Team:Bolivia/Design

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.

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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.

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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.

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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.

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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.