Difference between revisions of "Team:Bolivia/Design"

 
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                                 <li><a class="dropdown-item" href="#">INTEGRATED</a></p></li>
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                             </div>
 
                             </div>
 
                             <h1 class="text-center text-md-start text-white p-0 titulo-principal">DESIGN</h1>
 
                             <h1 class="text-center text-md-start text-white p-0 titulo-principal">DESIGN</h1>
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                             <p class="text-white text-white text-sm-center text-md-start" style="font-size: 20px;">The
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                             <p class="text-white text-white text-sm-center text-md-start" style="font-size: 20px;">A deep look
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                                work begins starting with
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                            at our constructs and how they work</p>
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                                the
+
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                                design of the bioparts</p>
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                     <div class="col-md-8  d-flex align-items-center mb-3">
 
                     <div class="col-md-8  d-flex align-items-center mb-3">
 
                         <p>
 
                         <p>
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                             The development of whole-cell biosensors for heavy metals is widely described in the
+
                             The development of whole-cell biosensors for heavy metals detection is widely described in the
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                             literature,
+
                             literature, and other iGEM teams explored this tools as a way to help with the enviromental   
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                            within the iGEM competition many teams have explored various well-established design
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                             pollution. <strong class="text-azul"> We, as the Bolivian team, consider that, a less explored,
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                             approaches.
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                             but more advantageous approach, is the use of whole-cell biosensors that work as semaphores but
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                            In an attempt to differentiate ourselves our team has decided to take a less explored
+
                            also can  report quantitative data. </strong> We present for competition a biosensor for arsenic
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                             approach
+
                            detection, based in part on the Wang et al [1] proposal, that relies mainly on 3 strategies:
−
                            for the design of <strong>our arsenic biosensor, based initalt on the work of Wang et
+
                     
−
                                al.1</strong> It relies mainly on 3 strategies:
+
 
                         </p>
 
                         </p>
 
                     </div>
 
                     </div>
 
                     <h2 class="text-azul mt-5 mb-4">
 
                     <h2 class="text-azul mt-5 mb-4">
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                         I. Control of intracellular density of arsR protein
+
                         I. Intracellular arsR density control
 
                     </h2>
 
                     </h2>
 
                     <p>
 
                     <p>
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                         Normally arsR regulates its own expression within the arsRDABC operon2 and in whole-cell
+
                         Normally arsR regulates its own expression within the arsRDABC operon [2] However, we can control the
−
                         biosensors
+
                        intracellular arsR by using constitutive promoters. Recent studies have shown the changes in arsR
−
                         with a more conventional design, <strong class="text-azul">recent studies have been conducted
+
                         expression related to the control of constitutive promoters [3].
−
                            where the expression
+
                         For instance, <strong class="text-azul">there will be lower levels of arsR protein inside the cell
−
                            of
+
                        when the expression is controlled by a weak promoter resulting  in changes in the minimum
−
                            arsR has been placed under the control of constitutive promoters of different strengths,
+
                         concentration for arsenic necessary to activate the genetic circuits. On the other hand, if an arsenic  
−
                            this
+
                        biosensor with sensitivity for higher concentrations is required, the promoter can be changed to one  
−
                            has resulted in a finer and more comprehensive control of outputs.(3) </strong>
+
                         of greater strength.</strong> In this way, we can manipulate the detection limits of our biosensor.
−
                    </p>
+
                 
−
                    <p>
+
−
                        A weak constitutive promoter controlling arsR expression results in a lower concentration of
+
−
                        arsR
+
−
                        protein in the bacterial cytosol, loosening the control of the expression that it exerts and the
+
−
                         minimum concentration of arsenic necessary to activate the genetic constructs, in other words,
+
−
                        the
+
−
                        detection limit of the biosensor is improved. On the other hand, if an arsenic biosensor with a
+
−
                        sensitivity for higher concentrations is required, the promoter can be changed to one of greater
+
−
                         strength.
+
 
                     </p>
 
                     </p>
 
                 </div>
 
                 </div>
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                 <div class="row ">
 
                 <div class="row ">
 
                     <div class="col-md-5 d-flex align-items-center text-md-end mb-3">
 
                     <div class="col-md-5 d-flex align-items-center text-md-end mb-3">
−
                         <p class="txt"><strong>Figure 1.</strong>Adjusting the intracellular receptor concentration
+
                         <p class="txt"><strong>Figure 1. </strong>Adjusting the intracellular receptor concentration
 
