Engineering

Modularity and Standardisation in genetic engineering

The modularity and standardization of electronic components are central to the architecture of emerging electronic and digital technologies. Following engineering modularity and standardization principles, we have conceived and created a bidirectional bio-electrical communication platform by both genetic and electrical engineering.

In our bio-electronic platform, not only do we apply engineering principles to biological systems, but, we go a step further by engineering bacterial populations to act as electronic components. We have designed and tested a library of parts and constructs that, when transformed into bacterial populations, behave analogously to electronic components. Building on the analogy from the world of electronics, we see our collection like the electronic components on the shelves of a hardware workshop where electronic designers can swap resistors and capacitors in a circuit.

Our main Human Practices takeaway, from our interviews with Brian Ringley, Cesar Harada, and Xiao Xiao, was that to be useful and serviceable to the engineering community, our bio-electronic platform had to be as modular as possible. We have used engineering principles to achieve our engineering goal of modularity in our genetic constructs.

What

Our wetware platform is made of genetic constructs organized into three categories:
  • The INPUTs: electrical to biochemical signal transduction.
  • The PROCESSORs: biochemical to biochemical signal transduction performing genetic logic and/or biosensor operations.
  • The OUTPUTs: biochemical to electrical signal transduction.
Go to Parts Collection

The modularity of our composite parts is based on two systems:

  1. The three Input, Processor, and Output components depend on a 3 plasmid system.
  2. The connection between these components is established by a standardized signal transduction communication pipeline using a tunable combination of transcription factors, promoters, and their associated chemical inducers.
These properties are illustrated below:

Engeneering Diagram


1. Multiple Construct Combination - pDUET plasmids

The constructs in our collection can function independently or jointly, similar to electronic components that can be used in isolation or combined into electronic circuits.

We iterated through two cycles of the Design-Build-Test-Learn (DBTL) cycle which allowed us to demonstrate that our genetic constructs function independently using the V35 backbone vector - and jointly - using the pDUET backbone vectors.

Iteration 1 of the DTBL cycle:

In this iteration we demonstrate that our construct dpB.002 (Bba_K4216027) assembled in the Moclo-compatible V35 vector works as expected.

Design:
We designed constructs with the pyocyanin inducible promoter pSoxS (Bba_K4216006) upstream of a fluorescence report in the MoClo compatible V35 vector plasmid.
Build:
We transformed this plasmid in a E. coli DH5alpha strain.
Test:
We measured the dose response of the pSoxS promoter to the inducer pyocyanin.
Reflection:
The V35 backbone vector has the advantage of being a high-copy number plasmid, meaning the construct is highly expressed. The disadvantage is that adding other types of plasmids to an E. coli already containing the V35 would be too burdensome for the cell. However, we wanted to combine different types of plasmid within a cell to increase the modularity of our system. We enquired our instructors, who informed us of the existence of the pDuet vector plasmids designed the for coexpression of multiple plasmids at a time in one strain. We iterated through a second DTBL cycle to achieve our desired modularity using these vectors.
Responsive image
Responsive image

Iteration 2 of the DTBL cycle:

