Team:TU Delft-Leiden/Project/Life science/curli

Module ‘Conductive Curli'

Described are the general why and how of the practices Modeling as well as Microfluidics with respect to experimental work performed in the Module 'Conductive Curli'.

Integration of Modeling, Experimental Work and Microfluidics

Curli are bacterial protein chains that are formed extracellularly and are attached to bacterial cell walls. These chains play a role in biofilm formation, amongst others. This Module describes the implementation and emergence of Curli in E. coli. In addition, a link with the Module Electron Transport is made by binding conductive gold nanoparticles to the protein chains. Formation of Curli will thus improve conductivity of the medium.

Exemplary mathematical standards were implemented within the subdivision Modeling. Summarizing, modeling covers the generation of Curli utilizing parameters specified with respect to the genome of the single cell and implements these algorithms in determination of the emergence of amyloids. Following this second level of programming, the generated codes form the parameters for the next level: plotting the point(s) in time in which cultures of cells operating in an undefined space generate the expected quantifiable decrease in resistance. This latter level is shaped via the Graph Theory, which is accurately fit to the underlying biological problem. For future prospects, it is quite feasible to generate the data necessary to compare hypotheses derived from models of Curli to actual situations.

Sidetrack and useful in a more general sense is the implementation of Percolation Theory, which shows the transition in conductivity at a certain and definable point in time.

Escherichia coli natively carry genes for the production of the amyloid fibrils termed Curli. Experiments were designed centering on strain ΔCsgB. Theory states protein CsgB as the central factor in nucleation of amyloid monomers CsgA. Generating CsgA in a constitutively overexpressed sense and, at a defined point in time, transcribing specified amounts of CsgB will result in nucleation, generation of Curli and a measurable formation of biofilm. Curli monomers carry modified histidine tags in order to bind gold nanoparticles. Experimental characterization thus consists of three complementary aspects, being assaying biofilm formation via eGFP signals constitutively expressed in relevant cells, theory on speed of nucleation and measurements of conductivity, of which the results can be found under [.].

Implementation of a system of microfluidics within this Module is based on the aspiration to image biofilm formation accurately and, eventually, couple formation of biofilm to measurements of conductivity. Aspects central in development of this type of device are, amongst others, the speed of reactions, the possibility of in vivo measurements at all points in time and options for quantification of signal of eGFP.

Aim, construction and functionality of what is hereafter referred to as the mother machine are discussed under [.]. This type of microfluidic system is intended for single-cell experiments, utilizing nanoscale channels coupled to a central channel for flow of media. Team iGEM 2014 TU Delft – Leiden University has made use of the standard model developed at Massachusetts Institute of Technology.

Summarizing, the device is constructed via positive silicon waver coupled to a plastic mold. The team has constructed its negative with PDMS, generating channels. Size of channels and size of cells should be taken in consideration, the latter partly depending on growth conditions. Holes and tubes coupled to these holes, coupled to the central channel deal with flushing with medium, with respect to the Module Conductive Curli imaging of eGFP. Before application, the PDMS sample is connected to a glass slide by oxygen plasma and prepared for action by consecutive flushing with, amongst others, BSA. More information regarding the design and construction of the mother machine can be found in the section [.].

Conductivity determination was to be measured via the resistance of the generated biofilm as a result of binding of gold nanoparticles.

Background Information

In the module CONDUCTIVE CURLI we combine the advantages of live bacteria with the benefits of nonliving materials. A great advantage of bacteria is their ability to respond to the environment, but unfortunately, they have limited possibility to create new functionality all of the sudden. However, if you would find a way to combine bacteria with nonliving materials, you can choose your material in a way it meets your requirements. A beautiful example of these “living materials” has recently been shown by MIT engineers, who were able to reprogram E. coli in a way in which the bacteria were actually making gold nanowires and conducting biofilms when gold nanoparticles were present [1].

For the CONDUCTIVE CURLI part of the project we induce curli formation in E. coli. Curli are extracellular amyloids that form fibers. They are involved in adhesion, cell aggregation and biofilm formation [2]. The curli pathway involves the so called CsgA-G proteins (see figure 1). CsgA is the major curli subunit, with a CsgB subunit at the beginning of every fiber. CsgE, CsgF and CsgG are non-structural proteins involved in curli biogenesis and CsgD is a transcriptional regulator [3].

But how to make these curli conductive and thus make "living material" ourselves? Our curli subunit CsgA carries a histidine tag, which facilitates binding of gold nanoparticles to the curli [1]. The gold nanoparticles on their turn end up in the biofilm, improving the conductivity of the extracellular environment. This change in conductivity is easily measurable by our ELECTRACE device, but in combination with the extracellular electron transport module , it could even result in enhanced extracellular electron transport.

Cloning Strategy and Characterisation of this module

References

1. Chen et al., Synthesis and patterning of tunable multiscale materials with engineered cells. Nature Materials 13, 515–523 (2014)
2. M. M. Barnhart and M. R. Chapman, Curli Biogenesis and Function. Annu Rev Microbiol. 60, 131–147 (2006)
3. L.S. Robinson et al., Secretion of curli fibre subunits is mediated by the outer membrane-localized CsgG protein. Mol Microbiol. 59(3): 870–881. (2006)