                             allows manipulating the detection limit of the biosensor. a) Strong constitutive promoter:
 
                             allows manipulating the detection limit of the biosensor. a) Strong constitutive promoter:
 
                             higher concentration of ArsR results in a higher concentration of Arsenic for the activation
 
                             higher concentration of ArsR results in a higher concentration of Arsenic for the activation
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             <div class="container">
 
             <div class="container">
 
                 <h2 class="text-azul mb-4">II. Using simple and cascading transcriptional amplifiers</h2>
 
                 <h2 class="text-azul mb-4">II. Using simple and cascading transcriptional amplifiers</h2>
−
                 <p>Upon activation of the constructs, <strong class="text-azul">the transcriptional signal generated
+
                 <p>Upon activation of the constructs, <strong class="text-azul">the transcriptional signal
−
                        will be received by
+
                  will be received by the HrpRS and/or RinA amplifier systems greatly enhancing the output signal.</strong>  
−
                        the
+
                  This result is predictable, and it provides a new level of control on the genetic construct outputs where  
−
                        HrpRS and/or RinA amplifier systems and will be scaled by a gain factor to produce an enhanced
+
                  low-level or saturated signals must be scaled to increase the sensitivity. Like its electronic  
−
                        output signal.</strong> Being able to predictably scale a transcriptional signal provides a new
+
                  counterparts, amplifier engineering is highly valuable in customizing signal processing in cells for  
−
                    level of control and flexibility over the outputs of genetic constructs where low-level or saturated
+
                  various applications.[4] </p>
−
                    transcription signals must be scaled to increase sensitivity. Like its electronic counterparts,
+
−
                    amplifier engineering is critically valuable in customizing signal processing in cells for various
+
−
                    applications.4 4</p>
+
 
                 <p>
 
                 <p>
−
                     <strong>HrpRS system :</strong> it is composed by the genetic components <strong>(hrpRS
+
                     <strong>HrpRS system :</strong> Is composed by the genetic components <strong>(hrpRS
−
                         and PhrpL)</strong> of the regulatory network of the hrp gene (hypersensitive
+
                         and PhrpL)</strong> in the regulatory network of the hrp gene (hypersensitive
−
                     reaction and pathogenicity) for type III secretion in Pseudomonas
+
                     reaction and pathogenicity) for type III secretion system in <i>Pseudomonas
−
                     syringae . Activating hrpR and hrpS proteins results in the formation
+
                     syringae</i>. The activation of hrpR and hrpS proteins results in the formation
−
                     of an ultrasensitive complex which binds a sequence up-stream the
+
                     of an ultrasensitive complex that binds the up-stream sequence of the
−
                     hrpL promoter (σ54 factor dependent) for remodeling the closed
+
                     hrpL promoter (σ54 factor dependent). As a consequence, the the closed transcription σ54-RNAP-hrpL complex  
−
                    transcription σ54-RNAP-hrpL complex into a open one through ATP
+
                    is changed into an open one through ATP hydrolysis for promoting transcription. [5]
−
                    hydrolysis for finally promoting transcription. (5)
+
 
                 </p>
 
                 </p>
 
             </div>
 
             </div>
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         <div class="container mt-5 ">
 
         <div class="container mt-5 ">
 
             <p class="">
 
             <p class="">
−
                 <strong>RinA system : </strong> consists of a family of phage-encoded proteins that
+
                 <strong>RinA system : </strong> Is a family of phage-encoded proteins that
−
                 act as activators for the transcription of late operons in a large
+
                 act as activators for the transcription of late operons in the
−
                 group of Staphylococcus aureus phages . It has the ability to bind
+
                 group of Staphylococcus aureus phages. It has the ability to bind
 
                 to the PrinA_p80 promoter sequence where it promotes
 
                 to the PrinA_p80 promoter sequence where it promotes
−
                 transcription of down-stream elements in an ultrasensitive way. (6)
+
                 transcription of down-stream elements in an ultrasensitive way. [6]
 
             </p>
 
             </p>
 
         </div>
 
         </div>
Line 355: Line 368:
 
