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

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 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

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

Planning

Gantt Chart

Team

Our team is composed of seven members, 2 intercalating medical students and 5 biomedical engineering students.

Intercalating medical students

  • Lana Al-Nusair
  • Hamza Nawaz

Biomedical engineering students

  • Nicolae Barcaru
  • Dimitra Marmaropoulou
  • Lorenzo Mazzaschi
  • Ali Reza Moghaddam Nourani
  • Nicholas Ustaran-Anderegg

Task Allocation

The first thing we did as a team was to 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

Task: Design the parts and choose the inputs and outputs with computer modelling.
Team members: Nicolae Barcaru, Ali Reza Moghaddam Nourani, Nicholas Ustaran-Anderegg

Wet lab work

Task: DNA sequencing and gene synthesis.
Team members: Lana Al-Nusair, Dimitra Marmaropoulou, Lorenzo Mazzaschi, Hamza Nawaz

Literature review

Task: Read various papers on the subject of interest and create a summary for them.
Team members: Entire team

Wiki

Task: Document our findings regularly to keep track of progress.
Team members: Entire team

Computer labs

Differential equations

Simple Chain Reaction


[math]A \xrightarrow{k_{1}} B \xrightarrow{k_{2}} C [/math]


[math] \begin{alignat}{2} \frac{d[A]}{dt} & = - k_{1}*[A] \\ \frac{d[B]}{dt} & = k_{1}*[A] -k_{2}*[B] \\ \frac{d[C]}{dt} & = k_{2}*[B] \end{alignat} [/math]

Enzymatic Reaction

[math] E + S \begin{matrix} k_1 \\ \longrightarrow \\ \longleftarrow \\ k_{2} \end{matrix} ES \xrightarrow{k_{3}} E + P [/math]

[math] \begin{alignat}{2} \frac{d[E]}{dt} & = k_{2}[ES] - k_{1}[E][S] + k_{3}[ES] \\ \frac{d[S]}{dt} & = k_{2}[ES] - k_{1}[E][S] \\ \frac{d[ES]}{dt} & = k_{1}[E][S] - k_{2}[ES] - k_{3}[ES] \\ \frac{d[P]}{dt} & = k_{3}[ES] \end{alignat} [/math]

Constitutive Gene Expression


[math] Gene \xrightarrow{k_{1}} mRNA \xrightarrow{k_{2}} Protein [/math]


[math] \begin{alignat}{1} \frac{d[mRNA]}{dt} & = k_{1} - d_{1}[mRNA] \\ \frac{d[Protein]}{dt} & = k_{2}[mRNA] - d_{2}[Protein] \\ \end{alignat} [/math]

Simplified Constitutive Gene Expression


[math] Gene \xrightarrow{k_{1}} Protein [/math]


[math] \begin{alignat}{2} \frac{d[Protein]}{dt} = s - d[Protein] \\ \end{alignat} [/math]

Repressed Gene Expression


[math] \begin{align} & Repressor \\ & \bot \\ Gene &\rightarrow mRNA \rightarrow Protein \end{align} [/math]


Hill function for transcriptional repression:

[math] \begin{align} \\ transcriptionRate=\frac{k_1.{K_m}^n}{{K_m}^n+R^n} \end{align} [/math]


[math]k_1[/math]: maximal transcription rate


[math]K_m[/math]: repression coefficient


[math]n[/math]: Hill coefficient


R = [repressor]


[math] \begin{alignat}{1} \frac{d[mRNA]}{dt} & = \frac{k_{1}.{K_m}^n}{{K_m}^n+R^n} - d_{1}[mRNA] \\ \frac{d[Protein]}{dt} & = k_{2}[mRNA] - d_{2}[Protein] \\ \end{alignat} [/math]

Activated Gene Expression


[math] \begin{align} & Activator \\ & \downarrow \\ Gene & \rightarrow mRNA \rightarrow Protein \end{align} [/math]


Hill function for transcriptional activation:

[math] \begin{align} \\ transcriptionRate=\frac{k_1.{A}^n}{{K_m}^n+A^n} \end{align} [/math]


[math]k_1[/math]: maximal transcription rate


[math]K_m[/math]: activation coefficient


[math]n[/math]: Hill coefficient


A=[activator]


[math] \begin{alignat}{1} \frac{d[mRNA]}{dt} & = \frac{k_{1}.A^n}{{K_m}^n+A^n} - d_{1}[mRNA] \\ \frac{d[Protein]}{dt} & = k_{2}[mRNA] - d_{2}[Protein] \\ \end{alignat} [/math]

Repressilator


[math] \begin{alignat}{1} \frac{d[mRNA]_{i}}{dt} & = \frac{a}{1+{[Protein]_{j}}^n} - [mRNA]_{i} \\ \frac{d[Protein]_{i}}{dt} & = b[mRNA]_{i} - b[Protein]_{i} \\\\ \ i=1,2,3; \\ \ j=3,1,2; \\ \end{alignat} [/math]


Toggle Switch


[math] \begin{alignat}{1} \frac{d[A]}{dt} & = \frac{\alpha}{1+[B]^4} - \gamma*[A] \\ \frac{d[B]}{dt} & = \frac{\beta}{1+[A]^4} - \gamma*[B] \end{alignat} [/math]

Responses to various promoters


[math] \begin{alignat}{1} f([I]) & = \frac{\alpha+[I]^{n_i}}{[K_1]^{n_i}+[I]^{n_i}}*k \end{alignat} [/math]


[math][I][/math]: concentration of the inducer


[math]K_1[/math]: Hill constant


[math]n_i[/math]: Hill coefficient


[math]k[/math]: the maximum expression level due to induction


[math]\alpha[/math]: constant relating to the basal level of the promoter due to leakage


AND gate


[math] \begin{alignat}{1} f([R], [S]) & = \frac{[G]}{[G]_{max}} & = \frac{{(\frac{[R]}{K_R})^{n_R}}*{(\frac{[S]}{K_S})^{n_S}}}{{(1+\frac{[R]}{K_R})^{n_R}}*{(1+\frac{[S]}{K_S})^{n_S}}} \end{alignat} [/math]


[math][R],[S][/math]: concentration of the activator proteins


[math][G][/math]: output


[math]K_R,K_S[/math]: Hill constants


[math]n_R, n_S[/math]: Hill coefficients


Overview

GitHub

GitHub link

Schedule

Week 1

Monday Tuesday Wednesday Thursday Friday
11:00am: Group Meeting 11:30am: Lab Induction 02:00pm: Tutorial 1
  • Derived and simulated mass action and
    enzymatic reactions from first principles

Week 2

Monday Tuesday Wednesday Thursday Friday
11:00am: Group Meeting 02:00pm: Tutorial 2
  • Derived and created standard models for constitutive
    gene expression
  • Derived and created standard models for activated
    and repressed gene expression

Week 3

Monday Tuesday Wednesday Thursday Friday
11:00am: Group Meeting 02:00pm: Tutorial 3
  • Derived and simulated a repressilator from first principles


Week 3

Monday Tuesday Wednesday Thursday Friday
11:00am: Group Meeting 02:00pm: Computational Modelling
  • Modelled Hill functions using parameters describing the
    best fits of the characterised promoter responses using
    different RBS found by Wang et al. 2011

Week 3

Monday Tuesday Wednesday Thursday Friday
11:00am: Group Meeting 02:00pm: Computational Modelling
  • Modelled transfer function describing biological AND gate
    for differing inducer concentrations

Computational Modelling

We replicated the best fits of the characterised promoter responses, using different RBSs, from the parameters described by Wang et al. in Engineering modular and orthogonal genetic logic gates for robust digital-like synthetic biology, 2011.