Design:
In our design, we selected three pDuet vector plasmids - pCOLADuet, pCDFDuet and pACYDuet - and inserted three constructs - dpB.003 (Bba_K4216028), dpB.011 (Bba_K4216036), and dpB.013 (Bba_K4216038) - in each of the vectors (see pDuet combinaison optimizationation . The genetic constructs carried by the vectors activate each other through a combination of transcription factors, promoters and inducers inspired by the Marionette system (2).
Build:
We modified the pDuet vectors to make them MoClo compatible. To achieve this, we extracted, by PCR amplification, both the pDuet vectors without the LacI expression cassette and the sfGFP expression cassette from the V35 vector. Simultaneously, using designed primers with floating overhangs in our extractions, we added restriction sites for BsaI with overhangs compatible with the CIDAR moclo to all pDuet vectors. We assembled the now MoClo-compatible pDuet vectors with the extracted sfGFP transcriptional unit using Gibson assembly. Then, we assembled theMoClo-compatiblee pDuets with our constructs using Golden Gate cloning.
Test:
We tested the expression of different combinations of the pDuet vectors.
Learn:
Modular gene expression is possible using the pDuet vectors (figure 1). The combination here is the pACYDuet_Bettencourt for the Input, the pCDFDuet_Bettencourt for the Processor, and the pCOLADuet_Bettencourt for the Output. This figure shows the successful induction of three pDuet plasmids as a dose response of the Input activation. This shows that the Output expression correlates positively with the level of induction in the Input plasmid.
Reflection:
Using the DTBL cycles we achieved our engineering goal of modularity allowing the constructs of our toolkit to be expressed together or independently. Based on the highest expressing combination, we chose to establish a standard for the pDuets for our Input, Processor, and Output components.
Modulation:

We assembled our constructs into three interchangeable vector plasmids, that can be combined together or used seperately.

Standardisation:

Each genetic component is associated with a pDuet vector

  • pACYDuet for the INPUT
  • pCDF for the PROCESSOR
  • pCOLA for the OUTPUT.

2) Tunability of Expression

In our three constructs three plasmid system, each component is tunable using chemical inducers. This means that the expression of each construct can be fine-tuned to transmit the signal more or less strongly. Like knobs on a modular synthesizer, multimodal modes of control of our circuits is possible. We designed the signal transmission of the 3 plasmids via the Processor component based on the Marionette strains developed by Meyer et al. (2). This system consists of transcription factors, promoters and inducers with high range and specificity. Out of the 12 sensor circuits established in this system we selected two for our system:

  • The CinR+OCH14+ pCin for the communication between the Input and the Processor
  • and the LuxRQ + OC6 + pLux for the communication between the Processor and the Output

We selected these systems because of their compatibility with the CIDAR MoClo kit (they can be found in the CIDAR MoClo extension kit) (1).

Our engineering goal was to achieve modularity in the Processor component, the “computation” module of our system. As the logic operator, this module can perform common genetic logic on the signal transmition, such as amplification, resistance, inhibition, and biosensing.

In our Signal Modulation experiment we show the use of this module which can act as a biosensor -activates gene expression at the presence of a chemical- or as a signal amplificator/resistor -tunes the signal strength.

To the Signal Modulation Experiment

The Processor component can be interchanged with any synthetic biology logic operation of choice. This allows for a variety of biosensors to be built and added to the collection of Processor components.

In summary, the goal of modularity and standardisation in our system was achieve through the applications of engineering principles.

To the Proof of Concept
Modulation:

We assembled our constructs into three interchangeable vector plasmids, that can be combined together or used seperately.

Standardisation:

Each genetic component is associated with a pDuet vector

  • pACYDuet for the INPUT
  • pCDF for the PROCESSOR
  • pCOLA for the OUTPUT.

References

[1] **CIDAR MoClo: Improved MoClo Assembly Standard and New E. coli Part Library Enable Rapid Combinatorial Design for Synthetic and Traditional Biology. Iverson SV, Haddock TL, Beal J, Densmore DM. ACS Synth Biol.* 2015 Nov 4. doi: 10.1002/bit.25814. PubMed [PMID 26479688](https://www.ncbi.nlm.nih.gov/pubmed/26479688).

[2] Meyer, A.J. et al. (2019) ‘Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors’, Nature Chemical Biology, 15(2), pp. 196–204. Available at: [https://doi.org/10.1038/s41589-018-0168-3.](https://doi.org/10.1038/s41589-018-0168-3)

[3] “Modular.” *Merriam-Webster.com Dictionary*, Merriam-Webster, https://www.merriam-webster.com/dictionary/modular. Accessed 11 Oct. 2022.



Access Wiki Tools View Source Code