Polymerase Chain Reaction - for Construction

New England Biolabs Phusion High-Fidelity DNA Polymerase

Composition:

Component	50 µl Reaction
Phusion HF Buffer (5x)	10 µl
dNTPs (10 mM)	1 µl
Forward Primer (10 µM)	2.5 µl
Reverse Primer (10 µM)	2.5 µl
Template DNA	variable
DMSO (optional)	up to 3%
Nuclease-free water	up to 50 µl
Phusion Polymerase	0.5 µl

Thermocycling Conditions:

Temperatur	Time
98°C	30 seconds
98°C 45-72°C 72°C	5-10 seconds 10-30 seconds 15-30 seconds per kb
72°C	5-10 minutes

The appropriate annealing temperature was calculated from NEB's Tm Calculator

Kapa Biosystems Hifi Hotstart Ready Mix

Component	50 µl Reaction
Master Mix (2x)	25 µl
Forward Primer (10 µM)	2.5 µl
Reverse Primer (10 µM)	2.5 µl
Template DNA	variable
Nuclease-free water	up to 50 µl

Thermocycling Conditions:

Temperatur	Time
95°C	3 minutes
98°C 55-75°C 72°C	20 seconds 15 seconds 15-60 seconds per kb
72°C	1 minute per kb

Kapa Hifi Hotstart has similar annealing temperatures as Phusion DNA polymerase, even slightly higher. Only in very few cases a lower annealing temperature was found to be better.

Polymerase Chain Reaction - for Analysis

Solis BioDyne FirePol DNA Polymerase

Composition:

Component	8 x 20 µl Reaction (+ 10 µl excess)
FIREPol DNA Polymerase	0.85 µl
MgCl2 (25 mM)	10.2 µl
Reaction Buffer B (10x)	17 µl
dNTPs (10 mM)	3.4 µl
Forward Primer (10 µM)	3.4 µl
Reverse Primer (10 µM)	3.4 µl
Template DNA	1 µl per 20 µl reaction
Nuclease-free water	123.25 µl

Thermocycling Conditions:

Temperatur	Time
98°C	3-5 minutes
95°C 50-72°C 72°C	30-60 seconds 30-60 seconds 1 minute per kb
72°C	5-10 minutes

New England Biolabs Taq DNA Polymerase

Composition:

Component	8 x 20 µl Reaction (+ 10 µl excess)
Taq Reaction Buffer (10x)	17 µl
dNTPs (10 mM)	3.4 µl
Forward Primer (10 µM)	3.4 µl
Reverse Primer (10 µM)	3.4 µl
Template DNA	1 µl per 20 µl reaction
Taq DNA Polymerase	0.85 µl
Nuclease-free water	133.45 µl

Create a mastermix, aliquote into PCR tubes and add 1 µl the template (resuspended colony from plate in 20 µl LB medium).

Thermocycling Conditions:

Temperatur	Time
95°C	5 minutes
95°C 45-68°C 68°C	30 seconds 20 seconds 1 minute per kb
72°C	5 minutes

The appropriate annealing temperature was calculated from NEB's Tm Calculator

Site-Directed Mutagenesis (QuickChange)

Component	50 µl Reaction
Kapa Hifi Hotstart Master Mix (2x)	25 µl
Forward Primer (10 µM)	2.5 µl
Reverse Primer (10 µM)	2.5 µl
Template DNA	variable
Nuclease-free water	up to 50 µl

Thermocycling Conditions:

Temperatur	Time
95°C	3 minutes
98°C 65°C 72°C	20 seconds 15 seconds 15-60 seconds per kb
72°C	1 minute per kb

Primers were designed according to the guidlines of The Richard Lab ¹

• The targeted mutation should be included into both primers.
• The mutation can be as close as 4 bases from the 5-terminus.
• The mutation should be at least 8 bases from the 3-terminus.
• At least eight non-overlapping bases should be introduced at the 3-end of each primer.
• At least one G or C should be at the end of each primer.
• Design your primers (including the mutations) to have a Tm >=78°C.

