GOALS OF THE EXPERIMENTS
The goals of our experiments were the following:
- Confirm the strain chosen for the biosensor can survive in wastewater
- Assemble and test functionality of the genetic circuit
- Ensure ermC provides resistance to macrolide antibiotics
- Correlate amount of erythromycin to GFP expression
- Improve the sensitivity of the biosensor
- Find potential immobilization methods for our biosensor
- Assess whether isoelectric focusing is a suitable method for concentrating macrolides in a sample
Detailed protocols can be found here.
Our lab notebook is available here.
All sequences used can be downloaded here.
VIABILITY TESTS
Plate-Reader Experiment
In order for our biosensor to work, it is crucial that the organism we selected is capable of surviving in wastewater. For that, BL21 cells were transformed with GFP-expression plasmid (pET28a-sfGFP, Addgene) and the fluorescence produced by them was measured for 20 h in different growth media: Luria Bretani (LB) as a positive control, milli-Q as a negative control, wastewater, LB and wastewater 1:1, 1:3 and 3:1. The GFP values were normalized using OD600 measurements. Protocol: Plate-reader experiment for cell viability.
Flow Cytometry Experiment
To confirm the plate-reader experiment results and obtain more precise measurements concerning cell survival and viability, we have performed flow cytometry for cells that have been grown in following experimental set-ups for 3 h: LB as a positive control, milli-Q as a negative control, wastewater, LB and wastewater 1:1, 1:3 and 3:1. The cells were induced with IPTG 1.5 h before the measurement. A viability dye was added to distinguish dead from living cells. Protocol: Flow cytometry experiment for cell viability.
CONSTRUCTING AND TESTING THE GENETIC CIRCUIT
Modular Cloning System
MoClo is a Golden Gate based cloning system utilizing BsaI and BbsI restriction enzymes [1]. This method was chosen, because it allows for addition of more parts than RFC10 in a single reaction and is compatible with iGEM standards. Another advantage is its modularity. This feature was very relevant for this project, since it is easier to troubleshoot if one part is not functional, as well as replace elements with altered sequences. More about MoClo can be found here. All of the constructed assemblies were sequences to confirm the sequence is correct (See: Sequencing protocol).
Constructing Backbones for MoClo
Since most of the backbones available from iGEM distribution kits were compatible for RFC10 cloning, it has been decided that RFC10 backbones (pBS1C3 containing chloramphenicol resistance gene and pSB1K3 with kanamycin resistance gene) will be adjusted for MoClo by inserting a sequence, containing RFC94 suffix and prefix, as well as a red fluorescence protein gene (RFP) for easy selection of successful transformants (BBa_K2560001). Since we are cloning two level 1 assemblies and one level 2 assembly, we needed to construct three different backbones (Fig. 1). Protocols: Digestion & Ligation. The enzymes used for digestion of pSB1C3 backbone and RFP insert were XbaI and SpeI. These enzymes have the same sticky ends: initially targeted cut sites were PstI and EcoRI, but due to insufficient overhang length we had to choose XbaI and SpeI (see Plasmid design troubleshooting on our contribution page). Phosphatase was used to limit backbone re-ligation.
To initially assess whether the ligation was successful, we performed colony PCR (protocol: Colony PCR) with FW2 and RV primers from selected colonies that expressed RFP after 48 h incubation and ran the DNA in a 0.7% agarose gel (protocol: Agarose gel electrophoresis). The bands were visualized with SYBR Safe DNA Gel Stain (ThermoFisher Scientific). The expected lengths of successfully assembled backbones were around 3000 bases. Re-ligation of backbone would result in around 2000 bp band for pSB1C3 and 2200 bp for pSB1K3 backbone.
Level 1 Assemblies
Since MoClo allows for reliable insertion of up to 5 fragments, our final optical device has been divided into two level 1 assemblies: called level 1 repressor cassette (or level 1 MphR) and level 1 output cassette (or level 1 EGFP). The inserts required to construct the level 1 repressor cassette included: MphR (BBa_K3386001) [2] and ermC (BBa_K3386000) [3] genes under a constitutive promoter (BBa_J23106) and a terminator (BBa_B0029). Level 1 output cassette comprises EGFP (BBa_K3386003) gene under an inducible promoter (pMphR - BBa_K3386002) negatively controlled by MphR and a terminator (BBa_B0029). All sequences containing proteins also included ribosomal binding sites (BBa_B0015) in order to decrease the number of elements in one MoClo reaction, except for egfp which was preceded by BBa_K3386005. Both of level one assemblies were ligated into a level 1A and level 1B chloramphenicol selection backbones containing different suffixes with different overhangs (Fig. 2). Protocol: Modular Cloning. The enzyme used for digestion of inserts and backbones were BbsI.
To initially assess whether the cloning was successful, we performed colony PCR (protocol: Colony PCR) with FW2 and RV primers from selected colonies that did not express RFP after 48 h incubation and ran the DNA in a 0.7% agarose gel. The bands were visualized with SYBR Safe DNA Gel Stain (ThermoFisher Scientific). The expected length of successful assembly was around 3000 bp.
