IC.Y3.AND.Gate.with.RiboJ:Wet labs

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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 (2015)
    • Review paper that describes many strategies for engineering synthetic gene circuits
    • Detailed a large variety of techniques, below are those relevant to our project
    • Modulation of transfer functions beyond transcriptional and translational control
      • HrpRSV system is a protein/protein interaction that can amplify and diminish a signal
      • HrpR + HrpS from a heterohexamer that activates the sigma54-dependant transcription from the PhrpL promoter, allowing AND logic function
      • Conversely, HrpV negatively modulates promoter output, therefore reducing the gain and dynamic range
      • Illustrates an important strategy to lower the OFF level of a signal and increasing the sensitivity of a response
    • Principles for robust and scalable gene circuit design - modularity and orthogonality
      • Physical composition
        • Parts can be sensitive to sequence changes at their boundaries
        • From their analysis of 12,563 promoter/RBS combinations, Kosuri et al. found that nearly 17% of the variation in protein levels could not be predicted from the promoter or RBS strength
        • A solution to this is to expand the definition of a part to include the regions that influence its function
        • E.g. promoter/UTR junction and UTR/Gene of interest (GOI)junction are important sources of variability in gene expression
        • Mutalik et al. created an EOU (expression operating unit) comprising of a promoter and a 5' UTR adjacent to the transcription start site to remove unwanted contextual effects
        • Another approach to improve modularity is to insulate parts from their sequence contexts. At the DNA level, this might include adding standardised spacers between components
      • Functional composition
        • Failure can occur when individual parts are connected to form a circuit, as the output range of an upstream part may not match the input range of a downstream component (retroactivity)
        • Retroactivity can be mitigated by characterising each part in their functional context, or by increasing downstream loads by through the use of an insulating kinase/phosphorylase buffer module
        • There may also be unwanted cross-talk between components
        • This can be avoided using part families that have been very well characterised
        • RNA-based part families are advantageous in that cross-talk can be eliminated during in silico modelling
      • Host context
        • E.g. Availability of cellular resources (ribosome availability and growth rate) are key factors in determining the effectiveness of a gene circuit in differing E. Coli strains
        • If the gene circuit has a burden on the host exceeding homeostatic limits, this will lead to altered behaviour or even failure of the cell and circuit
        • Direct cross talk can also occur between host components and the gene circuits, leading to interference of toxicity (at high expression levels)
        • Parts are often sourced from unrelated organisms in order to avoid this


  • 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

DNA Sanger Sequencing

Overview Input Protocol Output
  • Also known as the chain termination method
  • Method for determining the nucleotide sequence of DNA
  • Uses three main stages
    • Chain termination PCR
    • Size separation by gel electrophoresis
    • Gel analysis and determination of DNA sequence
  • Sequence of DNA to be sequenced
  • Complementary primer at a location an adequate distance from the desired portion of DNA to be sequenced
  • All the inputs for PCR with one major difference - ddNTPs (dideoxyribonucleotides) as well as dNTPs
  • Chain termination PCR
    • Performed in a similar manner to standard PCR however the user mixes in a low ratio of ddNTPs with the dNTPs
    • ddNTPs lack the 3'-OH group required for phosphodiester bond formation; therefore, when DNA polymerase incorporates a ddNTP at random, extension ceases
    • In manual Sanger sequencing, four PCR reactions are set up, each with a different ddNTP (A, T, C, G)
    • In automatic sequencing, all four ddNTPs are mixed in a single reaction, and each has a unique fluorescent label
  • Gel electrophoresis
    • The chain terminated sequences are separated according to size
    • The experimental protocol is the same as standard gel electrophoresis
    • In manual sequencing, this results in four parallel lanes, each with one type of ddNTP
    • In automatic sequencing, all oligonucleotides are run in a single capillary gel
  • Gel analysis and determination of DNA sequence
    • Since the electrophoresis results in bands arranged from largest to smallest reading from top to bottom, and since DNA polymerase only adds in the 5' to 3' direction, each ddNTP corresponds to a specific nucleotide in the sequence, and we can therefore read the sequence from smallest to largest, giving us the exact sequence from the 5' to 3' direction
    • In manual sequencing, the user reads all four lanes at once
    • In automated sequencing, the computer reads the lane from top to bottom. A laser excites each of the bands in turn, and since each band has a specific ddNTP, it emits a specific wavelength of light. This can then be directly tied to the identity of each ddNTP and therefore provides you with a chromatogram outlining the specific DNA sequence
 A chromatogram, which shows the fluorescent peak of each nucleotide along the length of the template DNA

Procedure from SigmaAldrich

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 11
Dimitra 12:00pm: Wet Lab Session 13
Hamza 12:10pm: Wet Lab Session 9 9:30am: Wet Lab Session 12
Lana 3:00pm: Lab Session 10

Week 6

Assigned Monday Tuesday Wednesday Thursday Friday
Everyone 11:30am: Group Meeting Work on report
Lorenzo 3:00pm: Lab Session 15
Dimitra 8:00am: Lab Session 14
Hamza 8:00am: Lab Session 14
Lana 3:00pm: Lab Session 15
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