The resulting PCR product has to be purified (e.x. Agencourt AMPure XP) and digested with DpnI (NEB) for 4 hours and gel-purified. In case Phusion polymerase is used, DpnI can directly be added to the PCR. The product can then immediately be used for transformation. With proper removal of template plasmid DNA the efficiency of exchanged bases is very high.

Isothermal "Gibson" Assembly

Recipe for Ready-to-Use Isothermal Assembly Mixes

5x Isothermal Reaction Buffer (6ml)

• 3 ml of 1 M Tris-HCl pH 7.5
• 150 µl of 2 M MgCl2
• 60 µl of 100 mM dGTP
• 60 µl of 100 mM dATP
• 60 µl of 100 mM dTTP
• 60 µl of 100 mM dCTP
• 300 µl of 1 M DTT
• 1.5 g PEG-8000
• 300 µl of 100 mM NAD

This buffer can be aliquoted and stored at -20 °C.

Isothermal Assembly Master Mix

• 320 µl 5x isothermal reaction buffer
• 0.64 µl of 10 U/µl T5 exonuclease
• 20 µl of 2 U/µl Phusion DNA polymerase
• 160 µl of 40 U/µl Taq DNA ligase
• Fill up with water to a final volume of 1.2 ml

The master mix is devided into aliquots of 15 µl and stored at -20 °C.

Protocol for Isothermal Assembly

• 15 µl aliquot of master mix
• 0.02-0.5 pmol DNA in total for 2-3 fragments
  or
• 0.2-1 pmol DNA in total for 4-6 fragments
• Fill up with water to 20 µl

Consider the following:

• 2-3 times more insert than backbone (molar ratio)
• 5 times more insert for fragments < 200 bp (molar ratio)

The assembled mix is then incubated for 60 minutes at 50 °C. It can be directly transformed with chemically competent cell (volume of assembly reaction not exceeding 10% of the volume of the competent cells). Otherwise, the reaction mix is purified and desalted (e.x. Agencourt AMPure XP) and a fraction of it transformed with electrocompetent cells.

Preparation of Competent Cells

Preparation of Chemically Competent Cells

• Inoculate 100 ml of prewarmed LB medium with 1 ml overnight culture and grow the bacteria to an OD₆₀₀ of 0.5.
• Cool the culture on ice, transfer the cells into cetrifugation tubes and harvest them by centrifugation for 5 min (4000xg, 4°C)
• Carfully discard supernatant, keep cells always on ice.
• Resuspend cells in 30 ml cold TFB1 and incubate on ice for 90 minutes.

• Thanks to the sponsors that supported our project:

  • 
  • 
  • 
  • 
  • 
  • 
  • 
  • 
  • 
  • 
  • 
  • 
  • 
  • 
  • 
  • 

  
  

  
  

  
  

  
  

  
  

  Contents

  • 1 Nitric Oxide
  • 2 N-Acyl Homoserine Lactones
  • 3 Associative Learning Circuit
    • 3.1 Overview
    • 3.2 Biological Implementation: Recombinase
      • 3.2.1 Sensor AND-gate:
      • 3.2.2 Learning:
      • 3.2.3 Reporter AND-gate:
    • 3.3 Biological Implementation: CRISPR/Cpf1
      • 3.3.1 Sensor AND-gate:
      • 3.3.2 Learning:
      • 3.3.3 Reporter AND-gate:
  • 4 Directed Evolution of EsaR
    • 4.1 Dual Selection Procedure
      • 4.1.1 Negative Selection:
      • 4.1.2 Positive Selection:
      • 4.1.3 Potential Delivery Method of Reporter Strains
  • 5 References:

  Nitric Oxide

     <a href="https://2016.igem.org/File:T--ETH_Zurich--NorR_draft_vector.svg">
         <img src="https://static.igem.org/mediawiki/2016/c/c0/T--ETH_Zurich--NorR_draft_vector.svg">
     </a>
  

  Figure 1: NorR constitutively binds to DNA. Only when NO^• is present it activates the transcription of the gene under control of the norVW promoter (adapted from <a href="#green2014transcriptional" >Green et al.</a>)