Dose responses of Plac, Pbad and Plux using different RBSs

Using parameters KR =206.1±32.5, KS=3135±374, nR =2.381±0.475, and nS=1.835±0.286 we then replicated the output response of the AND gate for different inducer concentrations.

Output of engineered AND gate for different inducer concentrations

Wet labs

Overview

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

Additional Papers

  • Design, construction and characterization of a set of insulated bacterial promoters
    • Joseph H. Davis et al., Nucleic Acids Research (2011)
    • Generated variable-strength constituituie bacterial promoters that are predictable in different contexts using insulator sequences
    • Protein level control can be achieved using different promoter strength to change transcription rates, using different RBS (to change translation efficiency) and by using degradation tags however very labour intensive
    • Steady-state protein levels can be controlled by using libraries of variable-strength promoters to change transcription rates, by employing different ribosome-binding sites to alter translation efficiency, and by appending degradation tags to adjust rates of protein turnover
    • Wanted to design a set of variable-strength constitutive bacterial promoters that are insulated from influences of genomic context
    • Bacterial RNA polymer- ase (RNAP) is composed of a core polymerase – important for transcriptional elongation, and a sigma subunit, to determine promoter specificity during binding and initiation but unimportant for elongation
    • Promoter strength is influenced by neighbouring sequences which is why the use of insulators is important – they showed that their acitivity is highly predictable independent of environment – but important to keep promoter size manageable


  • A second paradigm for gene activation in bacteria
    • M. Buck1 et al., PLOS (2020)
    • specialized molecular machinery that utilizes ATP hydrolysis to initiate DNA opening and permits a description of how the events triggered by ATP hydrolysis within a transcriptional activator can lead to DNA opening and transcription. The bacterial EBPs (enhancer binding proteins) that belong to the AAA+ (ATPases associated with various cellular activities) protein family remodel the RNAP (RNA polymerase) holoenzyme containing the σ54 factor and convert the initial, transcriptionally silent promoter complex into a transcriptionally proficient open complex using transactions that reflect the use of ATP hydrolysis to establish different functional states of the EBP.
    • A molecular switch within the model EBP called PspF (phage shock protein F] is evident, and functions to control the exposure of a solvent-accessible flexible loop that engages directly with the initial RNAP promoter complex. The σ 54 factor then controls the conformational changes in the RNAP required to form the open promoter complex.
    • enormous potential for exploitation in areas of synthetic biology where new regu- latory devices can be assembled from existing components
    • looked at multisubunit DNA-dependent RNAP (RNA polymerase) to melt out DNA so that gene expression can occur
    • All AAA+ proteins share highly conserved motifs known as Walker A (consensus sequence GXXXXGK [T/S]) and Walker B (con- sensus sequence hhhhDE, where ‘h’ represents a hydrophobic amino acid) motif, which are involved in ATP binding and hydrolysis respectively
    • AAA+ proteins usually form hexameric rings in their active conformation, often assembled from inactive dimers
    • In AAA+ proteins that activate the Eσ54, nucleotide binding occurs at the interface between subunits, thereby permitting determinants from adjacent sub- units to contribute to nucleotide sensing and hydrolysis
    • The energy derived from nucleotide hydrolysis is usually co- upled with substrate remodelling and functional output
    • AAA+ proteins that activate transcription by Eσ54 are known as EBPs (enhancer binding proteins). EBPs often bind to enhancer sequences which are located approx. 100–150 base-pairs either upstream (usually) or downstream (rarely) from the transcription start site and interact with the initial Eσ54–promoter complex (referred to as the closed complex) by a DNA looping event
    • The ATPase activity of EBPs is used to regulate the activity of Eσ 54 at the DNA open- ing step: EBPs couple the energy derived from nucleotide hydrolysis to remodel the Eσ54 closed complex and trigger a cascade of protein and DNA isomerization events that result in the formation of a transcriptionally proficient open complex
    • In the open complex, the DNA strands are separ- ated and the template DNA strand has ‘loaded’ into the 54catalytic cleft of Eσ .
    • EBPs consist of 3 functional domains.
      • highly conserved central domain is referred to as the AAA+ domain - responsible for nucleotide interactions and energy coupling to the Eσ54 closed complex for transcription activation. The AAA+ domain contains the signature GAFTGA sequence, which is directly involved in contacting the Eσ 54 closed complex *** C-terminal domain contains a helix–turn–helix DNA- binding motif and is responsible for binding to specific enhancer sequences.
      • N-terminal domain has a regulatory role and controls the activity of the AAA+ domain in response to environmental cues
    • different nucleotide states (ATP, the ATP hydrolysis transition state and ADP) present during ATP hydrolysis cycle are sensed by an atomic switch pair (Asn64 –Glu108 ) in the AAA+ domain of PspF and relayed through a conformational signalling pathway within PspF to the Eσ54 interacting loops L1 (which contains the GAFTGA sequence) and L2
    • that nucleotide binding to PspF-(and, by extension, other EBPs) occurs in a stochastic fashion and nucleotide hydrolysis in a co-ordinated manner to allow proper control of the atomic switch pair Asn64-Glu108 for transcription activation.


  • A Modular Cloning System for Standardized Assembly of Multigene Constructs
    • Ernst Weber et al., PLOS (2011)
    • Paper describes the development of the MoClo assembly standard and operation
    • MoClo enables directional, hierarchical assembly of up to 6 DNA fragments in a one-pot reaction using type IIs restriction enzyme and T4 DNA ligase
    • Researchers demonstrate the method by constructing a 33kb DNA sequence with 11 transcriptional modules
    • Type IIs restriction enzymes can cleave outside of their recognition sites, leaving 4bp overhangs that can be designed to be complementary between consecutive fragments, enabling directional assembly
    • 4bp overhangs are known as fusion sites


  • CIDAR MoClo: Improved MoClo Assembly Standard and New E. Coli Part Library Enable Rapid Combinatorial Design for Synthetic and Traditional Biology
    • Sonya V. Iverson et al., ACS Synthetic biology (2016)
    • Developed a publicly available collection of modular DNA parts and enhanced MoClo protocols to enable rapid one-pot, multipart assembly, combinatorial design, and expression tuning in Escherichia coli
    • The Cross-disciplinary Integration of Design Automation Research lab (CIDAR) MoClo Library is openly available and contains promoters, ribosomal binding sites, coding sequence, terminators, vectors, and a set of fluorescent control plasmids.
    • Optimized protocols reduce reaction time and cost by >80% from that of previously published protocols
    • MoClo is a one-pot digestion and ligation multipotent assembly method – used Golden Gate (user-defined overhangs specific to a part create interchangeable DNA modules in the form of plasmids allowing library propagation and combination) – MoClo allows for up to 6 parts


