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Team:Tuebingen/Design

PacMn

Design

Motivation

Biosensors are analytical tools made up of biological components with the competence to sense substances in the sensor’s environment. Due to their versatility and the increasing toolbox provided by the characterization of the original organisms, especially microbes, biosensors have become very popular in synthetic biology, and thus also in the iGEM competition. The exploitation of biological features has led to superiority of biosensors in terms of specificity and sensitivity, compared to state-of-the-art chemical analysis tools [1].
Since the design of a general biosensor has been explored in the iGEM competition quite intensively throughout the years, we decided to increase the yield of our project by developing a bifunctional biosensor which does not only detect the respective substance, but is also able to remove it from the environment. Until now, some iGEM teams have already conducted research on biosensensing of heavy metals, an exceptional example being the iGEM Team Bielefeld 2015 who has even provided a cell-free system to circumvent biosafety shortcomings of GMO sensors. However, there is not a lot of data regarding the biosensing of the heavy metal manganese and its subsequent retention, which is why we have chosen to develop the biosensor PacMn: Phytochelatin-actuated complexation of Manganese.
Briefly, the heart of the design of PacMn lies within three genetic elements: a manganese (II) ion sensing riboswitch, a FAST-tag for fluorescent signalling, and a synthetic phytochelatin, which is able to chelate heavy metal ions.

The manganese riboswitch

Riboswitches are small cis-regulatory RNA elements that are co-transcribed with genes subjected to their regulation. Their specific recognition of a substance triggers a structural change in the molecule, which in return influences their interaction with the mRNA of the co-transcribed gene [2]. This may result in the activation or suppression of the translation of the regulated gene. In vivo, riboswitches are mainly found to be sensing metabolites for regulatory feed-back loops. However, RNA-thermometers, pH-sensors, and heavy metal sensors have also been described [2]. As the adaptation of the riboswitch sequence may affect its specific recognition, binding affinity, and the intensity of the gene regulation, they are recognized as versatile and strong regulatory elements [2], whose features can be beneficial in synthetic biology applications. Unlike regulatory repressor or activator proteins, riboswitches do not require translation, putatively providing a platform which may react more quickly to the introduction of a physiological substance.
As we are interested in the detection of manganese, we decided to use a manganese riboswitch which has already been introduced to the iGEM community as a BioBrick (BBa_K902074) by Team Calgary 2012 but has not been characterized yet. Dambach et al. have described this yybP-ykoY motif-based riboswitch to be a directly manganese-responsive element in E. coli and B. subtilis which is putatively involved in metal homeostasis [3]. Interestingly, the authors found that the manganese regulatory unit consists of two independently contributing elements (Figure 1), a manganese responsive promoter (activated by the regulatory proteins MntR and Fur), and a 5’ untranslated region (UTR); the riboswitch itself [3]. Both are described in this project. Mechanistically, the interaction of manganese with the riboswitch and the thus induced structural change in the element increases the ribosomal binding site accessibility for the co-transcribed genes. As a consequence, the translation of the co-transcribed gene is activated [3].
Figure 1: Regulation of manganese levels in E. coli. During high cellular manganese levels the regulators MntR (yellow) and Fur activate the promoter (green) of the mntP gene (purple). This allows for transcription, but binding of manganese to the riboswitch (orange)  is necessary for following translation and expression of the manganese efflux pump MntP (purple). This leads to export of manganese and lowers intracellular manganese concentrations. Additionally, MntR downregulates the expression of MntH (blue) a manganese importer. When little manganese is present in the cell, this downregulation stops and manganese is transported into the cell. At the same time, the riboswitch prevents translation of mntP and manganese export by MntP is decreased.

Figure 1: Regulation of manganese levels in E. coli. During high cellular manganese levels the regulators MntR (yellow) and Fur activate the promoter (green) of the mntP gene (purple). This allows for transcription, but binding of manganese to the riboswitch (orange) is necessary for following translation and expression of the manganese efflux pump MntP (purple). This leads to export of manganese and lowers intracellular manganese concentrations. Additionally, MntR downregulates the expression of MntH (blue) a manganese importer. When little manganese is present in the cell, this downregulation stops and manganese is transported into the cell. At the same time, the riboswitch prevents translation of mntP and manganese export by MntP is decreased.

The FAST2-tag

The FAST-tag is a small protein tag which leads to the immediate activation of an interchangeable fluorogen upon expression of the tag [4].

Phytochelatin

Phytochelatins are plant oligopeptides which bind heavy metal ions, such as lead, cadmium and manganese, thus resulting in detoxification of plants by transferring the pollutants into vacuoles [5]. In vivo, phytochelatins are formed by the transpeptidation of gamma-glutamylcysteine units from one glutathione molecule onto another via a phytochelatin synthase [7].
In synthetic biology and metabolic engineering, the ability to chelate and detoxify via phytochelatins has been vastly explored. Through sequence amendments, phytochelatins may be used for the bioremediation of heavy metal ions [6] or to increase heavy metal tolerance of plants (A. thaliana), single-cell eukaryotes (S. cerevisiae), and prokaryotes [7].

