Team:TU Delft-Leiden/Project/Life science/EET/integration

Electron Transport – Integration of Departments

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Described are the general why and how of the practices Modeling as well as Microfluidics with respect to experimental work performed in the module Electron Transport.

The team is developing a plug-and-play module to which any biosensor can be linked. This module generates a precisely measurable electrical signal, an improvement on existing outputs of biosensors, which often work with staining and / or fluorescence. As proof-of-concept, the biosensor from the Module Landmine Detection will be attached to this plug-and-play module. Employing, modeling and optimizing the electron transport Mtr system of bacterium Shewanella oneidensis in Escherichia coli forms the basis of our strategy. This system consists of a cluster of genes MtrC , MtrA and MtrB , that will be present in cell membranes of transformant bacteria. The complex of membrane proteins will receive and transport electrons. This module contains fundamental challenges. The functioning of the carbon metabolism of model organism E. coli is considered in Furthermore, (the production of) membrane proteins are looked into. Implementation of the system in this organism would result in the transportation of electrons. These three aspects are modeled and on the basis of the model, the system is optimized.

In general, reactions can be split into three consecutive sections, ie. carbon metabolism, assembly of the electron transport system and electron transport. Basics of the system are thus the enzymes involved in conversions in carbon metabolism, e.g. dehydrogenases, enzymes involved in assembly and transportation, e.g. CcmA-H as well as the membrane proteins that are termed the nucleus of this system. Carbon metabolism encompasses the oxidation of lactate to pyruvate by lactate dehydrogenase, generating ATP as well as NADH (via quinone pools, ie. menaquinone (MK) and dimethylmenaquinone (DMK)), the reducing factor influencing redox balance and essentially an intermediate in generation of EET. Pathways involved at this level are the respiratory chain and the electron transport pathway which is to be implemented. Regarding the assembly and functionality of the electron transport system, the scope of regulatory elements encompassing a project centered on life organisms in general is formed by the canon transcription, translation, enzymatic reactions and output. The proteins involved form the possibly most challenging part of the project ELECTRACE, as being influenced by multiple factors in which many unknowns exist. Factors include translation, ie. ribosome binding, folding; post-translational modifications; with regards to the underlying project, heme-loading and possibly heme availability as well as transportation of proteins; secretion; and finally, protein degradation.

As mentioned under Context, MtrA is a 32-kD periplasmic decaheme cytochrome C. MtrC is a 69-kD cell-surface-exposed lipid-anchored decaheme cytochrome C, MtrB is a 72-kD predicted twenty-eight strand β-barrel outer membrane protein. Also, several additional enzymes involved in electron transport, e.g. CymA, are possibly essential in generation of transport. Assembly of electron transport system consists of several parallel as well as consecutive steps. MtrC requires MtrB, for correct assembly in the outer membrane. MtrA also appears to be necessary for MtrB stability. MtrC and MtrA biogenesis requires the cytochrome c maturation (ccm) genes, which encode the eight membrane proteins CcmABCDEFGH. Ccm system translocates heme into the periplasm and catalyzes the formation of thioether bonds that link heme to two cysteine residues. The axial ligands (typically histidine) are then coordinated to the heme iron and the holocytochrome C is folded. MtrC and MtrB must be translocated to the outer membrane, presumably by type II secretion. Reducing equivalents resulting from L-lactate oxidation are delivered to the periplasmic reductase CymA via menaquinol. Electrons are transferred from CymA to MtrA. Electrons are transferred from MtrA to MtrC. From MtrC, electrons can be transported to terminal electron acceptors through redox shuttles, direct contact or possibly through other proteins on the cell surface, e.g., OmcA. The systems mentioned here will be taken into account as follows.

We will model the electron transport pathway engineered into E. coli and use the results to improve the system biologically. Modeling consecutive transfers of electrons starting from Cym A / NapC up to reduction of a terminal electron acceptor and changes in fluxes as a result of in- or decreasing ‘pools’ of proteins involved would pose the possibility of prioritizing bottlenecks or clusters of bottlenecks, and eventually guiding wet lab workers towards the extra amount of a factor needed for optimization. Cooperation within this Module between the Departments Life Science andMmodeling was straightforward, intriguing and essentially useful. The team has considered parameters that can be optimized within a certain range and conducted a search for optima within the post-translational modifications, including secretion. As cited in literature [Jensen] wanted was an “appropriate balance between the rates and levels of polypeptide synthesis, heme biosynthesis, polypeptide secretion, and cytochrome c maturation.” Eventually, there must also be a relationship established between input and output strength of the biosensory plug-and-play system. In this subdivision of modeling, the team aims to charter potential bottlenecks in the system, focus on one of several components (transcription, translation, PTM, breakdown, etc.). Also, on a next level synthetic biology, additional parameters could well be generated via the model, for instance, a model pointing to a necessary shunt between protein x and protein z to bypass protein y. Modeling relevant aspects of carbon metabolism proved to be challenging, as E.coli is quite the versatile organism with regards to use of different sources for carbon for growth and proliferation. As an example, in converting lactate to pyruvate as a sole source of carbon, the cellular system generates minor amounts of the necessary ATP. This resulst in a relatively large influence of the so-called household levels.

Regarding the experimental setup, the basic system will consist of MtrCAB (S. oneidensis), Ccm (E. coli) and NapC (native). Improvements to this system are CymA and / or an alternative to NapC, At a later stage, it might prove feasible to (possibly) increase the amount of type-II secretion systems, and addition of Curli. Steps included in optimization are the separation of the the Mtr genes and implementation of different ratios of transcription / translation. Also, (possible) flavins and / or alternative shuttles are included via plasmids carrying the relevant genes. In addition, genes resembling the S. oneidensis MtrCAB system have been taken into account. OmcA of S. oneidensis could form an addition to MtrCAB. OmcS, OmcE, OmcT Geobacter sp. are possible alternative to MtrCAB.

The aim of including Microfluidics in this Module is to be able to create a hand-held biosensory device and making measurements possible in a straightforward manner. What is needed is an anaerobe surrounding, applied potential and the possibility to flush with medium. The device Dropsens coupled to a potentiostat proved to serve as such. Three electrodes (counter and working) are attached to a microfluidic space considered to be anaerobic. Flushing with relevant medium is possible, so is addition of an analyte of interest. Cells are flushed and caught in the Dropsens. The Dropsens coupled to a potentiostat is essentially the microfluidic device of choice for this Module.