  • Regulation of the co-evolved HrpR and Hrps AAA + proteins required for Pseudomonas syringae pathogenicity
    • Milija Jovanovic et al 2010 DOI: 10.1038/ncomms1177
    • Bacterial pathogens use a type 3 secretion system(T3SS) to deliver proteins into host cells. hrp and hrc genes establish the regulatory and structural functions associated with the T3SS from the group I-type hrp/hrc cluster of Pseudomonas
    • hop genes encode secreted pathogenicity effectors and the majority of hop genes and hrp/hrc cluster genes are regulated by the extracytoplasmic func- tion σ-factor HrpL, which is regulated by the σ54-dependent hrpL promoter
    • Regulation of σ54-RNA polymerase (σ54-RNAP) activ- ity is achieved by the action of specific enhancer-binding proteins
    • hrpL expression is activated by a co-dependent pair of EBPs; HrpR and HrpS
    • EBPs are molecular machines belonging to the AAA + (ATPases associated with various cellular activities) superfamily
    • They remodelled σ54-RNAP at its promoter sites and, via a carboxy (C)-terminal HTH domain, bind promoter upstream activator sites (UASs). The ATPase activity of EBPs relies on the forma- tion of hexameric ring-like assemblies
    • hrpR and hrpS are arranged in tandem (hrpRS), transcribed as a single operon8 and exhibit high sequence similarity, suggesting that they have evolved from a single ancestral gene duplication even
    • co-expressed HrpR and HrpS (expressed simultaneously from different plasmid con- structs) or HrpRS (co-expressed as a single operon), but not HrpR or HrpS alone (expressed singly), resulted in elevated β-galactosi- dase activity—in line with the strict requirement for both HrpR and HrpS to activate hrpL transcription.
    • EBPs contact σ54 via the consensus GAFTGA motif27. Such inter- actions depend on the nucleotide-bound state28–30 and rely on the integrity of the ‘F’ and ‘T’ residues27,31. In HrpR, this sequence is pre- dominantly GAFTGV and in HrpS is always GAYTGA (including in singly acting HrpS
    • Regulation of EBPs is commonly achieved by altering higher- order oligomer formation (and hence ATPase activity
  • The Genetic Insulator RiboJ Increases Expression of Insulated genes
    • Kalen P. Clifton et al., Journal of Biological Engineering (2018)
    • Regulatory sequence downstream of transcriptional start site - when transcribed you get RNA leaders in 5' end
    • RNA leaders alter stability & secondary structure of mRNA
    • Nature of alterations specified by interactions between given RNA leader and downstream sequence of transcript
    • Genetic insulators prevent unintended RNA leaders by isolating parts from unwanted interactions with neighboring parts
    • RiboJ
      • Self-cleaving ribozyme
      • Insulates circuit from promoter-dependent effect
      • 75 nucleotide sequence
      • Satellite RNA of tobacco ringspot virus (sTRSV) derived ribozyme followed by 23 nucleotide hairpin
      • Inserted at junction between promoter and downstream sequence
      • Removed upstream sequences
      • After cleavage only hairpin containing sequence remains upstream and gene of interest
      • 5' end of insulated gene identical always
      • Standardise behavior of promoters
    • Constructed 2 measurement constructs for each promoter tested, on with RiboJ and one without
      • RiboJ upstream of RBS sequence
    • used low copy plasmid backbone p5B3k3 and NEB HiFi DNA assembly method
    • Transformed in 5-alpha E. Coli
    • Tested with different promoters
    • RiboJ had higher absolute florescence which means more gene expression
    • Stronger promoters felt the effect of riboJ more
    • RiboJ found to increase protein expression and transcript abundance
    • Increased stability of mRNA which leads to slower degradation of transcript, increased transcript abundance which in turn means more protein expression
    • Need to account for increased gene expression with insulator use


  • Recognizing and engineering digital-like logic gates and switches in gene regulatory networks
    • Robert W. Bradley et al., Current Opinion in Microbiology (2016)
    • Want to create biological versions of logic gates
    • Reduce noise by longer difference of output
    • Threshold and output of gate should be tuneable
    • Needs to be orthogonal
    • Want to characterise components because
      • dynamic range
      • activation threshold
      • transfer function steepness
    • Can usually fit components to Hill function
    • Gates usually have a high ON:OFF ratio when there is low intrinsic leakiness
    • Integrate multiple circuits by picking components with activating/repressing partners
    • Looked at Pseudomonas syringae hypersensitive response pathway regulatory components as model for orthogonal gates
    • Must have little cross talk and low toxicity to host
    • Usually tune through changes in transcription or translation
    • RBS design is efficient screening of sequences to achieve desired component levels
    • One can add buffer of decoy binding sites to lower OFF state
    • Cascade means ultra sensitivity
      • signal amplification which increase ON:OFF ratios


  • Rewiring cell signalling through chimaeric regulatory protein engineering
    • Baojun Wang et al., Biochemical Society Transactions (2013)


  • Rapid engineering of versatile molecular logic gates using heterologous genetic transcriptional modules
    • Baojun Wang et al., The Royal Society of Chemistry (2014)


  • Tools and Principles for Microbial Gene Circuit Engineering
    • Robert W. Bradley et al., Elsevier


  • A modular cell-based biosensor using engineered genetic logic circuits to detect and integrate multiple environmental signals
    • Baojun Wang et al., Elsevier
    • an Escherichia coli consortium-based biosensor has been constructed that can detect and integrate three environmental signals (arsenic, mercury and copper ion levels) via either its native two-component signal transduction pathways or synthetic signalling sensors derived from other bacteria in combination with a cell-cell communication module.
    • a cellular signalling network normally consists of three interconnected modules – the input sensors, internal processing and regulatory circuits and output actuators
    • input sensors are receptors, either embedded in the cell membrane (e.g. sensor kinases) or located freely in the cytoplasm (e.g. ligand responsive allosteric proteins)
    • engineered several E. coli-based logic-gated cellular biosensors by connecting the AND gate two inputs to a set of synthetic sensors for detecting and integrating the levels of arsenic, mercury, copper, zinc ions and bacterial quorum sensing molecules in an aqueous environment leading to a quantitative fluorescent output
    • a triple-input AND logic gated biosensor comprising two cell consortia was also constructed which can sense and integrate three environmental signals (As3 þ , Hg2 þ and Cu2 þ levels) via a synthetic cell–cell communication module driving the cooperation between the two cell populations
    • To design a logic AND-gated cellular biosensor, we connected two transcriptional inputs of the single input sensors to a modular and orthogonal genetic AND gate we have engineered in E. coli recently (Wang et al., 2011) and used gfp or rfp as the output readout. The modular two-input AND gate comprises two heterologous genes, hrpR and hrpS, and one s54-dependent out- put promoter, hrpL, from the hrp (hypersensitive response and pathogenecity) regulatory system of the plant pathogen P. syr- ingae
    • The hrpR and hrpS encode two regulatory enhancer binding proteins that act synergistically by forming a heteromeric protein complex to co- activate the tightly regulated hrpL promoter
    • Both the inputs and output of the AND gate were designed to be promoters to facilitate their connection to different upstream and downstream transcriptional modules. Due to this modularity, the inputs can be rewired to different input sensors and the output can be used to drive various cellular responses.
    • the sensor output is high only when both arsenic and mercury are above a certain threshold, similar to that of the single input sensors, sufficient to activate the two inputs of the genetic AND gate.
    • a second double-input AND gated biosensor that can detect and integrate Cu2 þ and the quorum sensing molecule 3OC6HSL. Using a similar architecture, the sensor circuit comprises the copper and 3OC6HSL sensors connected to the AND gate inputs and the rfp connected to the AND gate output promoter.
    • successful engineering of the double-input AND gated biosensors demonstrates the possibility of using synthetic sen- sory and gene regulatory circuits to rewire host cell native signalling and so to generate novel customized behaviour.