                 <h2 class="text-azul mb-4">III. Chromoprotein as a reporter</h2>
 
                 <h2 class="text-azul mb-4">III. Chromoprotein as a reporter</h2>
 
                 <p>
 
                 <p>
−
                     After processing the transcriptional signal, it is necessary to convert it to a
+
                     The described transcriptional apparatus should be translated into a measurable signal.
−
                     measurable type of signal,<strong class="text-azul">in our case this is possible through the use of
+
                     <strong class="text-azul"> In our case, this is possible through a
−
                        a
+
                    reporter gene that produces a colored protein observable to the naked eye.</strong>
−
                        reporter gene that produces a colored protein observable to the naked eye.</strong>
+
 
                     Chromoproteins have certain advantages over other fluorescent proteins,
 
                     Chromoproteins have certain advantages over other fluorescent proteins,
−
                     such as having dark colors under ambient light that allow inexpensive
+
                     for its dark colors easily distinguishable under ambient light without the necessity of additional
−
                     analysis without specialized instruments. They also avoid problems due to
+
                     equipment. They also help us to avoid problems present in fluorescence based assays such as the
−
                     background fluorescence, UV-induced bleaching, cell damage, and the
+
                     background noise, UV-induced bleaching, cell damage, and the
−
                     need for eye and skin protection. Applications of chromoproteins include
+
                     need for eye and skin protection. Chromoproteins are commonly used as markers in  
−
                    markers in living organisms for cloning (7), teaching (8) and biosensors. (9)
+
                    living organisms for cloning [7], teaching [8] and biosensors. [9]
 
                 </p>
 
                 </p>
 
             </div>
 
             </div>
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             <div class="container">
 
             <div class="container">
 
                 <div class="row ">
 
                 <div class="row ">
−
                     <div class="col-md-5 d-flex align-items-center text-md-end mb-5">
+
                     <div class="col-xs-12 col-sm-12 col-md-5 d-flex align-items-center text-md-end mb-5">
−
                         <p class="txt"><strong>Figure 3.</strong> Adjusting the intracellular
+
                         <p class="txt"><strong>Figure 3.</strong> mRFP1_Violet is a monomeric chromoprotein with a GFP analog structure. It has low molecular weight (33 kDA), fast maturation in the presence of oxygen and presents a deep violet color to the naked eye, it is also fluorescent with peak excitation and emission at 586 and 619 nm correspondingly.</p>
−
                            receptor concentration allows manipulating
+
−
                            the detection limit of the biosensor. a)
+
−
                            Strong constitutive promoter: higher
+
−
                            concentration of ArsR results in a higher
+
−
                            concentration of Arsenic for the activation
+
−
                            of the constructs. b) Weak constitutive
+
−
                            promoter: lower ArsR concentration results
+
−
                            in a lower Arsenic concentration for the
+
−
                            activation of the constructs.</p>
+
 
                     </div>
 
                     </div>
−
                     <div class=" offset-md-1 col-md-6">
+
                     <div class="col-xs-12 col-sm-12 offset-md-1 col-md-6">
  
  
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         <div class="container mt-5">
 
         <div class="container mt-5">
 
             <p class="">
 
             <p class="">
−
                 <strong>mRFP1E Chromoprotein:</strong> its a variant of the monomeric red fluorescent
+
                 <strong>mRFP1E Chromoprotein:</strong> Is a variant of the monomeric red fluorescent
−
                 protein 1 (mRFP1) gene that was codon optimized for E. coli (abbreviated
+
                 protein 1 (mRFP1) optimized for <i>E. coli</i> expression (abbreviated
−
                 mRFP1E). <strong class="text-azul">It produces a dark red color observable to the naked eye and
+
                 mRFP1E). <strong class="text-azul">It produces a dark red color observable to the naked eye, it
 
                     has a fluorescent capacity with an excitation spectrum at 582 nm and an
 
                     has a fluorescent capacity with an excitation spectrum at 582 nm and an
−
                     emission spectrum at 606 nm.</strong> Is low molecular weight (55 kDa ) and it has
+
                     emission spectrum at 606 nm.</strong> It also has low molecular weight (55 kDa), and reports indicated
−
                 been reported that its toxicity on E. coli is considerably lower than other
+
                 that mRFP1E is less toxic to <i>E. coli</i> in comparison with other chromoproteins, which is an
−
                chromoproteins, which is a great advantage for its use as a reporter, its
+
                advantage for its use as a reporter. The maturation time is 18 - 24 hours in the presence of oxygen. Orange,  
−
                maturation time is 18 - 24 hours in the presence of oxygen. Orange, pink,
+
                 pink, magenta and violet color variants have also been generated with the same characteristics. [10]
−
                 magenta and violet color variants have also been generated with the same
+
            </p>
−
                characteristics (10)
+
            <p>Red fluorescent proteins such as mRFP1 could interfere the measurement of optical density in cultures at 600 nm
 +
              (OD600) due to their absorption spectrum, causing interference and overestimations. [11]
 +
              <strong class="text-azul">For this reason we choose for the violet variant of the chromoprotein, mRFP1-Violet,
 +
                which has an absorption spectrum ranging from 350 - 450 nm that eliminates interference with OD600.</strong>
 +
                mRFP1-Violet was generated via mutations of the original chromoprotein fluorophore site and             
 +
                <strong class="text-azul"> it retains its fluorescence, maturation time and low toxicity.[10] </strong>
 