  Beside the penetration of immunogenic antigens across the epithelial layer, there is also non-normal leakage of inflammation markers into the gut lumen. One of these molecules is nitric oxide (NO^•, t_1/2 < 6 seconds^{<a href="#kochar2011nitric" class="cit">7</a>}) and is one of the molecules we are going to sense with our system. The sensing of NO^• with E. coli has already been described by Archer et al.^{<a href="#archer2012engineered" class="cit">8</a>} in 2012. This work provides us with the relevant genetic elements and helps us to design this system for our purpose. Additionally, they present their system as a rapid detection system for IBD related disease flare-ups which would allow for an immediate intervention.
  NorR is capable of binding NO^• with its mononuclear non-heme iron center. While other sensor proteins are not only specific for NO^• but also for other NO_x species, NorR binds specifically the NO^• radical. NorR is constitutively bound as a hexamer upstream of the norVW promoter but inhibiting transcription in absence of NO^•. Once NO^• binds to NorR, its ATPase activity is triggered and provides energy to form a productive interaction with the σ⁵⁴ - RNA polymerase holoenzyme^{<a href="#green2014transcriptional" class="cit">9</a>}.

  </div> </div>

  N-Acyl Homoserine Lactones

     <a href="">
         <img src="">
     </a>
  

  Figure 2: DNA- and 3-oxo-C6-HSL bound, dimeric form of TraR, a close homolog of EsaR (PDB: <a href="http://www.rcsb.org/pdb/explore.do?structureId=1l3l" >1L3L</a>, edited with UCSF Chimera^{<a href="#chimera" class="cit">1</a>})

  In addition to a general inflammation marker we want to sense molecules secreted by the microbiota in order to identify the bacteria. One well-known class of molecules secreted by many bacterial species belongs to the quorum sensing (QS) system. QS molecules act as bacterial hormones among and between species which control for example the formation of biofilms and growth behaviour. Furthermore, QS molecules can alter the microbiota's composition^{<a href="#thompson2015manipulation" class="cit">1</a>}. The best known subclass of QS molecules are the N-acyl homoserine lactones (AHL) which will be identified by our living biosensor.
  One of the AHLs to be found upregulated in IBD^{<a href="#landman2013sa1804" class="cit">1</a>} is 3-hydroxy-hexanoyl-HSL (3-OH-C6-HSL). A well characterized regulatory protein that senses a very similar HSL (3-oxo-C6-HSL) is EsaR from Erwinia stewartii that was used by a <a href="https://2015.igem.org/Team:Manchester-Graz/Project/Vectordesign" >previous iGEM team</a>. The special feature of EsaR is its regulatory behaviour: while most HSL-responsive elements are inducible activators, EsaR is a repressor that dissociates from the DNA in presence of HSL. This is important for our circuit as a repressor is thought to be less leaky than an activator.
  As our target HSL is not the natural ligand for EsaR, we applied a directed evolution strategy to change its specificity.

  Associative Learning Circuit

  Overview

  To serve as a diagnostics and research tool, our system should not only be able to sense a single molecule alone but should associate an inflammation marker - in our case NO^• - with a potential trigger of the inflammation itself. Thus, we implemented an associative learning circuit that allows for the detection of the temporal and spatial presence of two markers.
  Nitric oxide and 3-OH-C6-HSL are only two possbile markers of IBD. There exist many more that are definitively worth to be further investigated and are ideally observed in parallel. This is why we extended the AND-gate by a learning component. While the number of distinguistable reporters (e.g. fluorophores) is limited, our system allows for simultanious observation of a multitude of parallel measured markers. Our Pavlov's Coli learn the occurence of the presence of two markers and store this information in their DNA until readout.
  We designed our system in a way that allows fast and easy demultiplexing of a complex mixture of different reporter strains. If the reporter strains encounter again the with inflammation associated marker, they generate an easily observable output: fluorescence. This was achieved by integrating a second AND-gate that relies on the successful learning process.
  