Functionality Test
In order to confirm the ermC gene is functional, TOP10 cells transformed with level 1 MphR assembly were grown in LB with various concentrations of erythromycin (10, 50 and 100 μg/ml). Optical density was measured after 20 h of growth. The cells were also grown in clarithromycin, which is structurally similar to erythromycin. The aim was to assess the resistance that ermC provides to other macrolide antibiotics. As a negative control, cells were grown in media containing spectinomycin, which belongs to a different class of antibiotics and has a different mechanism of action. A positive control comprised cells grown in LB. A similar experiment was performed using non-transformed TOP10 cells, to ensure the difference in growth rates and survival was not a result of metabolic burden due to transformation with high copy number plasmid. All values were normalized. We did not perform western-blotting to confirm MphR is expressed, but since mphr gene was expressed under the same promoter as ermC and had the same RBS, it is very likely this protein is properly produced as well.
Optical Device Assembly
When two level 1 assemblies (containing MphR and EGFP constructs) were correctly ligated, a level 2 assembly could be cloned into a level 2 kanamycin selection backbone (Fig. 3). Protocol: Modular Cloning. The enzymes used for digestion of inserts and the backbone was BsaI.
To initially assess whether the cloning was successful, we performed colony PCR (protocol: Colony PCR) with F_kan and R_kan primers primers from selected colonies that did not express RFP after 48 h incubation and ran the DNA in a 0.7% agarose gel. The bands were visualized with SYBR Safe DNA Gel Stain (ThermoFisher Scientific). The expected length of successful assembly was around 4500 bp.
Functionality Test
We performed a plate-reader experiment, where we grew cells transformed with the optical device in various erythromycin concentrations. Non-transformed E. coli, as well as cells transformed with the optical device grown in LB with 100 µl kanamycin were positive controls. A negative control comprised non-transformed cells grown in LB with no antibiotic. Optical density and fluorescence were measured every 15 minutes over 15 h. Experiments were performed at 37°C. The GFP intensity was normalized using OD600 measurements. Protocol: Plate-reader experiment for the optical device.
OPTICAL DEVICE FINE-TUNING
Stronger Output Signal
We performed a plate-reader experiment, where we compare fluorescence signal obtained from cells transformed with the optical device grown for 20 h in various erythromycin concentrations to cells transformed with level 1 output cassettes (both the original one containing egfp, as well as newly assembled one where egfp was replaced with sfgfp) grown in lack of repressor (no MphR present). Non-transformed E. coli grown in LB with 100 µl erythromycin were a positive control. A negative control comprised non-transformed cells grown in LB with no antibiotic. Optical density and fluorescence were measured every 15 minutes over 15 h. Experiments were performed at 37°C. The GFP intensity was normalized using OD600 measurements. Protocol: Plate-reader experiment for the output signal comparison.
Altered Optical Device Assembly with Inducible Promoter
The altered optical device was assembled analogically to the initial optical device (protocol: Modular Cloning), but constitutive promoter was replaced with pBAD promoter. See section: Optical device assembly. In addition to an inducible optical device containing the output cassette with EGFP, another assembly was constructed with sfGFP. See section: Optical Device Assembly. To initially assess whether the cloning was successful, we performed colony PCR (protocol: Colony PCR) with FW2, RV primers (for level 1 assembly), as well as F_kan and R_kan primers (for level 2 assembly) from selected colonies that did not express RFP after 48 h incubation and ran the DNA in a 0.7% agarose gel. The bands were visualized with SYBR Safe DNA Gel Stain (ThermoFisher Scientific). The expected length of successful assembly was around 3700 bp.
Functionality Test
WWe performed a plate-reader experiment, where we grew cells transformed with the optical device in various combinations of erythromycin and arabinose concentrations (Table 1). Non-transformed E. coli, as well as cells transformed with the inducible optical device grown in LB with 100 µl erythromycin were positive controls. A negative control comprised non-transformed TOP10 cells grown in LB with no antibiotic. Optical density and fluorescence were measured every 15 minutes over 15 h. Experiments were performed at 37°C. The GFP intensity was normalized using OD600 measurements. More detailed protocol: Plate-reader experiment for the inducible optical device.
| Arabinose by weight [%] | 0,1µg/ml Erythromycin | 1µg/ml Erythromycin | 10µg/ml Erythromycin | 50µg/ml Erythromycin | 100µg/ml Erythromycin |
|---|---|---|---|---|---|
| 0,005 | x | x | x | x | x |
| 0,01 | x | x | x | x | x |
| 0,05 | x | x | x | x | x |
| 0,1 | x | x | x | x | x |
Improving Sensitivity - MphR Mutants
In order to see if alterations to MphR modelled by our team improve sensitivity of the biosensor, we have used MoClo to construct new optical devices (analogically to the original optical device, see section: Optical device assembly), using 5 different MphR mutants (Table 2). More about our modelling can be found here.
| MphR Mutant | Chosen characteristic |
|---|---|
| MphR common mutations Erythromycin and Clarithromycin | M60A, M94V, V127A, T155I |
| MphR Erythromycin 1 | V67A, V127A, T155A, M156I |
| MphR Erythromycin 2 | V67A, M94V, V127A, T155I, M156I |
| MphR Clarithromycin 1 | M60A, M94V, V127A, T155I |
| MphR Clarithromycin 2 | M60A, Y104Q, V127A, T155Y, M156A |
To initially assess whether the cloning of level 1 assemblies was successful, we performed colony PCR (protocol: Colony PCRColony PCR) with F_kan and R_kan primers from selected colonies that did not express RFP after 48 h incubation and ran the DNA in a 0.7% agarose gel. The bands were visualized with SYBR Safe DNA Gel Stain (ThermoFisher Scientific). The expected length of successful assembly was around 3700 bp.
