Team:Macquarie Australia/Composite Part
COMPOSITE PARTS
Cyclic-di-GMP riboswitch with eGFP reporter BBa_K3151011
The expression of green fluorescent protein depends on the intracellular concentration of cyclic-di-GMP. Our riboswitch plasmid contains a riboswitch, terminator stem, and is coupled with eGFP and BBa_K3151009 BBa_J45992 BBa_K1094400 BBa_B0010 comprises this part.
The regulation of green fluorescent protein expression is dependent on the interactions between the terminator stem-loop region of the riboswitch and the activation or inactivation of this terminator stem-loop by the presence or absence of cyclic-di-GMP. When the terminator stem-loop is active and cyclic-di-GMP is bound, it prevents the expression of green fluorescent protein through premature termination of emerging transcripts. From this, we hypothesized that the riboswitch suppresses the expression of green fluorescent protein in the presence of cyclic-di-GMP, hence, this part will express less eGFP than the riboswitch without the terminator stem loop.
Nissle 1917 E. coli cells transformed these plasmids were spotted onto LB agar plates supplemented with Congo Red and Coomassie Blue. We compared the effect of induction of the yhjH phosphodiesterase between the yhjH+RON and yhjH+ROFF transformants. Induction of yhjH appeared to have no effect on the fluorescence of yhjH+RON transformants (Figure 1A, Figure 1C), which corroborates our hypothesis that its expression of eGFP is not influenced by the intracellular concentration of c-di-GMP. Induction of yhjH appeared to significantly increase the fluorescence of yhjH+ROFF transformants (Figure 1B, Figure 1D), again corroborating our hypothesis.
In order to characterise the interaction between the function of our riboswitch and phosphodiesterases, we constructed two plasmids containing the coding sequences both the riboswitch/eGFP operon (either RON and ROFF) under the stationary phase promoter and the yhjH gene under the Lac promoter. We confirmed the assembly through restriction digestion as well as sequence confirmation. These plasmids were then transformed into the E. coli strains DH5α and Nissle 1917.
E.coli DH5α cells transformed with these plasmids we used to perform fluorescence assays using the BMG Pherastar plate reader to measure eGFP (Ex485nm Em520nm) and OD (600nm) shown in Figure 2. These results confirm our hypothesis that the ROFF construct will produce significantly lower levels of eGFP due to inhibition from cellular cyclic-di-GMP.
Figure 1: LB agar plate supplemented with Congo Red and Coomassie blue, spotted with yhjH+RON induced (A), yhjH+ROFF induced (B), yhjH+RON uninduced (C), and yhjH+ROFF uninduced (D).
Figure 2: The constitutive riboswitch was expressed in DH5ɑ cells. The blue lines represent RON samples, the black lines represent ROFF samples. All RON samples were observed to express more Green Fluorescent Protein (GFP) than corresponding ROFF samples. The effect of sucrose is shown as inhibiting the expression of GFP, shown in the decrease of lower.
Riboswitch without a terminator loop, coupled with eGFP BBa_K3151002
The expression of green fluorescent protein depends on the presence of cyclic-di-GMP. Our riboswitch plasmid contains a riboswitch, terminator, and is coupled with eGFP and BBa_J45992 BBa_K3151009 http://parts.igem.org/Part:BBa_K1094400">BBa_K1094400 BBa_B0010 comprises this part.
The regulation of green fluorescent protein expression is dependent on the interactions between the terminator stem-loop region of the riboswitch and the activation or inactivation of this terminator stem-loop by the presence or absence of cyclic-di-GMP. When the terminator stem-loop is active and cyclic-di-GMP is bound, it prevents the expression of green fluorescent protein through premature termination of emerging transcripts. From this, we hypothesized that the riboswitch suppresses the expression of green fluorescent protein in the presence of cyclic-di-GMP, hence, this part will express more eGFP than the riboswitch with the terminator stem loop.
Lac + YHJH Phosphodiesterase BBa_K3151008
This part utilizes the yhjH cyclic-di-GMP phosphodiesterase BBa_K861090, and improves upon the Tec Chihuahua composite part BBa_K2471001 by adding an inducible Lac promoter BBa_R0010and double terminator (BBa_B0015).
The native activity of cyclic-di-GMP as a secondary messenger in bacteria is primarily to regulate the transition between motile and sessile biofilm-forming behaviour. Synthesis of cyclic-di-GMP itself is regulated by the stationary-phase sigma factor RpoS (σS), which when induced results in an increase in cyclic-di-GMP concentration, and subsequent downstream increase in the production of cellulose and curly fimbriae, the primary components of Escherichia coli biofilm.
