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

Integration of Departments

In the wet lab we integrated the Electron Transport pathway of Shewanella oneidensis into Escherichia coli. 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 has developed 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 is to be attached to the plug-and-play module. Employing, modeling and optimizing the extracellular electron transport (EET) Mtr system of bacterium Shewanella oneidensis in Escherichia coli formed the basis of our strategy [1, 2]. This system consists of a cluster of genes MtrC , MtrA and MtrB , of which the products are present in cell membranes of transformant bacteria. The complex of membrane proteins receive and transport electrons.

This module contains fundamental challenges. The functioning of the carbon metabolism of model organism E. coli has been considered in Carbon Metabolism and Electron Transport. Furthermore, (the production of) membrane proteins has been 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.

Challenges for Modeling and Experimental Work center on carbon metabolism and protein chemistry

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 was to be implemented. Regarding the assembly and functionality of the electron transport system, the scope of regulatory elements encompassing a project centered on living organisms in general is formed by the canon transcription, translation, enzymatic reactions and output. The proteins involved form possibly the 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, heme-loading and possibly heme availability as well as transportation of proteins, secretion; and finally, protein degradation.

Relevant details of the extracellular electron transport system

As mentioned under Context, MtrA is a 32-kD periplasmic decaheme cytochrome C. MtrC is a 69-kD decaheme cytochrome C exposed to the cell surface, MtrB is a 72-kD predicted outer membrane protein. Also, several additional enzymes involved in electron transport, such as CymA, are possibly essential in generation of electron transport. Assembly of the 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 CcmA-H. The Ccm system translocates heme into the periplasm and subsequently catalyzes the formation of intermolecular bonds that link heme to two cysteine residues, after which coordination to the heme iron takes place and the holocytochrome C is folded. MtrC and MtrB must be translocated to the outer membrane. This is expected to involve type II secretion. Reducing equivalents resulting from carbon source oxidation are delivered to the periplasmic reductase CymA via menaquinol. Electrons are then transferred from CymA to MtrA, from MtrA to MtrC, from MtrC to terminal electron acceptors through redox shuttles, direct contact or possibly through other proteins on the cell surface, such as OmcA [1, 2]. The systems mentioned here will be taken into account as follows.

Bypassing bottlenecks in EET via interactions between Modeling and Experimental Work

We have modeled the electron transport pathway engineered into E. coli and have used 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, we assumed, 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 and Modeling was straightforward, intriguing and essentially useful and included Deterministic Modeling of EET Complex Assembly . The team has considered parameters that can be optimized within a certain range and conducted a search for optima within the modifications proteins undergo, including secretion. As cited in literature [2] wanted was an “appropriate balance between the rates and levels of polypeptide synthesis, heme biosynthesis, polypeptide secretion, and cytochrome c maturation”. 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. Eventually, the team also wanted to establish a relationship between input and output strength of the biosensory plug-and-play system. Modeling relevant aspects of carbon metabolism in Flux Balance Analyses, which can be found under Modeling: Flux Balance Analysis of the EET Module proved to be challenging, as E.coli is quite the versatile organism with regards to use different sources of 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 results 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 that might be 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. We urge teams of the upcoming years to consider picking up where this team left off.

Combining electrochemistry with Microfluidics for an improved biosensor

The aim of including Microfluidics in this Module was to be able to create a hand-held biosensory device and make measurements possible in a straightforward manner. What was needed is an anaerobe surrounding, applied potential and the possibility to flush with medium. The microfluidic device, of which details can be found under Microfluidics: Microfluidics Device for Electron Transfer 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 microfluidic channels. When coupled to a potentiostat, this is essentially the microfluidic device of choice for this Module.

References

1. C.P. Goldbeck et al., Tuning promoter strengths for improved synthesis and function of electron conduits in E. coli ACS Synth. Biol. 2 (3), pp 150–159 (2013)
2. H.M. Jenssen et al., Engineering of a synthetic electron conduit in living cells. PNAS ∣ vol. 107 no. 45 (2010)