User:Nb2817/Notebook/Engineering a library of biological logic gates using synthetic biology

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Aim

The overall objective of this group project was to engineer a library of biological logic gates which replicate the behaviour of digital AND gates in a biological context. The library has been made with the motivation to create a tool to aid the development of more complex biological systems in future research by easing the selection process of correct parts for the operating conditions and required characteristics of the systems. The AND gate that constitutes the main subject of our tests and characterizations was engineered by Wang et al. in 2011 and detailed in a paper published in Nature Communications.

Background

Synthetic biology is the application of engineering principles in biology.

The major applications are in:

  • Medicine
  • Chemistry
  • Sustainability


Medical applications

  • Analytical devices (e.g. biosensors)
  • Treatment of cancer and tumours
  • Tissue engineering
  • Biomaterials


Chemical applications

  • Protein synthesis (e.g. insulin, enzymes, therapeutics)
  • Material synthesis (e.g. spider silk)


Sustainability applications

  • Pollution (biodegradable plastic; decontamination of water, soil, air)
  • Food (increasing yield; surviving tough conditions)


An example of a logic system in biology is a repressilator.


Literature Review and Preliminary Findings

List of reviewed papers:

  • Arkin et al., Environmental signal integration by a modular AND gate
  • Collins et al., Synthetic Gene Networks That Counts
  • Collins et al., Bistable genetic toggle switch
  • Collins et al., Complex cellular logic computation using ribocomputing devices
  • Dixon et al., Biotechnological solutions to the nitrogen problem
  • Fussenegger et al., Programming mammalian gene expression with the antibiotic simocyclinone D8 and the flavonoid luteolin

2 modular and orthogonal transcriptional gene switches triggered by Antibiotic simocyclinone D8 and luteolin were built. These can be combined to build AND and OR gates. SD8 is harmful to its producing cell, which has developed a transcription repression mechanism that removes the SD8 when it is produced. Luteolin is a antiallergic, anticancer, anti-inflammatory plant flavonoid. Binding of SD8 and luteolin to their respective repressors reduces affinity with DNA and releases them. Switches can be SD8 (and luteolin) inducible, or SD8 (and luteolin) repressible, but the performance of the inducible ones is much better: they are non-toxic and highly specific and reliable.

  • Fussenegger et al., BioLogic Gates Enable Logical Transcription Control in Mammalian Cells
  • Poole et al., Symbiotic Nitrogen Fixation and the Challenges to Its Extension to Nonlegumes
  • Poole et al., The Rules of Engagement in the Legume-Rhizobial Symbiosis
  • Silver et al., A tunable zinc finger-based framework for Boolean logic computation in mammalian cells
  • Silver et al., Two- and three-input TALE-based AND logic computation in embryonic stem cells
  • Voigt et al., Genetic programs constructed from layered logic gates in single cells
  • Voigt et al., Cellular checkpoint control using programmable sequential logic
  • Voigt et al., Dynamic control of endogenous metabolism with combinatorial logic circuits
  • Weiss et al., Multi-Input RNAi-Based Logic Circuit for Identification of Specific Cancer Cells

A 5-input highly specific logical AND gate to identify and destroy HeLa cancerous cells was engineered. Inputs are miRNA markers which are either expressed (2 of the 5) or not (3 of the 5) in HeLa cancer cells. The specific combination of the 2 HIGH inputs being ON and the 3 LOW inputs being OFF triggers apoptosis (cell destruction). Although there still are challenges to implement DNA delivery to cells in vivo, this is an example of how logic gates can be used for a sensing-processing-acting application. Such logic-based cancer identification mechanisms have the potential to aid greatly not only in recognising but also in curing tumours.

  • Weiss et al., The Device Physics of Cellular Logic Gates

Efficient gene expression-regulating bioLogical gates were and are essential for the development of novel biological organisms. This experiment consisted of building synthetic gene circuits using lacI, tetR and cI repressors, to insert them in E. Coli and communicate with programmable and programmed cells. With these, in vivo signals can be controlled by external inputs (e.g. by IPTG diffusing into the cell as the input to a combination of NOT and IMPLIES gates). Mutations are required for an effective combination of 2 separate natural mechanisms, and in this case they were done by varying the RBSs of the plasmids used. The larger goal of this experiment was to build a library of standardized biological components that could be efficiently combined together.

After conducting the literature review, we identified factors that are important in the design of our circuit such as: orthogonality, modularity, leakage and clearly identifying precise ON/OFF states. Furthermore, we came to realise that the forward-engineering approach which utilises quantitative characterisation and mathematical modelling before building the circuits were quite important for our group project timeline.

Following the review, the group read the paper that forms the starting point of this project: Engineering modular and orthogonal genetic logic gates for robust digital-like synthetic biology, by Wang et al. A presentation about the paper was also made and can be found

Planning

Guntt Chart

Task Allocation

The first thing we did as a team is conduct a literature review to identify how logic gates are constructed and how they can be used. We then broke down the main tasks around this project into four parts:

  • Mathematical modelling of the the genetic circuit
    This represents designing the parts and choosing the inputs and outputs.
  • Wet lab work
    It mainly involved DNA sequencing and gene synthesis.
  • Literature review
    Each person in the group was allocated papers to read.
  • Wiki
    We documented our findings regularly to keep track of our progress.


The tasks have been split in the following way:

  • Mathematical Modelling: Nicholas Ustaran-Anderegg, Nicolae Barcaru, Ali Nourani
  • Wet-lab work: Lana Al-Nusair, Hamza Nawaz, Dimitra Marmaropoulou, Lorenzo Mazzaschi
  • Literature review: Everyone's responsibility
  • Wiki: Everyone's responsibility

Computer labs

GitHub link

Wet labs

We have taken a bottom up approach to design the gates, following parts-based characterisation and mathematical modelling.

The following information about each AND gate produced has been recorded:

  • The specific design of the gate detailing the specific parts used
  • The experimental behaviour of the gate implemented in a prokaryotic cell detailing its dynamic range and success to replicate the characteristics of digital AND gate with a sharp and distinct change in state, in response to the correct inputs
  • Comparison of the experimental data with derived models to draw a conclusion on the predictability of each gate with computational modelling