  • Engineering modular and tunable genetic amplifiers for scaling transcriptional signals in cascaded gene networks
    • Baojun Wang et al., Nucleic Acids Research (2014)
    • Paper describes the construction of 2 pairs of modular tunable and non-tunable genetic amplifiers
    • Operation of the devices is illustrated in conjunction with an arsenic-sensing module with a weak output
    • Amplifiers can be used to detect environmental pollutants or other parameters even at low levels, enabling early identification of problems
    • The amplifiers exploit the hrp regulatory network from Pseudomonas Syringae
    • The pair of 2-terminal (transcriptional input - fluorescent output) non-tunable amplifiers only used the hrpR and hrpS genes which produce the respective activator proteins HrpR and HrpS
    • The pair of 3-terminal (transcriptional input - tuning input - fluorescent output) tunable amplifiers also used the hrpV gene, which codes for the HrpV inhibitor protein (HrpV binds to HrpS, preventing the operation of the HrpRS complex)
    • It consists of 2 pairs since 2 RBSs were used: rbs30 (stronger, more amplification) and rbs32 (weaker, less amplification)
    • Results confirm the outcome of computational modelling and simulations (conducted in accordance with the forward-engineering approach fundamental to synthetic biology)
    • Similar steady state saturated amplification for rbs30 and rbs32, but the rbs30 amplifier reaches saturation earlier, indicating stronger amplification as expected
    • This was also confirmed by the non-saturated transfer function, which shows linear amplification for both rbs30 and rbs32, with rbs30 having a steeper slope/higher gradient
    • Such results were also observed in the tunable amplifiers
    • Tunable amplifiers showed a decrease in amplification following an increase in arabinose, i.e. the hrpV inducer, as expected
    • Modularity of the constructs was proved by wiring them to 6 constitutive promoters, of different strengths: in all 6 circuits, the desired amplification was observed
    • In addition, the amplifiers were demonstrated not to introduce any significant time delay in the output expression, enabling fast detection of any input of interest


  • Synthetic Biology Enables Programmable Cell-Based Biosensors
    • Maggie Hicks et al., ChemPhysChem Reviews (2019)
    • Paper is a review of the current state of cell-based biosensors and the advances in this field in the last few years
    • Cell-based biosensors with customisable performance ideally offer a cheap and efficient way of detecting molecules of interest
    • Issues lie in the general public's biosecurity concerns and the sensitivity of such sensors; a lot has been done to overcome both problems
    • Biosensors have a lot of areas of application, namely in environmental and medical monitoring and bioproduction
    • A biosensor is usually a combination of a sensing component (which detects the molecule of interest) and a reporter protein output component, whose expression is regulated by a transcription factor expressed by the sensing component
    • Paper section on tuning the dynamic range is relevant to this project, even if the latter is not technically about sensing
    • Using the riboJ to improve AND gate operation is effectively a way of tuning the dynamic range (on top of insulating adjacent promoter and rbs, of course)
    • hrp regulatory network from Pseudomonas Syringae can also be used as a highly specific metal pollutant sensor thanks to its AND gate operation (more detail in Elsevier by Baojun Wang et al.)


Procedures

Polymerase Chain Reaction (PCR)

Overview Input Protocol Equipment Output
  • PCR is an in vitro reaction/technique used for DNA amplification
  • It uses Taq DNA polymerase as the replication enzyme
  • It requires primer (replication-initiating single-stranded DNA fragment) pairs to be initiated
  • Primers are specifically tailored to the region of interest

DNA fragments (RBSs,
promoters, coding sequences,
plasmid backbones)

  • All reaction components (listed below for a 50µl reaction) are to be mixed in tubes on ice and then transferred to the thermocycler, preheated at 94°C.

5µl 10X pfu Buffer
1µl 10 mM dNTPs
2µl 10 µM Forward Primer
2µl 10 µM Reverse Primer
1µl Template DNA
1µl pfu DNA Polymerase
38µl distilled H2O

  • The thermocycling steps, temperatures and durations:
Initial denaturation 94°C 2 minutes
Denaturation 94°C 30 s
Annealing Tm - 4°C /
Extension 72°C 60 s
Final extension 72°C 10 minutes
  • Important specifications:

good to use high-quality, purified templates;
primers of roughly 20 bp in length and 40% GC content;
pfu DNA polymerase is a proofreading DNA polymerase;
Tm is the calculated melting temperature of applied primers;
the 3 middle steps should be repeated 25 to 35 times for significant results;
products contain dA overhangs at 3' end.

  • Thermocycler
  • Pipettes
  • Tubes
  • Centrifuge
Amplified DNA fragments (RBSs,
promoters, coding sequences,
plasmid backbones)

Procedure from New England Biolabs and Dr. B. Wang Thesis

Cell Culture growth

Overview Medium Use Protocol
  • There are 2 stages of cell culture one for the growth of bacteria in liquid and solid media and the other is for the characterisation of the fully constructed plasmids and AND gate.
  • Electric field moves negatively charged DNA towards positive end through gel
  • Shorter fragments move more quickly
  • Use DNA ladder to determine approximate size of fragments
  • LB (Luria-Bertani Broth)
  • LB agar
  • M9 minimal media with 0.4% glycerol
  • M9 minimal media with 0.01% glucose
  • Growth in liquid media
  • Growth in solid media
  • Characterisation
  • Characterisation
  1. Bacterial strains are streaked for single colonies from glycerol stocks onto solid agar media and grown overnight at 37 °C. Liquid cultures were inoculated with single bacterial colonies and grown overnight at 37 °C with shaking (200 rpm). The day liquid cultures were then inoculated from the overnight cultures and grown at 37 °C or 30 °C with shaking (200 rpm).
  2. Cell were prepared using CaCl method for heat transformation.
  3. A colony of the bacteria was inoculated in 5 ml LB media with 14ml Falcon tube containing appropriate antibiotic at 37 degreess with 200rpm shaking
  4. The culture was diluted 100x to 200 mL fresh LB media + antibiotic and grown to a mid log phase (OD600 between 0.3-0.4) at 37 degrees
  5. The culture was then transferred to pre-cooled 50 ml Falcon tubes and incubated on ice for 10 min before being centrifuged (5578 × g, 6 min, 0 °C)
  6. Harvested cells are then washed gently with cold CaCl2 sol (50mM), then pelleted by centrifugation (5578 × g, 6 min, 0 °C)
  7. The washed pellet is then resuspended in cold CaCl2 solution (50 mM), left on ice for 30 min and pelleted again
  8. The pellet is then resuspended in 1 ml cold solution (50 mM CaCl2, 15% (v/v) glycerol) and incubated on ice for 2 hours
  9. The cells were then dispensed into 1.5 ml microtubes in 100 μl aliquots and stored at -80 °C for later heat shock transformation
  10. For characterisation using a fluorometric assay of GFP synthesis, day-cultures were grown and monitored in 96 well micro-assay-plates
  11. Load day cultures in wells with different amounts of inducers to have a final volume of 200 microlitres per well
  12. The plate is then immediately incubated in the microplate reader (BMG POLARstar Omega) at 30/37 degrees with shaking (200rpm) between each 20min cycle of absorbance and fluorescence readings

Procedure from Dr. B. Wang Thesis

Agarose Gel Electrophoresis

Overview Equipment Reagents Protocol
  • Separating DNA by size for visualization and purification
  • Electric field moves negatively charged DNA towards positive end through gel
  • Shorter fragments move more quickly
  • Use DNA ladder to determine approximate size of fragments
  • Casting tray
  • Well combs
  • Voltage source
  • Gel box
  • UV light source
  • Microwave
  • Thermometer
  • Microwavable flask
  • Scale
  • Tape
  • Pipette
  • Agarose
  • TAE
    • Tris-base: 242g
    • Acetate (100% acetic acid): 57.1 mL
    • EDTA: 100 mL 0.5M sodium EDTA
    • Add dH2O up to one liter
    • To make 1xTAE from 50X TAE stock, dilute 20mL of stick into 980 of DI water
  • SYBR Safe DNA Gel Stain
  • Loading buffer
  • DNA ladder (Invitrogen)
  1. Measure 1g of agarose
  2. Mix powder with 100 mL of 1xTAE in a microwavable flask
  3. Microwave for 1-3 minutes, stopping every 30-45 seconds to swirl, until fully dissolved
  4. Let solution cool to about 50 degrees Celsius, until you can hold it comfortably with your hand, about 5 mins
  5. Add the SYBR Safe DNA Gel Stain with a final concentration of 1:10,000
  6. Tape edges of the gel tray to create a closed container
  7. Place the well comb in the gel tray
  8. Pour the solution into the gel tray
  9. Let rest for 20-30 mins at room temperature or 5 mins in a fridge until the gel has solidified
  10. Add loading buffer to each of the DNA samples
  11. Remove tape from edges of gel tray and place into gel box with the wells by the negative end
  12. Fill gel box with 1xTAE until gel is covered
  13. Choose a well for the DNA ladder to go and pipette some slowly into the well - this will be used to compare the rest of the bands
    • Be careful not to pierce the gel and do not release the pipette contents until you are inside the well
  14. Add the rest of the DNA samples into the wells of the gel, spacing them out accordingly
  15. Plug in the gel box to the voltage source
  16. Run the gel at 100V until the dye has moved about 75% of the way down the gel, usually 1-1.5 hours
  17. Once the run has finished, power off the voltage source and remove the electrodes
  18. Remove the gel from the gel box
  19. To visualize the DNA fragments, use the UV light to give an image of the bands on the gel
    • In Wang's paper it says to visualize under blue light using a Bio-Rad gel imaging system