             </p>
 
             </p>
−
            <p>Red fluorescent proteins such as mRFP1 have an absorption spectrum
 
−
                that can overlap with optical density measurements of cultures at 600 nm
 
−
                (OD600), causing interference and overestimations.111 <strong class="text-azul">For this reason we
 
−
                    have opted for the violet variant of this chromoprotein, mRFP1-Violet , its
 
−
                    absorption spectrum is in the region of 350 - 450 nm, eliminating the
 
−
                    interferences with OD600.</strong> It has been generated by mutations of the
 
−
                original chromoprotein fluorophore site and <strong>retains its characteristics such
 
−
                    as fluorescence, maturation time and low toxicity. (10)</strong></p>
 
 
         </div>
 
         </div>
  
 
         <section>
 
         <section>
 
             <div class="container">
 
             <div class="container">
−
                 <h2 class="text-center text-azul mb-4">ARSENITO IN DETAIL</h2>
+
                 <h2 class="text-center text-azul mb-4">ARSEMAPHORE IN DETAIL</h2>
−
                 <p>We apply the aforementioned strategies to generate 4 genetic constructs
+
                 <p>We apply the mentioned strategies to generate 4 genetic constructs
−
                    that offer different ranges of sensitivity to arsenic. <strong>Transformed into 4
+
                  that offer different ranges of sensitivity to arsenic. <strong class="text-azul">By transforming
−
                        different E. coli BL21 strains, we can generate traffic light patterns visible
+
                <i>E. coli</i> BL21 strains with each construct, we can generate traffic light easy-to-interpret patterns  
−
                        to the naked eye and easy to interpret.</strong></p>
+
                  visible to the naked eye. </strong></p>
 
             </div>
 
             </div>
 
         </section>
 
         </section>
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                 <div class="row ">
 
                 <div class="row ">
 
                     <div class="col-md-5 d-flex align-items-center text-md-end mb-3">
 
                     <div class="col-md-5 d-flex align-items-center text-md-end mb-3">
−
                         <p class="txt"><strong>Figure 4.</strong>The different arsenic
+
                         <p class="txt"><strong>Figure 4. </strong>The different arsenic
 
                             sensitivities of the constructs
 
                             sensitivities of the constructs
 
                             allow semi-quantitative
 
                             allow semi-quantitative
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         <section>
 
         <section>
 
             <div class="container">
 
             <div class="container">
−
                 <h2 class="text-azul mb-4">1. As0 Construct (≥ 0.5 ppb [As] sensitivity):</h2>
+
                 <h2 class="text-azul mb-4">1. As0 Construct (detection limit ≥ 0.5 ppb [As]):</h2>
 
                 <p>The detection module consists of a weak constitutive promoter
 
                 <p>The detection module consists of a weak constitutive promoter
 
                     <strong>(BBa_J23109)</strong> that is in charge of the expression of the arsR protein
 
                     <strong>(BBa_J23109)</strong> that is in charge of the expression of the arsR protein
Line 505: Line 505:
 
         <section>
 
         <section>
 
             <div class="container">
 
             <div class="container">
−
                 <h2 class="text-azul mb-4">2. As2 Construct(≥ 3 ppb [As] Sensitivity):</h2>
+
                 <h2 class="text-azul mb-4">2. As2 Construct (detection limit ≥ 3 ppb [As]):</h2>
 
                 <p>The detection module consists of a weak constitutive promoter
 
                 <p>The detection module consists of a weak constitutive promoter
 
                     <strong>(BBa_J23109)</strong> that is in charge of the expression of the arsR protein
 