  Biological Implementation: Recombinase

  Sensor AND-gate:

  
  

  At an inflammation spot, nitric oxide activates NorR and triggers the transcription of the bxb1 integrase gene. The transcription can only proceed if 3-OH-C6-HSL is present. The HSL lets the repressor EsaR dissociate from the regulatory element (esaBox) on the DNA and thus annihiliates its roadblock activity.
  

  Learning:

  
  

  Once the bxb1 gene is successfully transcribed and translated, Bxb1 binds to the attP and attB recombination sites flanking a constitutive promoter and inverts it. As attP and attB are destroyed through inversion, Bxb1 mediated recombination acts as a one-way switch.
  

  Reporter AND-gate:

  
  

  The constitutive promoter, now being placed upstream of the reporter protein GFP, is further under control of another esaBox, the binding site of EsaR.
  After the system now has learnt to respond to the associated stimulus alone, the expression of GFP can easily be induced by just exposing it to the stimulus again, e.g. EsaR's ligand.

  Biological Implementation: CRISPR/Cpf1

  An alternative to a recombinase-based switch is the usage of the 2015 characterised <a href="#cpf1" class="cit">CRISPR/Cpf1</a> system. Instead of cutting both DNA strands at the same position, Cpf1 cuts the DNA with an offset of four or five nucleotides, thus producing single-stranded overhangs. It is suggested that this is advantageous for genome editing via non-homologous end-joining.
  We will use the features of Cpf1 to create an AND-gate controlled one-way switch with finally the same functionality as the recombinase-based switch.
  For this, a reporter construct is stably integrated into the genome of E. coli whereas Cpf1 and its guide RNAs will be expressed from a plasmid.
  

  Sensor AND-gate:

  
  

  At an inflammation spot, nitric oxide activates NorR and triggers the transcription of Cfp1. The transcription can only proceed if 3-OH-C6-HSL is present. The HSL lets the repressor EsaR dissociate from the regulatory element (esaBox) on the DNA and thus annihiliates its roadblock activity. The guide RNAs are expressed constitutively at a high level.
  

  
  

  Learning:

  
  

  Once Cfp1 is expressed, it is brought to the cutting-sites by the two distinct guide RNAs. There, Cfp1 cuts out the mNectarine gene while creating sticky ends. These will then be ligated by endogeneous ligases by NHEJ which reconstitutes the GFP gene.
  

  
  

  Reporter AND-gate:

  
  

  The reconstituted GFP gene is now under the control of a constitutive promoter regulated be an esaBox, the binding site of EsaR.
  After the system now has learnt to respond to the associated stimulus alone, the expression of GFP can easily be induced by just exposing it to the stimulus again, e.g. EsaR's ligand.

  Directed Evolution of EsaR

  In order to change EsaR's specificity towards an IBD related HSL, we need to apply directed evolution on this repressor protein. The variant of EsaR that we used was already an improved version with a D91G mutation that has an increased signal sensitive compared to the wildtyp^{<a href="#esar" class="cit">X</a>}. We combine several approaches to find new variants of EsaR that are responsive to our target. To select for these variants we have different constructs that form a dual selection system. This systems allows for negative selection ("killing") of variants that still react to the former HSL (3-oxo-C6-HSL) and positive selection ("survival") of variants that respond to the new target HSL (3-OH-C6-HSL). It consists of a fusion protein that is composed of an antibiotic resistance and an enzyme that converts a non-toxic compound into a cellular toxin. We test the combination of the chloramphenicol acetyltransferase (CAT) and the uracil phosphoribosyltransferase (UPRT)^{<a href="#rackham2005network" class="cit">1</a>} as well as the herpes simplex virus thymidine kinase (hsvTK) fused to the aminoglycoside phosphotransferase (APH)^{<a href="#tominaga2015rapid" class="cit">1</a>}. Whereas CAT and APH confer resistance for the positive selection step, UPRT and hsvTK are necessary for the negative selection.
  UPRT normally converts uracil into uridine monophosphate (dUMP). 5-fluorouracil is metabolised by UPRT to 5-fluoro-dUMP which irreversibly blocks the thymidylate synthase (thyA), a key enzyme for the production of pyrimidine nucleosides in the cell, what finally leads to cell death^{<a href="#hartmann1961studies" class="cit">1</a>}.
  The herpes simplex virus thymidine kinase has a less stringent substrate specificity than normal thymidine kinase and thus also metabolises ganciclovir, a guanin analogue. The metabolised ganciclovir is then treated by the cells as guanin but finally inhibits DNA replication by chain termination.
  To generate a library of high diversity, we use site directed mutagenesis as well as random mutagenesis with Taq polymerase and manganese.
  