Procedure from Addgene and Dr. B. Wang Thesis

DNA Purification

Overview Equipment Reagents Protocol
  • isolate and purify DNA fragments based on size
  • helpful for PCR or restrictive enzyme-based cloning
  • razor blade or scalpel
  • UV box
  • microfuge tube
  • scale
  1. Complete a Agarose Gel Electrophoresis with the following adjustments in mind
    • Make sure there are crisp bands - using a wider comb or running with a lower voltage can help
    • Should be space around each band to cut it out - easily achieved by skipping lanes
    • Limit UV exposure of DNA - don't want to take a picture of it before cutting out the bands
  2. Remove the gel from the gel tray and place in an open UV box on a glass plate
    • Use long-wave UV and be quick with any actions in the UV box
  3. Slice the desired fragment from the gel
    • Try to reduce the extra gel around the band if possible
    • Trim the top, bottom and sides
  4. Place gel in labeled microfuge tube
  5. Weigh the gel fragment making sure to zero the scale with an empty microfuge tube
    • Make a note of the weight as it will be used for the DNA isolation step
  6. Isolate the DNA from the gel using a QlAquick PCR Purification Kit according to manufacturer's instructions
  7. Elude DNA in 30μL distilled water

Procedure from Addgene and Dr. B. Wang Thesis

Gibson Assembly Cloning

Overview Equipment Reagents (2-3 fragment reaction) Protocol
  • Relatively new assembly/cloning method
  • Independent of fragment length or compatibility
  • Isothermal reaction joins multiple linear overlapping DNA fragments
  • Uses 3 different enzymes
  • Efficient and effective plasmid assembly
  • Ice Bath
  • Thermocycler
  • Tubes
  • Gibson Master Mix 10µl
    • T5 Exonuclease
    • Phusion DNA Polymerase
    • Taq DNA ligase
  • Deionized H2O (10-X)µl
  • DNA fragments (total amount) 0.02-0.5mol (Xµl)
  • Prepare reaction on ice
  • Incubate tubes contaning samples at 50°C in the thermocycler for 15 minutes
  • Store product on ice

Procedure from New England Biolabs

Heat Shock Transformation

Overview Equipment Reagents Protocol
  • Creation of pores in bacterial cell membrane
  • Bacterial cells able to take up foreign DNA from environment
  • Uses calcium chloride to counteract the electrostatic repulsion between the plasmid DNA and bacterial cellular membrane
  • Shaking incubator at 37°C
  • Stationary incubator at 37°C
  • Water bash at 42°C
  • Ice
  • Microcentrifuge tubes
  • Sterile spreading device
  • DNA to transform
  • LB agar plate (with appropriate antibiotic)
  • LB or SOC media
  • Competent cells
  1. Thaw competent cells on ice for about 20-30 min
    • It is recommended to use the manufacturer's directions if they were bought from a company
  2. Allow agar plates to warm up to room temperature
    • Optional: Incubate in 37°C incubator
  3. Measure out 1-5 μL of the DNA and 20-50 μL of competent cells
  4. Mix the DNA and competent cells in a microcentrifuge tube
    • Mix by gently flicking the bottom of the tube
  5. Incubate the mixture on ice for 20-30 mins
  6. Heat shock each tube by placing 1/2 to 2/3 of the tube in a 42°C water bath for 30-60 secs (45 secs is ideal, but it will depend on competent cells)
  7. Ice the tubes for 2 mins
  8. Add 250-1000 μL LB or SOC media to bacteria and grow in 37°C shaking incubator for 45 mins
  9. Plate some of the transformation onto a 10 cm plate with LB agar containing antibiotic
    • For better results, put 50 μL on one plate and the rest on another
  10. Incubate overnight at 37°C
    • If the cell culture is too big, collect cells by centrifugation and resuspend in a smaller volume of LB
    • When the agar plate doesn't dry out properly, it could prevent bacteria from growing into colonies

Procedure from Addgene

Flow Cytometry

Overview Equipment Reagents Protocol
  • Technique used to detect and measure physical and chemical characteristics of a population of cells or particles
  • In our case, used for fluorescence measurement
  • Fluid suspension containing stained cells of interest is injected into the cytometre
  • Cells pass through a laser beam one at a time: precise measurement method
  • Flow cytometre
  • Laser light source
  • Tubes
  • Phosphate Buffer Saline (PBS)
    • 10% FCS
    • 1% sodium azide
  • Conjugated Primary Antibody
  • (Both reagents needed for staining)
  • Prepare suspension with a concentration of 1-5 x 10^6 cells/mL of PBS
  • Add 0.1-10 µg/mL of CPA
  • Incubate suspension for 30 minutes at 4°C
  • Wash cells by centrifugation and resuspend in fresh PBS
  • Store cells at 4°C before analysis
  • Inject suspension into cytometre

Procedure from Abcam

List of parts to order

Ribosome-binding sites

  • BBa_B0033 (rbs33)
  • rbsH

Coding Sequences

  • BBa_E0840 (GFP coding sequence)
  • hrpR
  • hrpS

Completed Plasmids

  • pBW400hrpL-gfp (output plasmid)
  • pBW115lac-hrpR (input plasmid 1)
  • pBW213ara-hrpS (input plasmid 2)

Schedule

Week 1

Monday Tuesday Wednesday Thursday Friday
  • Look into protocols
11:00am: Group Meeting
12:00pm: Benchling Tutorial
4:30pm: Workflow Session 11:30am: Lab Induction
  • Investigate other DNA assembly methods
  • Come up with a work flow to build, test, and
    characterize the different parts if we have the
    fully assembled plasmid

Week 2

Assigned Monday Tuesday Wednesday Thursday Friday
Everyone 11:00am: Work flow Session 2
  • Create a small presentation for long and short-term goals
  • Update wiki
11:00am: Group Meeting
Lorenzo Read papers 2:00pm: Start in wet lab
Dimitra Update Wiki Read papers Read papers
Hamza Read papers 2:00pm: Start in wet lab
Lana Read papers Read papers Read papers

Week 3

Assigned Monday Tuesday Wednesday Thursday Friday
Everyone 10:30am: Group meeting about report
11:00am: Group Meeting
  • Start report
11:00am: Tutorial with Charles
Lorenzo Read papers Read papers
Dimitra Read papers Read papers Read papers
Hamza Read papers Read papers Read papers
Lana 9:00am: Mini-prep Read papers Read papers

Week 4

Assigned Monday Tuesday Wednesday Thursday Friday
Everyone 11:00am: Group Meeting Work on report 12:00pm: Group Meeting
  • work on report
Lorenzo 2:00pm: Wet Lab Session 3
Dimitra 3:00pm: Wet Lab Session 4 8:00am: Wet Lab Session 5


