Engineering bacteria to control electron transport altering the synthesis of non-native polymer

Abstract

The use of bacteria as catalysts for radical polymerisations of synthetic monomers has recently been established. However, the role of trans Plasma Membrane Electron Transport (tPMET) in modulating these processes is not well understood. We sort to study this by genetic engineering a part of the tPMET system NapC in E. coli. We show that this engineering altered the rate of extracellular electron transfer coincided with an effect on cell-mediated polymerisation using a model monomer. A plasmid with arabinose inducible PBAD promoters were shown to upregulate NapC protein upon induction at total arabinose concentrations of 0.0018% and 0.18%. These clones (E. coli _{(IP_0.0018%)} and E. coli _{(IP_0.18%)}, respectively) were used in iron-mediated atom transfer radical polymerisation (Fe ATRP), affecting the nature of the polymerisation, than cultures containing suppressed or empty plasmids (E. coli _{(IP_S)} and E. coli _(E), respectively). These results lead to the hypothesis that EET (Extracellular Electron Transfer) in part modulates cell instructed polymerisations.

Fig. 1. Assembly of inducible promoter vector containing promoter P_BAD for NapC overexpression and control. PCR was carried out with specific primers to extract and amplify regions (1) P_BAD_araC and (2) napC. Region 1 was obtained from the plasmid pMTL71101_P_BAD_araC and region 2 was obtained from E. coli gDNA. The plasmid pMTL83153 was digested with REs NotI and SalI to remove the Pfdx promoter, resulting in region 3, digested plasmid. The three regions were ligated together with Hifi assembly to create the completed vector. Gel electrophoresis of colony PCR products for DNA regions making up the inducible promoter vector, against 1 KB Plus DNA ladder P_BAD_araC DNA region (1278 bps) and napC DNA region (633 bps) are as expected. Sanger sequencing diagrams for inducible promoter vector showing matching DNA regions of sequencing with forward and reverse primers compared to a model sequence.

Fig. 2. Fe ATRP polymerisations activated by E. coli cultures harbouring different plasmids to compare the effects of NapC protein upregulation. (a) ¹H NMR spectra of Fe ATRP activated by E. coli_(E) (black), or inducible promoter plasmids, E. coli_(IP) either (i) suppressed by addition of glucose E. coli_{(IP_S)} (red), (ii) activated by 0.0018% total arabinose concentration E. coli_{(IP_0.0018%)} (blue) or (ii) activated by 0.18% total arabinose concentration E. coli_{(IP_0.18%)} (purple). (b) Possible rate limiting steps in Fe(iii) reduction including electron transfer (ET) via NapC, ET via cascade proteins and ET via mediator molecules.

Fig. 3. Electrochemical detection of ferrocyanide using linear sweep voltammetry. (a) First derivative function applied to linear sweep voltammogram of ferricyanide/ferrocyanide redox couple (Fig. S6†) to determine d(I_ss)/dt values. (b) Concentrations (%) of ferrocyanide detected in the supernatant of samples incubated for 1 hour with E. coli_(IP) or E. coli_(E). Results are expressed as mean ± S.D. ** P < 0.005 vs. E. coli_(E), obtained using 1-way ANOVA with a Dunnett's post-test. LSV was used to analyse the supernatant of incubated samples (N = 2, n = 6) and the first derivative function was applied to resulting voltammograms. The concentrations were determined using the calibration graph showed in Fig. S7.