  
  

  Dual Selection Procedure

  Negative Selection:

  
  

     <a href="https://static.igem.org/mediawiki/2016/8/89/T--ETH_Zurich--NegativeSelection.svg">
         <img src="https://static.igem.org/mediawiki/2016/8/89/T--ETH_Zurich--NegativeSelection.svg">
     </a>
  

  In a first step, the created library of variants is grown in presence of the old inducer 3-oxo-C6-HSL and a toxic precursor. Variants whose expression is still induced by the old HSL or have a non-functional repressor (EsaR) express the fusion protein which converts the toxic precursor into a toxin (A), non-responsive repressors stay bound to the DNA and inhibit protein expression (B).
  After a certain time, the surviving variants are transfered into culture medium without the toxic precurser and without HSL in order to eliminate the fusion protein.

  Positive Selection:

  
  

     <a href="https://static.igem.org/mediawiki/2016/8/87/T--ETH_Zurich--PositiveSelection.svg">
         <img src="https://static.igem.org/mediawiki/2016/8/87/T--ETH_Zurich--PositiveSelection.svg">
     </a>
  

  In a second step, these variants now undergo a round of positive selection to select for variants that are responsive to the new HSL 3-OH-C6-HSL.
  The bacteria are cultured in medium containing the new HSL and the antibiotic whose resistance is part of the fusionprotein. Inducible variants express the resistance protein and survive (A). Variants that are non-responsive do not express it and can not grow (B).
  Afterwards, the variants can be plated and analyzed or undergo further rounds of positive / negative selection to enrich for suitable variants.

  Potential Delivery Method of Reporter Strains

  The idea to administer genetically modified bacteria in the context of IBD was published by Steidler et al. in 2000^{<a href="#steidler2000treatment" class="cit">1</a>}. Generally, to use engineered probiotic bacteria as a delivery vector for in vivo produced therapeutic agents has been described multiple times^{<a href="#bermudez2009lactococcus" class="cit">1</a>,<a href="#bermudez2013engineering" class="cit">1</a>,<a href="#wells2008mucosal" class="cit">1</a>,<a href="#deming2013genetically" class="cit">1</a>}.
  As the stomach and the gastrointestinal tract are rough environments for non-adapted (probiotic) bacteria, we suggest to encapsulate the bacteria in a hydrogel. This protects the bacteria and ensures the recovery of the reporter strain. The method of encapsulation is well known for oral administration in animal models (e.g. Prakash et al.^{<a href="#prakash1996microencapsulated" class="cit">1</a>}) and is summarised in several reviews (e.g. Prakash et al. (2008)</i>^{<a href="#prakash2008colon" class="cit">1</a>} and Tomaro et al. (2012) ^{<a href="#tomaro2012microencapsulation" class="cit">1</a>}).

  
  

  
  

  References:

  • <a name="richard" class></a>[1] Tom Richard, Department of Agricultural and Biological Engineering, Penn State University.
  • <a name="scaldaferri2015gut" class></a>[1] Scaldaferri, F., et al. "Gut microbiota molecular spectrum in healthy controls, diverticular disease, IBS and IBD patients: Time for microbial marker of gastrointestinal disorders?." Journal of Crohns & Colitis. Vol. 9. Oxford Univ Press, 2015.

  
  

  
  

  
  

  
  

  
  </body> </html>

  Thanks to the sponsors that supported our project:

  • 
  • 
  • 
  • 
  • 
  • 
  • 
  • 
  • 
  • 
  • 
  • 
  • 
  • 
  • 
  •