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.
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.
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).
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.
