IC.Y3.AND.Gate.with.RiboJ:Background
Overview
Synthetic biology is the application of engineering principles in biology.
The major applications are in:
- Medicine
- Chemistry
- Sustainability
An example of a logic system in biology is a repressilator.
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)
Literature Review
- 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
Gates have parts that can be diversified to build multiple orthogonal gates. Inputs and outputs had common signal carriers so they can be layered – output of one is input of another. They built 2 input AND gate – one input is TF another is a chaperone protein – cahperone is needed to turn on the output promoter therefore – output promoter only active when both promoters are active and found a genetic circuit within salmonella and built it – 3 parts that form core of AND gate – the activator, the chaperone and inducible promoter. Salmonella was selected due to its orthogonality and dynamic range. A saturation mutagenesis library was designed to change the −10 region and screened to identify a mutant with a decreased background and higher dynamic range. They also tested for orthogonality – looked at interaction between activators and chaperones, TF and promoter. A problem with the gate characterization is that the data are presented where the input is an inducer concentration and the output is fluorescence. To guide the connection of circuits, the data needed to be in a form in which the inputs and the outputs have the same units. This was achieved by using a mathematical model combined with additional experiments. The 2 input AND gates – permutations – created 2/4 input AND gates – alternaitve logic combinations – can produce same funciton – those design were chosen with the purpose of studying gate layering. A potential problem in layering genetic logic gates is that the resulting programs are asynchronous. Because there are delays at each layer, this can lead to transient errors in the output, known as faults
- Voigt et al., Cellular checkpoint control using programmable sequential logic
Using NOT gates - an input promoter drives the expression of a repressor protein that turns off an output promoter. Each gate is characterized by measuring its response function, in other words, how changing the input affects the output at steady state. The circuits can be connected to genetic sensors that respond to environmental information. This is used to implement checkpoint control, in which the cell waits for the right signals before continuing to the next state.
Each latch requires 2 repressors – inhibit each others expression – 11 SR latches were designed using a phase plane analysis – 43 circuits were constructed – connects these latches to different combo of sesnsors that responsds to small mol – a gated D latch is constructed – u to 3 SR latches (based on 6 repressors) are combined in a single cell – 3 bits are reversibly stored. Checkpoint control leads to variability in the time spent in each stage but synchronizes the requirements for progression across cells and buffers against fluctuations. Latches are analogous to bistable switches and have two stable states that are used to store one digital bit of information – cross coupled NOR gates. The toggle switch has been connected to a single sensor by having the output promoter drive the expression of one repressor, and this has been used to remember transient exposure to a sugar, quorum signal, or an antibiotic. Repressor-based NOR gates are connected to each other and sensors by signal matching their response functions. Latches are designed by recognizing that these empirical functions can serve as nullclines to identify gate combinations that will exhibit bistability. Bistability is a necessary criterion for building an SR latch. This is achieved by arranging two repressors to regulate each other’s expression. For latch to be extensible – must have 2 promoter inputs and 2 promoter outputs – if have the same units – can be used to connect latch to genetic sensors.
- Voigt et al., Dynamic control of endogenous metabolism with combinatorial logic circuits
E coli sensors that respond to the consumption of feedstock (glucose), dissolved oxygen, and by product accumulation (acetate) are constructed and optimised - by integrating these sensors logic circuits implement temporal control over an 18h period. Two circuits are designed to control acetate production by matching their dynamics to when endogenous genes are expressed (pta or poxB) and respond by turning off the corresponding gene. However, an individual sensor can only implement a switch at a one defined cell state and cannot be used to drive a series of events. An alternative approach to modifying the sensors is to select a set of sensors that turn on at different times during a bioprocess and then use a genetic circuit that responds to a pattern of sensor activities to turn on at a defined point. During a bioprocess, many conditions change dynam- ically inside the reactor and inside of individual cells. Therefore, the same set of sensors can be integrated in different ways to generate different dynamic responses. The low oxygen sensor turns on first, followed by the turning off of the glucose sensor, and finally the acetate sensor turns on. Simulations of many genetic circuits implementing these sensors’ signals into different logic operations show that diverse responses are possible. From these, we select several based on layered AND and ANDN gates, construct them, and verify their temporal response. Over the course of a growth experiment, the output of the three sensor promoters is continuously changing. These promoters can be connected as inputs to a logic circuit that responds only when each sensor is at the correct level. Thus, by connecting the sensors to circuits that implement different logic operations (truth tables), the circuits will produce different responses over time. Because the circuits are based on the layered expression of regulators (a cascade), different circuits that encode the same truth table can result in different dynamics due to delays in signal propagation. To determine the range of possible dynamics, simulations were run for all possible 3-input logic circuits designed based on layered AND, ANDN, and NOR gates.Foremost is the problem of toxicity and stability. Even medium- sized synthetic circuits (≥4 regulators) can slow growth instability in the form of plasmid loss or mutations to the genome. Further, the slowing of growth can be devastating for bioproduction.
- 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.
Preliminary Findings
On top of all these papers, the whole group read the paper: Engineering modular and orthogonal genetic logic gates for robust digital-like synthetic biology
- Baojun Wang et al., Nature Communications (2011)
- Constructed an orthogonal AND gate in Escherichia coli using a hetero-regulation module from Pseudomonas syringae. The device comprises two co-activating genes hrpR and hrpS controlled by separate promoter inputs, and a σ54-dependent hrpL promoter driving the output. The hrpL promoter is activated only when both genes are expressed, generating digital-like AND integration behaviour. The AND gate is demonstrated to be modular by applying new regulated promoters to the inputs, and connecting the output to a NOT gate module to produce a combinatorial NAND gate.
- Most circuits are not modular - limited by having to use specific inputs and outputs
- Not insulated from host chasis – hence they have to operate in a specific genetic background to avoid cross talk affecting host machinery – hence cannot build larger biological systems.
- Ideally, a genetic logic device should be modular and orthogonal to their host chassis to facilitate its reuse and reliability in different contexts.
- Trial and error disadvantages: lack of predictability, and the long time and great effort taken to obtain a functional circuit and the circuit’s behaviour in a different context is unpredictable.
- They demonstrated that the resulting AND gate is modular by wiring the inputs to different input promoters and the output to a NOT gate module to produce a combinatorial NAND gate. The logic gates are shown to behave robustly across different cellular contexts.
- Two co-activating genes hrpR and hrpS and one σ54-dependent hrpL promoter, and can integrate two interchangeable environmental signal inputs to generate one interchangeable output. The output hrpL promoter is activated only when both the co-dependent HrpR and HrpS enhancer-binding proteins are present in a heteromeric complex
- Because of the requirement of modularity, both the inputs and output of the AND gate were designed to be promoters, allowing the inputs to be wired to any input promoters and the output to be connected to any gene modules downstream to drive various cellular responses. It is important to select the right RBS.
- IPTG inducible Plac, Arabinose inducible Pbad and AHL inducible Plux were the promoters that were selected.
- Characterised using 6 RBS, 6 E.Coli strains in M9-Glycerol/Glucose in 30/37 degrees.
- The order of the strengths of the six RBSs across these three promoters varies. This is largely due to the different 5′ untranslated region following each promoter
