incompatible with our Golden Gate assembly standard.

dpB.002

dpB.003

dpB.063

Plate reader pyocyanin dose response

Pyocyanin was purchased from Sigma Aldrich in solid form and diluted in DMSO to create a stock solution of 1mM. This stock solution was diluted in LB and antibiotic to achieve the desired experimental concentrations.

Cells were grown overnight in LB with Chloramphenicol 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 + Chloramphenicol with varying concentrations of H2O2 and grown for 12 hours in a Tecan Infinite® 200 PRO plate reader. The concentrations of pyocyanin tested were 0, 0.1, 0.5, 1, 5, 10, 15, 20, 25, 50, 75, and 100 uM. The final value of fluorescence was divided by OD600.

Pyocyanin dose response was performed on the Act106 and Inv106 genetic constructs sent to us by Joshua Lawrence and on the our pSoxS mScarlet (dpB.063 ) construct in the V35 vector.

Chronoamperometry electrical induction

We performed a chronoamperometry experiment on the Inverter construct dpB.063 in the pCOLA vector (Bba_K4216045). Cells were grown overnight and diluted 1:100 in media containing LB + Kanamycin and 10uM of pyocyanin.

We performed chronoamperometry experiments in anaerobic conditions to avoid ambient oxygenation of pyocyanin and allow the reduction of pyocyanin at the electrodes. Three electrodes - counter, working and reference electrodes- connected to the IO Rodeo potentiostat were sterilised with ethanol and placed inside one of the tubes containing our strain. We applied a -0.5V potential versus SHE for 16 hours and measured the final fluorescence and OD. For the anaerobic conditions we placed culture tubes in a hermetique jar and lit a candle inside, closed the jar and waited for the flame to consume the oxygen. The setup was placed inside an incubator and shaken at 160rpm.

Anaerobic Constant voltametry setup

Results

Plate reader pyocyanin dose response

Results of the pyocyanin dose response of dpB.002 (Bba_K4216027) show a high induction range with minimal effect on growth between 5 and 10 uM. This is in agreement the data from the 2018 Imperial iGEM Pixcell.

The pyocyanin dose response of the Act106 and Inv106 constructs sent by Joshua Lawrence both show an increased fluorescence expression at higher concentrations of pyocyanin. This is unexpected, as we would expect the Inv106 to show a repression of fluorescence expression with increased pyocyanin concentration as seen in the Lawrence et al. paper.

Chronoamperometry electrical induction

The results from this experiment can be considered preliminary. We observe a difference in fluorescence expression for the electrically induced Inverter construct. Fluorescence is higher in the Inverter without applied voltage potential, which is an unexpected result. This may be due to the sequence of the Inverter having a large mutation in the pPHIF promoter which was identified when sequenced. The promoter is meant to induce the expression of fluorescence when voltage is applied. Therefore this construct works in the opposite way, by being repressed when voltage is applied.

Future Work

Further work characterising the Inverter construct activity is needed. Our strain had a large deletion mutation in one of the promoters, which may have happened during the Gibson assembly step, which likely affected the induction system. Because of lack of time, we could not try this experiment again with non-mutated strains. The other explanation for the negative result is that the stress from the voltage potential affected the expression of the plasmid so it could not produce fluorescence. However, growth of the culture did not differ between the chronoamperometry and control conditions. Were we to continue these experiments, we would have also tried the chronoamperometry with the dpB.002 (Bba_K4216027) construct, to see whether fluorescence was repressed by an applied voltage.

References

1. Lawrence, J. M., Yin, Y., Bombelli, P. et al. Synthetic biology and bioelectrochemical tools for electrogenetic system engineering, Sc. Adv. (2022). DOI: 10.1126/sciadv.abm50