                     <strong>(BBa_J23109)</strong> that is in charge of the expression of the arsR protein
−
                     <strong>(BBa_J15101)</strong>, next is the processing module that is made up by
+
                     <strong>(BBa_J15101)</strong>, Followed by the processing module that is composed by
 
                     <strong>Pars</strong>
 
                     <strong>Pars</strong>
 
                     inducible promoter <strong>(BBa_K190015)</strong> , which receives the transcriptional
 
                     inducible promoter <strong>(BBa_K190015)</strong> , which receives the transcriptional
Line 551: Line 551:
 
         <section>
 
         <section>
 
             <div class="container">
 
             <div class="container">
−
                 <h2 class="text-azul mb-4">3. As4 Construct (≥ 10 ppb [As] Sensitivity)</h2>
+
                 <h2 class="text-azul mb-4">3. As4 Construct (detection limit ≥ 10 ppb [As]):</h2>
 
                 <p>The detection module consists of a constitutive promoter of medium
 
                 <p>The detection module consists of a constitutive promoter of medium
 
                     strength <strong>(BBa_J23115)</strong> that is in charge of the expression of the arsR
 
                     strength <strong>(BBa_J23115)</strong> that is in charge of the expression of the arsR
−
                     protein <strong>(BBa_J15101)</strong> , in this construct a processing module is not
+
                     protein <strong>(BBa_J15101).</strong> In this construct, processing modules are not
−
                     included and immediately afterwards is the output module made up by
+
                     included, and immediately afterwards is the output module made up by
 
                     <strong>Pars</strong> inducible promoter <strong>(BBa_K190015)</strong> that receives the signal
 
                     <strong>Pars</strong> inducible promoter <strong>(BBa_K190015)</strong> that receives the signal
 
                     from
 
                     from
Line 598: Line 598:
 
         <section>
 
         <section>
 
             <div class="container">
 
             <div class="container">
−
                 <h2 class="text-azul mb-4">4. As5 Construct (≥ 50 ppb [As] Sensitivity)</h2>
+
                 <h2 class="text-azul mb-4">4. As5 Construct (detection limit ≥ 50 ppb [As]):</h2>
 
                 <p>The detection module consists of a high-strength constitutive promoter
 
                 <p>The detection module consists of a high-strength constitutive promoter
 
                     <strong>(BBa_J23105)</strong> that is in charge of the expression of the arsR protein
 
                     <strong>(BBa_J23105)</strong> that is in charge of the expression of the arsR protein
−
                     <strong>(BBa_J15101)</strong> , a processing module is not included in this construct and
+
                     <strong>(BBa_J15101).</strong> Again, a processing module is not included in this construct and
 
                     immediately afterwards is the output module made up by the inducible
 
                     immediately afterwards is the output module made up by the inducible
 
                     promoter <strong>Pars (BBa_K190015)</strong> that receives the signal from the detection
 
                     promoter <strong>Pars (BBa_K190015)</strong> that receives the signal from the detection
Line 1,521: Line 1,521:
 
         </div>
 
         </div>
 
</div>
 
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Latest revision as of 17:48, 14 December 2021

Design

TEAM BOLIVIA

DESIGN

A deep look at our constructs and how they work

The development of whole-cell biosensors for heavy metals detection is widely described in the literature, and other iGEM teams explored this tools as a way to help with the enviromental pollution. We, as the Bolivian team, consider that, a less explored, but more advantageous approach, is the use of whole-cell biosensors that work as semaphores but also can report quantitative data. We present for competition a biosensor for arsenic detection, based in part on the Wang et al [1] proposal, that relies mainly on 3 strategies:

I. Intracellular arsR density control

Normally arsR regulates its own expression within the arsRDABC operon [2] However, we can control the intracellular arsR by using constitutive promoters. Recent studies have shown the changes in arsR expression related to the control of constitutive promoters [3]. For instance, there will be lower levels of arsR protein inside the cell when the expression is controlled by a weak promoter resulting in changes in the minimum concentration for arsenic necessary to activate the genetic circuits. On the other hand, if an arsenic biosensor with sensitivity for higher concentrations is required, the promoter can be changed to one of greater strength. In this way, we can manipulate the detection limits of our biosensor.