10:00am: Wet Lab Session 6

Hamza 2:00pm: Wet Lab Session 3
Lana 3:00pm: Wet lab Session 7

Week 5

Assigned Monday Tuesday Wednesday Thursday Friday
Everyone 11:00am: Group Meeting Work on report
Lorenzo 8:00am: Lab Session 8 4:00pm: Lab Session 12
Dimitra 9:30am: Wet Lab Session 13 12:00pm: Wet Lab Session 14
Hamza 12:10pm: Wet Lab Session 9
Lana 3:00pm: Lab Session 10 Morning: Lab Session 11


Wet lab Notebook

Original work flow diagram for laboratory

4/2/2020 Benchling introduction

  • went through how to use the Benchling
  • talked about creating primers - must be in pairs
  • were told to look on AddGene for some of the sequences we needed

5/2/2020 Work flow Session

  • started talking about what we will do in the wet lab
  • discussed different protocols
  • created a work flow diagram
    • large post-its represent actions, square post-its are protocols, and small post-its are inputs and outputs
  • decided that we will order the complete plasmids and try to assembly them in parallel
  • originally were going to use restrictive digestion technique, but discussed changing to Gibson Assembly
  • gained insight on how we will structure our wet lab work and will work into dividing all the steps between group members
  • will create schedule for next meeting

10/2/2020 Workflow Session 2

  • Showed the updated wiki and discussed protocols
  • We can use TAE instead of TBE
  • Charles will help us come up with what we will slice and insert into completed plasmid
  • Need to update cell culture on the wiki and finish heat transformation
  • Wanted us to look into more assembly techniques
  • Look into MoClo for tomorrow
  • Matt is sending us a paper for RiboJ - we can use this as an insulator, it works really well
  • Re-defined project
    • Order three complete plasmids, extract some parts (like promoters and coding sequences) from them and operate PCR
    • We will then perform electrophoresis to see if they are the correct length
    • If the part is what is expected, we will send off to get sequenced and characterized
    • Try inserting RiboJ into plasmids to see if AND gate operation is improved
  • Told to also look into primer design for PCR
  • Plan for this week is to make a mini presentation to update Kitney at group meeting on 11/2/2020 and in the labs we will work to familiarize ourselves with the assembly phase with parts the lab already has while we wait for the ones we want to be ordered
  • They said only 2 people in the lab at once - we will be in pairs

10/2/2020 Lab Session 1

  • Pipetting practice
  • Observing Charles and Alexis preparing the LB-Agar with antibiotic and streaking them with bacterial colonies grown in a glycerol stock:
    • LB-Agar mix was made by Charles and Alex beforehand, by melting some agar into LB broth
    • Then, 8 petri dishes were filled with LB-agar containing Ampicillin, to grow an Ampicillin-resistant colony, and 4 petri dishes were filled with LB-agar containing Kanamycin, to grow a Kanamycin resistant colony
    • The dishes were then left to dry, in order for the gel-like substance to fully solidify
    • Bacteria in glycerol stock come from CIDAR MoClo kit purchased from AddGene: the contents of the kit (different plasmids in E.Coli), which comes as a 96-well plate, are found here Addgene.
    • To streak the stocks on the LB-agar, Charles used an inoculating loop, and streaked the stocks in a spiral-like pattern on the LB-agar
    • The cultures were labelled and left to grow
    • They are stored on the lab bench, at room temperature

20/2/2020 Lab Session 2

Equipment used for Plasmid extraction

Lana went to the lab on Wednesday and after innoculation it was time for plasmid extraction - the following protocol was followed.

Monarch® Plasmid DNA Miniprep Kit Protocol (NEB #T1010)

Yield and quality of plasmid DNA is affected by plasmid copy number, plasmid size, insert toxicity, host strain, antibiotic selection, growth media and culture conditions. For standard cloning strains of E. coli, we recommend using a single colony from a freshly streaked selective plate to inoculate a standard growth media, such as LB (Luria-Bertaini) media. Cultures are typically grown at 37°C and 200–250 RPM in vessels that allow some aeration (Erlenmeyer flasks or culture tubes on a roller drum) and harvested after 12–16 hours as the culture transitions from logarithmic growth to stationary phase. This is the time at which the plasmid DNA content is highest. While cultures in LB often saturate with a final OD600 between 3–6, growth to saturation often leads to cell lysis. As a result, plasmid yields and quality are reduced and the likelihood of co-purifying unwanted host chromosomal DNA increases. Use of rich media, such as 2X YT or TB, produces higher biomass in a shorter time period. If chosen for growth, adjustments to the culture times and amount of cells used in the prep should be made to correct for these differences, and to avoid overloading the matrix and reducing DNA yield and quality.

PLASMID REPLICON COPY NUMBER CLASSIFICATION pUC and its derivatives pMB1* > 75 High copy pBR322 and its derivatives pMB1 15–20 Low copy pACYC and its derivatives p15A 10–12 Low copy pSC101 pSC101 ~5

  • pUC and its derivatives lack the Rop gene and contain a point mutation in the RNAII transcript. These changes result in higher copy number during routine growth with many sources reporting levels as high as 500 copies per cell.

Antibiotics for Plasmid Selection ANTIBIOTIC CONCENTRATION OF STOCK SOLUTION STORAGE TEMP. WORKING CONCENTRATION Ampicillin 100 mg/ml (H2O) –20°C 50–200 μg/ml Carbenicillin 100 mg/ml (H2O) –20°C 20–200 μg/ml Chloramphenicol 34 mg/ml (ethanol) –20°C 25–170 μg/ml Kanamycin 10 mg/ml (H2O) –20°C 10–50 μg/ml Streptomycin 10 mg/ml (H2O) –20°C 10–50 μg/ml Tetracycline 5 mg/ml (ethanol) –20°C 10–50 μg/ml

Buffer Preparation:

Add ethanol to Monarch Plasmid Wash Buffer 2 prior to use (4 volumes of ≥ 95% ethanol per volume of Monarch Plasmid Wash Buffer 2).

For 50-prep kit add 24 ml of ethanol to 6 ml of Monarch Plasmid Wash Buffer 2 For 250-prep kit add 144 ml of ethanol to 36 ml of Monarch Plasmid Wash Buffer 2 Always keep all buffer bottles tightly closed when not actively in use.

Protocol:

