Difference between revisions of "Team:Bolivia/Design"
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<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 | + | <p>Upon activation of the constructs, <strong class="text-azul">the transcriptional signal |
| − | + | will be received by the HrpRS and/or RinA amplifier systems greatly enhancing the output signal.</strong> | |
| − | + | 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] </p> | |
| − | + | ||
| − | + | ||
| − | + | ||
<p> | <p> | ||
| − | <strong>HrpRS system :</strong> | + | <strong>HrpRS system :</strong> Is composed by the genetic components <strong>(hrpRS |
| − | and PhrpL)</strong> | + | 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 . | + | syringae</i>. The activation of hrpR and hrpS proteins results in the formation |
| − | of an ultrasensitive complex | + | of an ultrasensitive complex that binds the up-stream sequence of the |
| − | hrpL promoter (σ54 factor dependent) | + | 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] | |
| − | + | ||
</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> | + | <strong>RinA system : </strong> Is a family of phage-encoded proteins that |
| − | act as activators for the transcription of late operons in | + | 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. | + | transcription of down-stream elements in an ultrasensitive way. [6] |
</p> | </p> | ||
</div> | </div> | ||
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<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> | ||
| − | + | The described transcriptional apparatus should be translated into a measurable signal. | |
| − | + | <strong class="text-azul"> In our case, this is possible through a | |
| − | + | 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, | ||
| − | + | 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 | + | background noise, UV-induced bleaching, cell damage, and the |
| − | need for eye and skin protection. | + | need for eye and skin protection. Chromoproteins are commonly used as markers in |
| − | + | living organisms for cloning [7], teaching [8] and biosensors. [9] | |
</p> | </p> | ||
</div> | </div> | ||
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<p class=""> | <p class=""> | ||
| − | <strong>mRFP1E Chromoprotein:</strong> | + | <strong>mRFP1E Chromoprotein:</strong> Is a variant of the monomeric red fluorescent |
| − | protein 1 (mRFP1) | + | 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 | + | 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> | + | emission spectrum at 606 nm.</strong> It also has low molecular weight (55 kDa), and reports indicated |
| − | + | that mRFP1E is less toxic to <i>E. coli</i> 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] | |
| − | magenta and violet color variants have also been generated with the same | + | </p> |
| − | + | <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> | ||
| − | |||
| − | |||
| − | |||
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<p>We apply the aforementioned strategies to generate 4 genetic constructs | <p>We apply the aforementioned 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>Transformed into 4 | ||
| − | different E. coli BL21 strains, we can generate traffic light patterns visible | + | different <i>E. coli</i> BL21 strains, we can generate traffic light patterns visible |
to the naked eye and easy to interpret.</strong></p> | to the naked eye and easy to interpret.</strong></p> | ||
</div> | </div> | ||
Revision as of 04:16, 20 October 2021
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.
Figure 1.Adjusting the intracellular 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.
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]
Figure 2. The HrpR and HrpS proteins form a hexameric complex that functions as an enhancer element that comes into contact with RNA polymerase and σ54 factor, which in turn are positioned on the PhrpL promoter, activating transcription in an ultra-sensitive and amplified way.
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]
Figure 3. Adjusting the intracellular 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.
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]
ARSENITO IN DETAIL
We apply the aforementioned strategies to generate 4 genetic constructs that offer different ranges of sensitivity to arsenic. Transformed into 4 different E. coli BL21 strains, we can generate traffic light patterns visible to the naked eye and easy to interpret.
Figure 4.The different arsenic sensitivities of the constructs allow semi-quantitative interpretation of results by means of traffic light patterns observable with the naked eye.
1. As0 Construct (≥ 0.5 ppb [As] sensitivity):
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.
2. As2 Construct(≥ 3 ppb [As] Sensitivity):
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 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.
3. As4 Construct (≥ 10 ppb [As] Sensitivity)
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 a processing module is 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.
4. As5 Construct (≥ 50 ppb [As] Sensitivity)
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) , 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.
5. Other considerations:
The detection module consists of a high-strength constitutive promoter
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Strong RBS sequences (BBa_J34801 and BBa_J34803) are located throughout the constructs coding genes.
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Terminator sequences (BBa_B1002) are positioned at the end of each coding gene.
-
All constructs are inserted into pIDTSMART-Kan high-copy plasmids.
References
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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Liljeruhm, J.; Gullberg, E.; Forster, A. C. Synthetic Biology: A Lab Manual; WORLD SCIENTIFIC, 2014. https://doi.org/10.1142/9061.
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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.
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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.
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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.