Cyclic-di-GMP is synthesised by diguanylate cyclases, and degraded by phosphodiesterases. Many enzymes which perform these functions are capable of both synthesis and degradation of cyclic-di-GMP, however, the yhjH phosphodiesterase is incapable of synthesis. Combined with the Lac promoter and double terminator, this Biopart allows for controlled expression of phosphodiesterase activity in a wide variety of E. coli strains such as DH5a, Nissle 1917 and BL21(DE3).
When combined with a cyclic-di-GMP riboswitch, such as BBa_K3151009 , this phosphodiesterase can be used to control the expression of reporter genes through modulating the intracellular concentration of cyclic-di-GMP.
Characterisation:As intracellular cyclic-di-GMP concentration is directly correlated with biofilm formation[1], we can utilise the biofilm-forming capability of the transformants as a proxy for intracellular cyclic-di-GMP concentration and phosphodiesterase activity. Nissle 1917 E. coli cells transformed with this Biopart were spotted onto LB agar plates supplemented with Congo Red and Coomassie Blue[2], and the cells transformed with the construct containing the promoter and terminator sequences produced significantly less cellulose than cells transformed with the construct without the promoter and terminator, as well as parental Nissle 1917 cells (Figure 3), indicating significant phosphodiesterase activity within the cell.
Figure 3: E. coli Nissle 1917 cells transformed with the yhjH phosphodiesterase with (A) and without (B) promoter and terminator, as well as negative control (C). Cells were spotted onto LB agar plates supplemented with Congo Red and Coomassie Blue, and visualised with Blue Light LEDs. Nissle 1917 was selected as it produces significantly higher levels of cellulose in its biofilm. The red colour observed in (B) and (C) is the Congo Red staining the cellulose.
When combined with a cyclic-di-GMP riboswitch + eGFP reporter, this Biopart reduces the inhibition of eGFP transcription due to a decrease in cyclic-di-GMP concentration.
Figure 4: GFP fluorescence assay performed on cells transformed with RONBBa_K3151011 and ROFF BBa_K3151002. Samples were measured with the BMG Pherastar plate reader to measure eGFP (Ex485 nm Em520 nm) and OD (600 nm).
Figure 5: GFP fluorescence assay performed on cells transformed with yhjH + RON BBa_K3151029and yhjH + ROFF BBa_K3151030. Samples were measured with the BMG Pherastar plate reader to measure eGFP (Ex485 nm Em520 nm) and OD (600 nm).
When compared with the Tec Chihuahua composite part BBa_K2471001, ours has the distinct advantage of being able to expressed in cells which lack T7 RNA polymerase. When transformed into the E. coli strain Nissle 1917, our device exhibited
Figure 6: Spot Assay of the negative control Nissle 1917 (A), Nissle 1917 with T7+yhjH (B), and Nissle 1917 with Lac+yhjH (induced with 1mM IPTG) (C). Plate observed under blue light to enhance visualisation of Congo Red-stained cellulose.
References:
- Weber H, Pesavento C, Possling A, Tischendorf G, & Hengge R. Cyclic‐di‐GMP‐mediated signalling within the σS network of Escherichia coli. Molecular microbiology. 2006 Nov;62(4):1014-34.
- Cimdins A, Simm R, Li F, Lüthje P, Thorell K, Sjöling Å, Brauner A, Römling U. Alterations of c‐di‐GMP turnover proteins modulate semi‐constitutive rdar biofilm formation in commensal and uropathogenic Escherichia coli. MicrobiologyOpen. 2017 Oct;6(5):e00508.
Lac Cyclic-di-GMP riboswitch BBa_K3151028
The expression of green fluorescent protein (GFP) relies on the action of the phosphodiesterase which decreases the intracellular concentration of cyclic-di-GMP available. This decrease in intracellular c-di-GMP then activates the riboswitch (Hengge, R. 2009). Our riboswitch plasmid contains a Plac promoter, the riboswitch, and a terminator. These three components form the riboswitch composite part. Combined, these catalyse the activation of the riboswitch, and consequently the production of green fluorescent protein.
Assembly and Design
Suitable inducible promoters were identified from the Parts Registry to be used in the part characterisation for the riboswitch. The Plac promoter was assembled with the riboswitch, which was originally ordered from Genewiz. The constitutive promoter BBa_J23100 has ~99% promoter efficiency and was tested using GFP assays in both induced and uninduced promoters.
YHJH + Riboswitch BBa_K3151029
The activity of the phosphodiesterase determines the concentration of the cyclic-di-GMP that is bound to the terminator stem of the riboswitch. This altogether then determines the production of the green fluorescent protein. The chief hypothesis formed was that the intracellular concentration of cyclic-di-GMP does not affect the activity of the riboswitch with no terminator stem-loop (RON) since there is no component to stop the transcription of the fluorescent protein. However, it does affect the Riboswitch with the terminator stem-loop (ROFF). As seen in (Figure 1) when YHJH (Phosphodiesterase) was induced and was activated, its activity had a decreasing effect on cyclic-di-GMP, hence, increasing the production of eGFP.