  1. All centrifugation steps should be carried out at 16,000 x g (~13,000 RPM).
  2. If precipitate has formed in Lysis Buffer (B2), incubate at 30–37°C, inverting periodically to dissolve.
  3. Store Plasmid Neutralization Buffer (B3) at 4°C after opening, as it contains RNase A.
  4. Pellet 1–5 ml bacterial culture (not to exceed 15 OD units) by centrifugation for 30 seconds. Discard supernatant.
    • Note: For a standard miniprep to prepare DNA for restriction digestion or PCR, we recommend 1.5 ml of culture, as this is sufficient for most applications. Ensure cultures are not overgrown (12-16 hours is ideal).
  5. Resuspend pellet in 200 μl Plasmid Resuspension Buffer (B1) (pink). Vortex or pipet to ensure cells are completely resuspended. There should be no visible clumps.
  6. Lyse cells by adding 200 μl Plasmid Lysis Buffer (B2) (blue/green). Invert tube immediately and gently 5–6 times until color changes to dark pink and the solution is clear and viscous. Do not vortex! Incubate for one minute.
    • Note: Care should be taken not to handle the sample roughly and risk shearing chromosomal DNA, which will co-purify as a contaminant. Avoid incubating longer than one minute to prevent irreversible plasmid denaturation.
  7. Neutralize the lysate by adding 400 μl of Plasmid Neutralization Buffer (B3) (yellow). Gently invert tube until color is uniformly yellow and a precipitate forms. Do not vortex! Incubate for 2 minutes.
    • Note: Be careful not to shear chromosomal DNA by vortexing or vigorous shaking. Firmly inverting the tube promotes good mixing, important for full neutralization.
  8. Clarify the lysate by spinning for 2–5 minutes at 16,000 x g.
    • Careful handling of the tube will ensure no debris is transferred - spun for 5 minutes to ensure efficient RNA removal by RNase A.
    • Also, longer spin times will result in a more compact pellet that lower the risk of clogging the column.
  9. Spin for two minutes only.
  10. Carefully transfer supernatant to the spin column and centrifuge for 1 minute. Discard flow-through.
  11. Spin for 30 seconds
  12. Re-insert column in the collection tube and add 200 μl of Plasmid Wash Buffer 1. Plasmid Wash Buffer 1 removes RNA, protein and endotoxin. (Add a 5 minute incubation step before centrifugation if the DNA will be used in transfection.) Centrifuge for 1 minute. Discarding the flow-through is optional.
  13. Ensure the column tip and flow-though do not make contact.
  14. Spin for 30 seconds,
  15. If using a vacuum manifold, add 200 μl of Plasmid Wash Buffer 1 and switch the vacuum on. Allow the solution to pass through the column, then switch the vacuum source off.
  16. Make sure to follow the manifold manufacturer's instructions to set-up the manifold and connect it properly to a vacuum source.
  17. Add 400 μl of Plasmid Wash Buffer 2 and centrifuge for 1 minute.
  18. When using a manifold add 400 μl of Plasmid Wash Buffer 2 and switch the vacuum on. Allow the solution to pass through the column, then switch the vacuum source off.
  19. Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column has not come into contact with the flow-through. If there is any doubt, re-spin the column for 1 minute before inserting it into the clean microfuge tube.
    • If using a vacuum manifold: Since vacuum set-ups can vary, a 1 minute centrifugation is recommended prior to elution to ensure that no traces of salt and ethanol are carried over to the next step.
  20. Add ≥ 30 μl DNA Elution Buffer to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA.
    • Note: Nuclease-free water (pH 7–8.5) can also be used to elute the DNA. Delivery of the Monarch DNA Elution Buffer should be made directly to the center of the column to ensure the matrix is completely covered for maximal efficiency of elution. Additionally, yield may slightly increase if a larger volume of DNA Elution Buffer is used, but the DNA will be less concentrated as a result of dilution. For larger plasmids (≥ 10 kb), heating the DNA Elution Buffer to 50°C prior to eluting and extending the incubation time after buffer addition to 5 minutes can improve yield.

21/02/2020 Assembly Tutorial

  • Short 1-hour tutorial by Charles about different assembly/cloning standards
  • Covered Site-directed Mutagenesis, Gibson assembly and Golden Gate assembly
  • For this project, Gibson assembly is the most important
  • Site-directed mutagenesis and Gibson assembly both require PCR, while Golden Gate assembly does not and works with type IIs restriction enzymes
  • SDM uses custom primers to insert or delete specific DNA sequences in plasmids, by first running PCR and then using P4 DNA Ligase
  • SDM is not directional
  • Gibson assembly requires 4 primers (2 pairs) per plasmid+insert construct
  • Primer sequences all cover both the insert and the plasmids, creating tails
  • After the PCR-based amplification of parts, T5 DNA Exonuclease chews back the 5' of these tails, revealing overlapping sites which enable directional assembly
  • Gibson assembly allows assembly of up to 5 fragments in an isothermal, one-pot reaction
  • Golden Gate assembly uses type IIs restriction enzymes which cut outside their recognition sites, providing scarless assembled sequences
  • Type IIs enzymes leave 4bp overhangs, known as fusion sites, which enable directional assembly when designed properly
  • [At the end of the tutorial, Charles also helped us with the in silico assembly of riboJ into our input plasmids on Benchling]

25/02/2020 Lab Session 3

frame
  • The following plasmids, which were ordered, were available for streaking:
    • pBW115lac-hrpR (Kanamycin resistant)
    • pBW213ara-hrpS (Chloramphenicol resistance)
    • pBW313lux-hrpR (Kanamycin resistant)
    • pBW400hrpL-gfp (Ampicillin resistant)
    • pBW412hrpL-cIgfp (Ampicillin resistant)
    • pBW414hrpL-cIgfp (Anpicillin resistant)
  • The plasmids all arrived in capsules filled with LB-agar
  • They arrived in E.Coli dh5 alpha strains which formed genetically heterogeneous clusters.
  • They were then streaked using an inoculating loop for single colonies on Petri dishes filled with LB-agar and their respective antibiotic
  • The bacteria with plasmid pBW213ara-hrpS were not streaked since the LB-agar containing Chloramphenicol had yet to be prepared
  • The capsules with the clusters were placed into the fridge at 4°C for storage
  • The Petri dishes with the streaked bacteria were left on the bench for storage at room temperature

27/02/2020 Lab Session 4

Picture of the tubes that will be incubated overnight
Picture of the incubator tubes were put in overnight
  • All of the streaks were successful
  • We looked at them under a blue light in the next room and oberved that those with gfp( might be a different one) were florescent
  • Charles identified one single colony on each plate which would be used for culturing overnight
  • We only looked at five plates, the sixth one he said wasn't for our group
    • I noticed that Lorenzo and Hamza did streak six when I was uploading this session
  • Started by getting 5 large tube to place the LB in.
    • Measured 3 (will get units later, but I think mL) using a smoothie pipette into each tube
    • This had to be done very carefully as we wanted to keep a sterile environment
    • Had a burner on around the equipment used to promote a sterile environment
      • Charles mentioned that it is good practice to do everything with one hand, ie remove the lid and hold it along with the flask in one hand
  • Labelled each tube with the appropriate name, both on the lid and on tubes
  • Charles took the antibiotic out of the freezer and explained we would have to add it if we wanted our cultures to grow successfully.
    • pBW115lac-hrpR needs Kanamycin
    • pBW213ara-hrpS needs chloraphenicol
    • pBW400hrpL-gfp needs ampicillian
    • pBW412hrpL-clgfp needs ampicillian
    • pBW414hrpL-clgfp needs ampicillian
  • Measured 30μL of the appropriate antibiotic into the respective tube
  • Mixed slightly by flipping tubes a few times
  • Scraped the single colony from the plate into the respective tube using a toothpick (it was like a toothpick with a hook - will note the name next time)
    • Put it in the mixture of the tube and stir a couple times, then dispose
  • Put the tubes in the incubator to grow overnight

28/02/2020 Lab Session 5

  • Got the tubes out of the incubator
    • The overnight cultures were successful - the liquid was cloudy as can be seen below.
Result of the overnight cultures
  • Performed a Mini-Prep of the cultures using the Monarch® Plasmid DNA Miniprep Kit Protocol (NEB #T1010)
    • See Lab Session 2 for the protocol and procedure - same steps were followed

28/02/2020 Lab Session 6

  • In this session we did PCR for the cultures
  • First put dNTP in a bunch of little tubes - we only used 1
  • Followed the protocol for routine PCR (explained in Protocols)
    • used 1 ng of template of DNA
  • Charles diluted the dry primers that were received from Addgene
  • We then diluted it even more, with 90uL of water and 10uL of the stock to make the working stock
  • Added all the necessary components to tiny tubes to put in PCR
    • You add the biggest volumes first and make sure to add the Phusion last
  • Changed the PCR settings accordingly
    • pBad Plasmid
      • 61 C annealing
      • 210 sec extension
    • pBad with RiboJ
      • 64 C
      • 10 sec extension
    • plac Plasmid
      • 58 C annealing
      • 240 sec extension
    • plac with RiboJ
      • 64 C annealing
      • 10 sec extension