PacMn: Phytochelatin-actuated complexation of Manganese

Taken together, in PacMn we will employ the manganese riboswitch (BBa_K902074) to regulate the expression of a FAST-tag on the translational level in order to yield a manganese-dependent fluorescent signal. Accordingly, the sequence of the fluorescent protein FAST-II by Twinkle Factory was placed in the cis-regulatory region of the riboswitch via Gibson Assembly. To render the biosensor bifunctional and thus allow for the chelation of manganese ions, a synthetic phytochelatin (BBa_K1321005) was additionally placed under the control of the riboswitch.
To generate a maximum yield of PacMn, four parallel constructs were designed, as is described in Engineering. As can be seen in Figure 2, the major construct, GA2, contains the manganese dependent promoter (BBa_K902073), a manganese riboswitch (BBa_K902074), the FAST-II tag (BBa_K3510000), as well as the phytochelatin (BBa_K1321005) for manganese chelation. GA1, GA3, and GA4 were designed as control constructs. Unlike GA2, GA1 carries a blue chromoprotein (BBa_K864401) instead of the phytochelatin. As described in Engineering, this was designed in an attempt to evaluate the effect of the phytochelatin on the riboswitch, as the chromoprotein should not interfere with manganese sensing. Compared to GA2, GA4 contains a constitutive Anderson promoter (BBa_J23102), which allows us to determine the efficiency of the riboswitch by itself, without the support of the manganese promoter. Finally, GA3 is the control for GA4, carrying a chromoprotein and no phytochelatin with the riboswitch under the control of the Anderson promoter.
Figure 2: Schematic figure of the PacMn constructs. Each Gibson Assembly consisted of two fragments and a pUC19 vector backbone. The dotted line indicates the ends of the two insert fragments. Here they are going to get connected by Gibson Assembly.

Figure 2: Schematic figure of the PacMn constructs.. Each Gibson Assembly consisted of two fragments and a pUC19 vector backbone. The dotted line indicates the ends of the two insert fragments. Here they are going to get connected by Gibson Assembly.

To conclude, PacMn aims to sense and retain manganese ions. While in the absence of the heavy metal the circuit should be repressed, the presence of manganese leads to the translational activation of FAST-II and phytochelatin, which in return induces a fluorescent signal and the complexation of the ions. It is an objective to analyse the linearity of the fluorescent signal of PacMn to determine its viability as a quantitative analytical tool.

Implementation

As can be seen in Figure 3, the application of the sensor includes two steps. In the first step, a sample is added into the PacMn mix. After some time, the construct carrier (e.g. E. coli) is isolated in step 2 and incubated in a labeling solution containing the fluorophore substrate (i.e. the fluorogen) of the FAST-II tag. In the third step, the labeled sample is transferred into a plate reader to measure the emitted fluorescence. Alternatively, for a point-of-care application, we considered using a qualitative tool for in-field detection by using the Chromoprotein in one of our approaches, or a cell-free system, as provided by the iGEM Team Bielefeld 2015.
Figure 3: Schematic figure of the PacMn biosensor test. (1) A water sample is added to the PacMn biosensor while also adding the fluorescent substrate. (2) The signal is measured using a plate reader for quantitative output (3).

Figure 3: Schematic figure of the PacMn biosensor test. (1) A water sample is added to the PacMn biosensor while also adding the fluorescent substrate. (2) The signal is measured using a plate reader for quantitative output (3).

Side project

In addition to our main project, we decided to modify an existing composite part; the vitamin B12 riboswitch with an inverter and reporter gene of the team Wageningen 2016 (BBa_K1913011). Their composite essentially promotes the production of an inducible mRFP (monomeric red fluorescent protein) based on the concentration of vitamin B12.
The idea was to exchange their reporter protein for GFP (green fluorescent protein), of which the expression can be measured reliably using the fluorescein-based iGEM fluorescence calibration protocol. Our idea was not only to present another option for future iGEM teams working with vitamin B12, but also to challenge ourselves by assembling the composite via Gibson Assembly® instead of via restriction-ligation, as originally done. For this, we designed a four-fragment assembly (three inserts, one vector backbone), once with mRFP (GA5) and once with GFP (GA6) to compare results (Fig. 4).
Figure 4: Schematic figure of the Vitamin B12 side project constructs. Each Gibson Assembly consisted of three fragments and a pUC19 vector backbone. The dotted lines indicate the intersections between the three insert fragments that were assembled

Figure 4: Schematic figure of the Vitamin B12 side project constructs. Each Gibson Assembly consisted of three fragments and a pUC19 vector backbone. The dotted lines indicate the intersections between the three insert fragments that were assembled.

References

  1. Kaur H, Kumar R, Babu JN, Mittal S. Advances in arsenic biosensor development--a comprehensive review. Biosens Bioelectron 2015; 63:533–45.
  2. Sherwood AV, Henkin TM. Riboswitch-Mediated Gene Regulation: Novel RNA Architectures Dictate Gene Expression Responses. Annu Rev Microbiol 2016; 70:361–74.
  3. Dambach M, Sandoval M, Updegrove TB, Anantharaman V, Aravind L, Waters LS et al. The ubiquitous yybP-ykoY riboswitch is a manganese-responsive regulatory element. Mol Cell 2015; 57(6):1099–109.
  4. The Twinkle Factory. The science behind FAST and SplitFAST [cited 2020 Oct 15]
  5. Gupta DK, Huang HG, Corpas FJ. Lead tolerance in plants: strategies for phytoremediation. Environ Sci Pollut Res Int 2013; 20(4):2150–61.
  6. Cahoon RE, Lutke WK, Cameron JC, Chen S, Lee SG, Rivard RS et al. Adaptive Engineering of Phytochelatin-based Heavy Metal Tolerance. J Biol Chem 2015; 290(28):17321–30.
  7. Biondo R, da Silva FA, Vicente EJ, Souza Sarkis JE, Schenberg ACG. Synthetic phytochelatin surface display in Cupriavidus metallidurans CH34 for enhanced metals bioremediation. Environ Sci Technol 2012; 46(15):8325–32.