Assembly and Design
The parts were digested using enzymes E and S and then ligated using standard assembly.
Characterization
Figure 7: Agarose gel of sequence confirmed parts, showing the size (bp) of ROFF.
Figure 8: The constitutive riboswitch was expressed in DH5ɑ cells. The blue lines represent RON samples, the black lines represent ROFF samples. All RON samples were observed to express more Green Fluorescent Protein (GFP) than corresponding ROFF samples. The effect of sucrose is shown as inhibiting the expression of GFP, shown in the decrease of lower.
YHJH + Riboswitch without terminator loop BBa_K3151030
The activity of the phosphodiesterase determines the concentration of the cyclic-di-GMP that is bound to the terminator stem-loop of the riboswitch. This altogether then determines the production of the green fluorescent protein. We hypothesized that the intracellular concentration of c-di-GMP does not affect the activity of the riboswitch with no terminator (ROFF), but affects the riboswitch with terminator (RON). As seen in (Figure 1) when YHJH (Phosphodiesterase) was induced and was activated, its activity had no effect on the Riboswitch with no terminator loop (ROFF) regardless of the cyclic-di-GMP levels, which confirms our hypothesis.
Characterisation
Figure 9: Agarose gel of sequence confirmed parts, showing the size (bp) of RON.
Figure 10:The constitutive riboswitch was expressed in DH5ɑ cells. The blue lines represent RON samples, the black lines represent ROFF samples. All RON samples were observed to express more Green Fluorescent Protein (GFP) than corresponding ROFF samples. The effect of sucrose is shown as inhibiting the expression of GFP, shown in the decrease of lower.
Hydrogenase cyclic-di-GMP phosphodiesterase biosensor BBa_K3151027
Without phosphodiesterase domain BBa_K3151025
Overview
The sensory Magnetospirillum magneticum [NiFe] hydrogenase operon consists of four parts: 1 kb hurS encoding a small subunit, 1.6 kb hurL encoding a large subunit, 0.5 kb hupD encoding maturation protease, and 2 kb hurR encoding cyclic-di-GMP phosphodiesterase.
Literature
[NiFe] hydrogenases show higher affinity for molecular hydrogen compared to [FeFe] hydrogenases [1]. One of the many advantages of [NiFe] hydrogenases is their fast response to the presence of molecular hydrogen. They usually have high turnover numbers (Kcat, s-1), which means they are capable of oxidising a large number of hydrogen molecules per second. In a previous study, Pershad et al. investigated the redox activity of [NiFe] hydrogenase from Chromatium vinosum using protein film voltammetry [2]. They found that the Kcat value was between 1500 – 1900 s-1 at 10% partial hydrogen pressure, 30°C, and pH 5 – 8, although there was a strong force driving the reverse reaction, i.e. reduction of H+ to H2 [2]. A number of enzyme kinetic studies also revealed high Kcat of [NiFe] hydrogenases under various reaction conditions [3, 4]. The high catalytic activity means that these hydrogenases can rapidly generate large amounts of H+, which in turn activate a series of downstream signalling events and cellular response.
The future of hydrogen gas biosensing lies in whole cell-based biosensors which use genetically modified living organisms to detect the presence of target substances. A few designs were introduced in the past for monitoring gaseous ammonia and carbon monoxide [5, 6]. It is expected that whole cell-based biosensors for hydrogen detection will be made available in the near future.
Furthermore, [NiFe] hydrogenases maintain high-affinity oxidative activity in a broad range of tropospheric hydrogen concentration [7], which cannot be achieved by physicochemical hydrogen sensors. This feature is crucial for Team Macquarie Australia 2019 project in building a hydrogen biosensor to meet the requirement of overcoming the cross-sensitivity problem faced by current hydrogen gas sensors.
Assembly and design
We added EcoRI (GAATTC), XbaI (TCTAGA), SalI (GTCGAC), SpeI (ACTAGT), and PstI (CTGCAG) restriction enzyme cleavage sites within the sequence.
All genes within this plasmid are sequences obtained from Magnetospirillum magneticum and codon optimised to be expressed in Escherichia coli.
Usage
Hydrogenase (Large subunit Hydrogenase + small subunit Hydrogenase + Hydrogenase maturation protease)The maturation protease will first cleave the C-terminus of [NiFe] hydrogenase large subunit after the Nickel insertion [8]. The large and small subunit will then come together to form the hydrogenase part of the biosensor.