II. Using simple and cascading transcriptional amplifiers

Upon activation of the constructs, the transcriptional signal will be received by the HrpRS and/or RinA amplifier systems greatly enhancing the output signal. This result is predictable, and it provides a new level of control on the genetic construct outputs where low-level or saturated signals must be scaled to increase the sensitivity. Like its electronic counterparts, amplifier engineering is highly valuable in customizing signal processing in cells for various applications.[4]

HrpRS system : Is composed by the genetic components (hrpRS and PhrpL) in the regulatory network of the hrp gene (hypersensitive reaction and pathogenicity) for type III secretion system in Pseudomonas syringae. The activation of hrpR and hrpS proteins results in the formation of an ultrasensitive complex that binds the up-stream sequence of the hrpL promoter (σ54 factor dependent). As a consequence, the the closed transcription σ54-RNAP-hrpL complex is changed into an open one through ATP hydrolysis for promoting transcription. [5]

RinA system : Is a family of phage-encoded proteins that act as activators for the transcription of late operons in the group of Staphylococcus aureus phages. It has the ability to bind to the PrinA_p80 promoter sequence where it promotes transcription of down-stream elements in an ultrasensitive way. [6]

III. Chromoprotein as a reporter

The described transcriptional apparatus should be translated into a measurable signal. In our case, this is possible through a reporter gene that produces a colored protein observable to the naked eye. Chromoproteins have certain advantages over other fluorescent proteins, for its dark colors easily distinguishable under ambient light without the necessity of additional equipment. They also help us to avoid problems present in fluorescence based assays such as the background noise, UV-induced bleaching, cell damage, and the need for eye and skin protection. Chromoproteins are commonly used as markers in living organisms for cloning [7], teaching [8] and biosensors. [9]

mRFP1E Chromoprotein: Is a variant of the monomeric red fluorescent protein 1 (mRFP1) optimized for E. coli expression (abbreviated mRFP1E). It produces a dark red color observable to the naked eye, it has a fluorescent capacity with an excitation spectrum at 582 nm and an emission spectrum at 606 nm. It also has low molecular weight (55 kDa), and reports indicated that mRFP1E is less toxic to E. coli in comparison with other chromoproteins, which is an advantage for its use as a reporter. The maturation time is 18 - 24 hours in the presence of oxygen. Orange, pink, magenta and violet color variants have also been generated with the same characteristics. [10]

Red fluorescent proteins such as mRFP1 could interfere the measurement of optical density in cultures at 600 nm (OD600) due to their absorption spectrum, causing interference and overestimations. [11] For this reason we choose for the violet variant of the chromoprotein, mRFP1-Violet, which has an absorption spectrum ranging from 350 - 450 nm that eliminates interference with OD600. mRFP1-Violet was generated via mutations of the original chromoprotein fluorophore site and it retains its fluorescence, maturation time and low toxicity.[10]

ARSEMAPHORE IN DETAIL

We apply the mentioned strategies to generate 4 genetic constructs that offer different ranges of sensitivity to arsenic. By transforming E. coli BL21 strains with each construct, we can generate traffic light easy-to-interpret patterns visible to the naked eye.

1. As0 Construct (detection limit ≥ 0.5 ppb [As]):

The detection module consists of a weak constitutive promoter (BBa_J23109) that is in charge of the expression of the arsR protein (BBa_J15101) , next is the processing module that is made up by Pars inducible promoter (BBa_K190015), which receives the transcriptional signal, and a double amplifying cascade (HrpRS-pHrpL-RinA), finally is the output module made up by an inducible promoter pRinA-p80 that receives the amplified signal of the cascade and promotes the expression of the reporter gene mRFP1 -Violet.

Click me, Please
Click me, Please

2. As2 Construct (detection limit ≥ 3 ppb [As]):

The detection module consists of a weak constitutive promoter (BBa_J23109) that is in charge of the expression of the arsR protein (BBa_J15101), Followed by the processing module that is composed by Pars inducible promoter (BBa_K190015) , which receives the transcriptional signal, and a simple transcriptional amplifier (HrpRS) , finally is the output module made up by an inducible pHrpL promoter that receives the amplified signal from the cascade and promotes the expression of the reporter gene mRFP-Violet.

Click me, Please
Click me, Please

3. As4 Construct (detection limit ≥ 10 ppb [As]):

The detection module consists of a constitutive promoter of medium strength (BBa_J23115) that is in charge of the expression of the arsR protein (BBa_J15101). In this construct, processing modules are not included, and immediately afterwards is the output module made up by Pars inducible promoter (BBa_K190015) that receives the signal from the detection module and promotes the expression of the reporter gene mRFP1-Violet.

