MerR activator under constitutive promoter and induceble merT promoter with 5 UTR -sfGFP

Mercury sensor activator under the control of the constitutive promoter (BBa_K608002) with mercury induceble promoter and 5´untranslated region which increses the output of the sfGFP which is used for detection.

Usage and Biology

For our sensor, we use parts ( BBa_K346002 and BBa_K346001) of the mercury sensor constructed by iGEM team Peking 2010. These Parts consist of the Mer operon from Shigella flexneri R100 plasmid Tn21, a mercury dependent operon. The expression of the Mer operon is regulated by the activator MerR. The MeR transcription however is regulated by itself. Mercury can bind to the C-terminal located cysteines and generates a conformal change to activate the expression (N.L. Brown et al.2003). Our mercury Sensor contains MerR, which is under control of a constitutive promoter and specific promoter MerT. sfGFP protein is used as measuring output signal and it´s transcription is controlled by the 5` untranslated region, which enhances the following reporter protein sfGFP .

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Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 462
Illegal NheI site found at 485
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 626

in vivo

One of the already existing sensors we used for our system is the mercury sensor consisting of MerR the activator and the mercury specific promoter pmerT. The promoter is regulated by the MerR, which binds Hg²⁺-ions. Similar to the former sensors we added a sfGFP for detection via fluorescence.

For our mercury sensor we used parts of the mercury sensor constructed by iGEM team Peking 2010. These parts consist of the mercury dependent mer operon from Shigella flexneri R100 plasmid Tn21. The expression of the genes in the mer operon depends on the regulation by MerR its activator and promoter PmerT. For our sensor we used the codon optimized activator (BBa_K1758340), under control of a constitutive promoter,(BBa_K346001). Additionally to this activator we designed and constructed the specific promoter PmerT(BBa_K346002)(figure 1). For our sensor we added a 5’-UTR downstreamd of this promoter, which increased the fluorscence of the used reporter protein sfGFP.

Figure 1: The concept of our *in vivo* mercury sensor ( BBa_K1758343), which consists of the activator under the control of a constitutive promoter BBa_K1758340)and the operator and promoter sequence of the mercury inducible promoter. An untranslated region in front of the sfGFP, which is used for detection, enhances its expression ( BBa_K1758342).

Adjusting the detection limit — Figure 2: During cultivation the sfGFP signal in reaction to different mercury concentrations was measured. The induction with mercury happened after 165 minutes. Error bars represent the standard deviation of three biological replicates.

We tested our mercury sensor with sfGFP as reporter gene, to test the functionality of the system. Moreover we tested different concentrations. The kinetic of our sensors response to different mercury concentrations is shown in figure 2. A strong increase in fluorescence levels is notecible after induction with mercury after 120 min. For better visualization the kinetics of figure 2 are represented as bars in figure 3. A fluorescence level difference for 120 min and 190 min is represented.

In vivo data show a highly significant, well working sensor, which even reacts to concentrations below the threshold of the water guidelines by the WHO (Figure 2 and 3).

The mercury detection was measured during the cultivation of E. coli KRX at 37 °C (Figure 2 and 3). The strain contained the plasmid with the activator merR under the control of a constitutive promoter and the specific promoter with an operator binding site, which reacts to the activator with bound Hg ²⁺-ions. The specific promoter is located upstream of the sfGFP CDS. Therefore, the mercury in the medium is detected via formation of sfGFP. In vivothis sensor devise shows a fast answer to occurrence of his heavy metal contrary to the other sensor systems In vivo.

Therefore we tested our sensor in vitro to check if an already functioning highly optimized sensor provides required data for guideline detections.

Refrences

Brown, Nigel L.; Stoyanov, , Jivko V.;Kidd,Stephen P.;Hobman; Jon L. (2003): The MerR family of transcriptional regulators. In FEMS Microbiology Reviews, 27 ( 2) pp.145-163.

BEAS_China 2019: Improvement

BEAS_China 2019 changed the merR promoter used in BBa_K1758343 to a weaker promoter, J23101 in our basic mercury sensor design (See BBa_K3143673). The results in Fig.4 showed that, compared to BBa_K1758343, BBa_K3143673 presented a higher fluorescence at the same mercury concentration.

Figure 4: Characterization of BBa_K1758343 and BBa_K3143673 (Flow cytometry data).

We fitted the sensors’ dose–response curves to a Hill function-based biochemical model to describe their input-output relationships. (Figure 5 & Table 1)

The Hill constant EC50 is the inducer concentration that provokes half-maximal activation of a sensor; EC50 is negatively correlated with sensitivity.
KTop is the sensor’s maximum output expression level; KTop is positively correlated with output amplitude.

Figure 5: The equation used to fit the sensors’ dose–response curves to a Hill function based biochemical model to describe their input-GFPput relationships

Table 1: Best fits for the characterized response of BBa_K1758343 and BBa_K3143673

Here, comparaed to BBa_K1758343, the EC50 of BBa_K3143673 showed a 1.3-fold decrease and the KTop of BBa_K3143673 showed a 2.23-fold increase(Table 1), confirming that the mercury sensor’s sensitivity and output amplitude were both increased in BBa_K3143673.

Team Florida 2019 Characterization Data

We took several metal promoter parts from the distribution kit given to us by iGEM. However most of the parts did not come with the repressor and therefore we only picked BBa_K1758333 (lead) and BBa_K1758343(mercury).

We used a 96-well plate to measure the RFU and GFP of the cultures containing the corresponding metal promoters.

96 Well Plate Set-Up

In 5 mL culture:

Hg/Pb

Low: 0.5mL of 10 mg/mL stock = 1ug/uL
Medium: 0.5mL of 1 mg/mL stock = 0.1ug/uL
High: 0.5mL of 0.1mg/mL stock = 0.01 ug/uL

Cr

Low: 2.5uL of 0.1 mg/mL stock = 0.05ug/uL
Medium: 2.5uL of 1 mg/mL stock = 0.5ug/uL
High: 2.5uL of 10mg/mL stock = 5 ug/uL

Cu

Low: 1uL of 10 mg/mL stock = 2ug/uL
Medium: 2.5uL of 10 mg/mL stock = 5ug/uL
High: 5uL of 10mg/mL stock = 10 ug/uL

Some cells with other metal promoters were emitting fluorescence even before we induced the cells with the toxins. As a result, the metal promoters are unaffected by the inducers and do not work effectively.

Looking at the graph, with increasing concentration of relevant levels of inducer(toxin) the relative fluorescence units are not increasing as expected.

As the inducer level increases we expect induction to increase. However for BBa_K1758343, all the induced RFU measurements for low, medium, and high concentration were less than the uninduced RFU measurements. The fold change was relatively the same for all levels of inducer which contrasts the trend that is shown on the Bielefeld 2015 website. Therefore, we can conclude that the mercury promoter did not work.

Part:BBa_K1758343