28/02/2020 Lab Session 7

  • In this session we transformed the cells using TOP10 E.Coli to try and combine all the parts together we followed the following protocol.
  • One Shot® TOP10 Chemically Competent E. coli with a transformation efficiency of 1 x 109 cfu/µg plasmid DNA and are ideal for high-efficiency cloning and plasmid propagation. They allow stable replication of high-copy number plasmids and are the same competent cells that come with many of our cloning kits
  • Centrifuge the vial(s) containing the ligation reaction(s) briefly and place on ice.
  • Thaw, on ice, one 50 μL vial of One Shot® cells for each ligation/transformation.
  • Pipet 1–5 μL of each ligation reaction directly into the vial of competent cells and mix by tapping gently.
  • The vials were then incubated on ice for 30 minutes.
  • The vials were then incubate for exactly 30 seconds in the 42°C water bath - this was the heat shock transformation
  • The vials were removed vial(s) from the 42°C bath and place them on ice.
  • 250 μL of pre-warmed S.O.C medium was added to each vial (rich nutrient material)
  • The vial(s) were placed in a microcentrifuge rack on its side and secure with tape to avoid loss of the vial(s). Shake the vial(s) at 37°C for exactly 1 hour at 225 rpm in a shaking incubator.
  • We had 200 μL from the transformation which was then spread on labeled LB agar plates.
  • The plates were then left to dry and incubated

3/3/2020 Lab Session 7

  • In this session, we prepared the gel for the electrophoresis of the following PCR products:
    • pBW115lac-hrpR
    • riboJ for pBW115lac-hrpR
    • pBW213ara-hrpS
    • riboJ for pBW213ara-hrpS
  • Electrophoresis is a first verification step to check that PCR of the fragments of interest has gone as expected
  • Because of the great size difference between the plasmids and the riboJs, we prepared 2 gels, one to visualize the 2 riboJs (short fragments) and one to visualize the 2 plasmids (long fragments)
  • The agarose gel mixture had been previously prepared, and we microwaved it for a few minutes before pouring to make sure it was not solid at all
  • The mixture was poured in the tray, onto which some DNA stain had been previously pipetted
  • The gel was then left to solidify at room temperature
  • In the meantime, we prepared the DNA fragments:
    • First, we added restriction enzyme ... to all DNA mixtures: that enzyme cuts at specific recognition sites which are only present in plasmid DNA and not in PCR products; that is useful because we don't want plasmid DNA in our assembly reaction
    • The tubes were then incubated for about 20 minutes
    • Then, we added 5µl purple loading dye to the 25µl DNA fragments mixture (to achieve a dye concentration of 1/6); the loading dye is useful because, due to similar viscosities, the DNA mixture and buffer that will be added on top of the electrophoresis trays mix together: the loading dye increases the viscosity of the DNA mixture, preventing such mixing from happening

3/3/2020 Lab Session 8

  • We launched electrophoresis, applying a voltage of 100V for 45 minutes. We loaded each tray with 2 1kb plus ladders, for appropriate DNA size identification (1kb plus ladders cover a 100bp-10000bp migration range)
  • While electrophoresis was running we performed a so-called "Clean and Concentrate" on our PCR products
    • For Gibson assembly, fairly precise quantities of DNA fragments in the reaction must be known
    • To obtain a precise estimate of these quantities, a machine, the NanoDrop spectrophotometre, is usually used
    • A drop of a DNA-buffer solution is dropped on a metal pedestal
    • The machine measures the absorbance of UV light by the whichever substance is on the pedestal as a function of wavelength
    • Since DNA has a specific absorbance wavelength for UV light, there is a typical target spectrum which should appear
    • The machine also gives the concentration of DNA in the drop
  • PCR products have many additional detached fragments (e.g. primers and single nuocleotides) floating around in their solution
  • Therefore, the spectrophotometre would show more than the actual quantity of DNA that actually matters in the assembly
  • PCR products must therefore be purified --> that is "Clean and Concentrate"
    • A specific kit, manufactured by NEB, is used
    • Special "columns" containing a piece of paper at the bottom end are placed in tubes
    • The paper binds specifically to single-stranded DNA of more than 200 bases, or double-stranded DNA of more than 50 bp, discarding all the rest
    • This is an effective method of discarding primers and other unwanted oligonucleotides from the mixture
    • After having thrown away the unwanted DNA by using a washing and a binding solution, the remaining amount is diluted, with water acting as the buffer
  • Its quantity is then measured with the spectrophotometre
  • Following the protocol, the Gibson assembly reaction was also set up
  • Electrophoresis results were also examined by looking at the gel first with a normal blue light emitter and then with a UV light machine
    • Unfortunately, pLac did not migrate by the desired amount, indicating a poor result for PCR
    • Due to this, we will only insert the riboJ sequence inside the pBad plasmid, which on the other hand gave the desired result
    • The 2 riboJs also had the desired migration characteristics
  • We then set up the Gibson assembly reaction
    • 4 reactions were setup
      • Gibson assembly of pBad with riboJ (5x more riboJ than pBad, according to the protocol for short length inserts)
      • Gibson assembly of pBad with riboJ (10x more riboJ than pBad)
      • Gibson assembly of pLac with riboJ (5x more riboJ than pLac)
      • Gibson assembly of pLac with riboJ (10x more riboJ than pLac)
    • Charles worked out the concentrations using an online calculator
    • The whole mixture containing also the assembly Master Mix was incubated for 15 minutes at 50°C for the cloning to happen

3/3/2020 Lab Session 9

Lab session 10 3/3/20

We used high efficiency transformation protocol For C2987H: Thaw a tube of NEB 5-alpha Competent E. coli cells on ice for 10 minutes.

  • We added 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. The tube should be flicked 4-5 times to mix cells and DNA. Do not vortex.
  • The mixture was then placed on ice for 30 minutes.
  • Heat shock at exactly 42°C for exactly 30 seconds then placed on ice for 5 minutes.
  • 950 µl SOC mixture Pipette of room temperature into the mixture.
  • the mixture was then placed at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
  • selection plates were warmed to 37°C.
  • the cells were mixed thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
  • 50-100 µl of each dilution were plated onto a selection plate and incubate overnight at 37°C.

Lab session 11

We verified the bacteria that Charles and Lana had streaked in the previous session (DH5-alpha E. Coli containing the Gibson assemblies)

    • 8 Petri dishes, 4 for pLac and 4 for pBad
      • 100µl of bacterial mixture, Gibson assembly with 5x more riboJ than plasmid
      • 900µl of bacterial mixture, Gibson assembly with 5x more riboJ than plasmid
      • 100µl of bacterial mixture, Gibson assembly with 10x more riboJ than plasmid
      • 900µl of bacterial mixture, Gibson assembly with 10x more riboJ than plasmid
    • The different Petri dishes were set up to make the process of picking a single colony easier
  • As expected, no colonies appeared in the pLac Petri dishes
  • That made sense since pLac plasmid PCR had failed
  • The dishes with 10x riboJ contained many less colonies
  • We then set up overnight liquid cultures based on a single colony
    • We prepared a mixture containing the medium and the appropriate antibiotic (Chloramphenicol in the case of pBad), in 3 different tubes
    • Using an inoculating loop, we then transferred one bacterial colony from the Petri dishes to each tube
    • Tubes were then incubated and left for overnight growth

6/3/2020 Lab Session 14

  • transforming three things
  • will be using heat shock transformation (view how to do it in protocols)
  • sequencing results came back! Seemed to be successful overall
  • RiboJ 3 seemed to have one base pair missing so we decided to discard it
  • decided to use RiboJ 1 with pBAD
  • the three things we will be transforming are
    • 1μL of pBW213ara-hrpS + RiboJ
    • 1μL of pBW115lac-hrpR plasmid
    • 5μL of pBW400hrpL-gfp plasmid (we are using more because it is a low copy plasmid

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