Click me, Please
Click me, Please

4. As5 Construct (detection limit ≥ 50 ppb [As]):

The detection module consists of a high-strength constitutive promoter (BBa_J23105) that is in charge of the expression of the arsR protein (BBa_J15101). Again, a processing module is not included in this construct and immediately afterwards is the output module made up by the inducible promoter Pars (BBa_K190015) that receives the signal from the detection module and finally promotes the expression of the reporter gene mRFP1- Violet.

Click me, Please
Click me, Please

5. Other considerations:

The detection module consists of a high-strength constitutive promoter

  • Strong RBS sequences (BBa_J34801 and BBa_J34803) are located throughout the constructs coding genes.

  • Terminator sequences (BBa_B1002) are positioned at the end of each coding gene.

  • All constructs are inserted into pIDTSMART-Kan high-copy plasmids.

References

  1. Wan, X.; Volpetti, F.; Petrova, E.; French, C.; Maerkl, S. J.; Wang, B. Cascaded Amplifying Circuits Enable Ultrasensitive Cellular Sensors for Toxic Metals. Nature Chemical Biology 2019, 15 (5), 540–548. https://doi.org/10.1038/s41589-019-0244-3.

  2. Li, S.; Rosen, B. P.; Borges-Walmsley, M. I.; Walmsley, A. R. Evidence for Cooperativity between the Four Binding Sites of Dimeric ArsD, an As(III)-Responsive Transcriptional Regulator *. Journal of Biological Chemistry 2002, 277 (29), 25992–26002. https://doi.org/10.1074/jbc.M201619200.

  3. Wang, B.; Barahona, M.; Buck, M. Amplification of Small Molecule-Inducible Gene Expression via Tuning of Intracellular Receptor Densities. Nucleic Acids Research 2015, 43 (3), 1955–1964. https://doi.org/10.1093/nar/gku1388.

  4. Wang, B.; Barahona, M.; Buck, M. Engineering Modular and Tunable Genetic Amplifiers for Scaling Transcriptional Signals in Cascaded Gene Networks. Nucleic Acids Res 2014, 42 (14), 9484–9492. https://doi.org/10.1093/nar/gku593.

  5. Jovanovic, M.; James, E. H.; Burrows, P. C.; Rego, F. G. M.; Buck, M.; Schumacher, J. Regulation of the Co-Evolved HrpR and HrpS AAA+ Proteins Required for Pseudomonas Syringae Pathogenicity. Nat Commun 2011, 2, 177. https://doi.org/10.1038/ncomms1177.

  6. Ferrer, M. D.; Quiles-Puchalt, N.; Harwich, M. D.; Tormo-Más, M. Á.; Campoy, S.; Barbé, J.; Lasa, Í.; Novick, R. P.; Christie, G. E.; Penadés, J. R. RinA Controls Phage-Mediated Packaging and Transfer of Virulence Genes in Gram-Positive Bacteria. Nucleic Acids Research 2011, 39 (14), 5866–5878. https://doi.org/10.1093/nar/gkr158.

  7. Andreou, A. I.; Nakayama, N. Mobius Assembly: A Versatile Golden-Gate Framework towards Universal DNA Assembly. PLoS One 2018, 13 (1), e0189892. https://doi.org/10.1371/journal.pone.0189892.

  8. Liljeruhm, J.; Gullberg, E.; Forster, A. C. Synthetic Biology: A Lab Manual; WORLD SCIENTIFIC, 2014. https://doi.org/10.1142/9061.

  9. Riangrungroj, P.; Bever, C. S.; Hammock, B. D.; Polizzi, K. M. A Label-Free Optical Whole-Cell Escherichia Coli Biosensor for the Detection of Pyrethroid Insecticide Exposure. Scientific Reports 2019, 9 (1), 12466. https://doi.org/10.1038/s41598-019-48907-6.

  10. Bao, L.; Menon, P. N. K.; Liljeruhm, J.; Forster, A. C. Overcoming Chromoprotein Limitations by Engineering a Red Fluorescent Protein. Analytical Biochemistry 2020, 611, 113936. https://doi.org/10.1016/j.ab.2020.113936.

  11. Hecht, A.; Endy, D.; Salit, M.; Munson, M. S. When Wavelengths Collide: Bias in Cell Abundance Measurements Due to Expressed Fluorescent Proteins. ACS Synth Biol 2016, 5 (9), 1024–1027. https://doi.org/10.1021/acssynbio.6b00072.