The hydrogenase with all three components was used in the hydrogenase consumption assay.
Phosphodiesterase
The mature hydrogenase (large and small subunits together) will come together with the phosphodiesterase to form the sensory component of the hydrogen biosensor for Team Macquarie Australia 2019. Cyclic-di-GMP phosphodiesterase breaks down cyclic-di-GMP [8]. The change in cyclic-di-GMP levels in the cell will affect the amount of biofilm produced.
Part Characterisation
We have successfully integrated the hydrogenase part Hyd A of M. magneticum [NiFe] hydrogenase into E. coli DH5α cells. The hydrogen consumption assay has confirmed that our hydrogenase is functional.
In order to test the functionality of the hydrogenase part of M. magneticum [NiFe] hydrogenase, we measured hydrogen desaturation rates (mV/s) in water and in E. coli DH5α Hyd A transformant cells (which contain hydrogenase) using Clark-type electrode. This test is called the hydrogenase consumption assay.
Testing the functionality of the hydrogenase part requires a sample that has all three components (small subunit, large subunit, and maturation protease). As seen in Figure 1, HydA 7 which has all three components present was chosen for the assay.
Figure 11: SDS-PAGE showing the three parts of the hydrogenase biosensor. HurL (large subunit), HurS (small subunit), and HupD (maturation protease). Four biological replicates of HydA 7, HydA 3, HydA 4, and HydA 2. HydA 7 was chosen for the assay.
As seen in Figure 2 and 3, the results showed higher rates of hydrogen saturation and desaturation for HydA 7 transformant than DH5α cells without [NiFe] hydrogenase. In the presence of hydrogen, HydA 7 cells appeared to consume hydrogen, which resulted in more hydrogen being absorbed into the solution and decreased of hydrogen concentration in the electrode chamber. In contrast, non-transformant DH5α cells did not show significant intake of hydrogen from the environment, resulting in lower hydrogen saturation rate. The results from this experiment demonstrated that our [NiFe] hydrogenase was functional and was capable of oxidising molecular hydrogen into protons and electrons.
Figure 12: Maximum hydrogen saturation rates of water (negative control), DH5α (positive control), and DH5α HydA 7 transformant. Standard deviation n=2.
Figure 13: Maximum hydrogen desaturation rates of water (negative control), DH5α (positive control), and DH5α HydA 7 transformant. Standard deviation n=2.
References
[1] Frey M. Hydrogenases: Hydrogen-Activating Enzymes. ChemBioChem. 2002;3(2-3):153-160.
[2] Pershad H, Duff J, Heering H, Duin E, Albracht S, Armstrong F. Catalytic Electron Transport inChromatium vinosum[NiFe]-Hydrogenase: Application of Voltammetry in Detecting Redox-Active Centers and Establishing That Hydrogen Oxidation Is Very Fast Even at Potentials Close to the Reversible H+/H2Value. Biochemistry. 1999;38(28):8992-8999.
[3] Preissler J, Wahlefeld S, Lorent C, Teutloff C, Horch M, Lauterbach L et al. Enzymatic and spectroscopic properties of a thermostable [NiFe]‑hydrogenase performing H2-driven NAD+-reduction in the presence of O2. Biochimica et Biophysica Acta (BBA) - Bioenergetics. 2018;1859(1):8-18.
[4] van der Linden E, Burgdorf T, de Lacey A, Buhrke T, Scholte M, Fernandez V et al. An improved purification procedure for the soluble [NiFe]-hydrogenase of Ralstonia eutropha: new insights into its (in)stability and spectroscopic properties. JBIC Journal of Biological Inorganic Chemistry. 2006;11(2):247-260.
[5] Bohrn U, Stütz E, Fuchs K, Fleischer M, Schöning M, Wagner P. Monitoring of irritant gas using a whole-cell-based sensor system. Sensors and Actuators B: Chemical. 2012;175:208-217.
[6] Bohrn U, Stütz E, Fuchs K, Fleischer M, Schöning M, Wagner P. Air Quality Monitoring using a Whole-Cell based Sensor System. Procedia Engineering. 2011;25:1421-1424.
[7] Greening C, Berney M, Hards K, Cook G, Conrad R. A soil actinobacterium scavenges atmospheric H2 using two membrane-associated, oxygen-dependent [NiFe] hydrogenases. Proceedings of the National Academy of Sciences. 2014;111(11):4257-4261.
[8] Greening C, Biswas A, Carere C, Jackson C, Taylor M, Stott M et al. Genomic and metagenomic surveys of hydrogenase distribution indicate H2 is a widely utilised energy source for microbial growth and survival. The ISME Journal. 2015;10(3):761-777.