Functionality Test
We performed a plate-reader experiment, where cells transformed with the inducible optical device comprising a modified MphR sequence were grown in various erythromycin concentrations (0 µg/ml, 1 µg/ml, 10 µg/ml and 100 µg/ml) and 0.1% of arabinose. Non-transformed TOP10 cells, as well as cells transformed with the optical device grown in LB with 50 µg/ml kanamycin were positive controls. A negative control comprised non-transformed TOP10 cells grown in LB with no antibiotic. Optical density and fluorescence were measured every 15 minutes over 15 h. Experiments were performed at 37°C. The GFP intensity was normalized using OD600 measurements. More detailed protocol: Plate-reader experiment for MphR mutants. An analogical experiment was performed with clarithromycin instead of erythromycin.
IMMOBILIZATION TESTS
In order to evaluate what immobilization technique we should use for our biosensor, we performed imaging using confocal microscopy. Immobilization can be achieved with different approaches from which we decided to try two: attachment and entrapment. For attachment, we had two matrices: Poly-D-Lysine and Poly-L-Lysine. For entrapment, we used three different matrices: alginate, egg white and Cultrex. As in our previous viability experiments, we used BL21 cells with and without pET28a-sfGFP (Addgene). Non-transformed BL21 cells were used to assure that fluorescence was due to GFP and not autofluorescence. Conditions were observed with 20X Live Preview to see movement of the cells and pictures were taken from each condition during a 10 minute time-frame for documentation. For each condition, viability dye was added to gain insight on the different matrices. The dye penetrates compromised plasma membranes and binds to nucleic acid with high-affinity, which exposes the less healthy cells.
Poly-D & L-lysine Immobilization
We prepared the polylysine plates one day in advance by pipetting 200 µL of 1 % polylysine solution into a 24-well plate. The plate was left to incubate overnight at room temperature. After this, wells were washed twice with PBS, and 50 µL of cells were added and left to settle for 30 minutes. 0.5 µL of viability dye was added 20 minutes before imaging. More detailed protocol: Immobilization experiments.
Alginate Immobilization
Alginate beads for cell entrapment were prepared using a 2 % sodium alginate solution. After the beads had hardened, they were transferred into a 96-well plate. Viability dye was added 30 minutes before imaging. More detailed protocol: Immobilization experiments.
Egg White & Cultrex Immobilization
A 96-well plate was prepared for cell entrapment experiments with egg white and Cultrex. We tested two different conditions: matrix on bottom and matrix on top. We prepared the plate by adding 10 µl of cells and 20 µl of matrix on top of the cells. For the other condition, only 20 µl of matrix was added to the wells. The plate was dried in 37°C for 30 minutes for the matrices to harden. After, 100 µl of cells were added to the wells with only matrix. Viability dye was added 20 mins before imaging. More detailed protocol: Immobilization experiments.
CONCENTRATION EXPERIMENTS
Isoelectric focusing for Macrolide Concentration
As a proof of concept for concentrating macrolide antibiotics in the sample, we performed an isoelectric focusing (IEF) experiment. The protocol can be found here. Specifications used were: for rehydration with the sample, 500 µl rehydration buffer was added; 17 cm IPT pH 7-10 gel was run at 10000V for a total of 50000 Vh. Two ReadyStrip immobilized pH gradient (IPG) pH7-10 gel strips were rehydrated utilizing sample rehydration with 1 mg of erythromycin and 1mg thymol blue (later replaced with cresol red, since it is only charged in high pH, while thymol blue is charged in the whole pH range of the IPG gel). Thymol blue is coloured and has a near identical pKa to erythromycin, enabling the localization of the invisible erythromycin band on the gel [4]. The rehydrated gels were positioned in the Biorad Protean IEF device and up to 10 000 volts was applied to move the analytes toward the pH of the gel matching their pKa. More detailed protocol: Isoelectric focusing for erythromycin.
Modified E. coli Strain
We performed a plate-reader experiment, where we grow TOP10 and GKCW104 modified E.coli strain cells
transformed with the constitutive optical device in various erythromycin concentrations for 20 h.
GKCW104 has deleted tolC genes, which are proteins forming an outer membrane channel in several
multidrug resistance efflux pumps. Moreover, this strain has open FhuA proteins, which leads to
hyperporination of the cell. GKCW104 strain has to be induced with arabinose for the cells to start
producing the modifications to cell membrane. It is also worth noting, that the strain is
intrinsically resistant to kanamycin [5].
GKCW104 transformed with the constitutive optical device were grown in LB containing 0.1% arabinose
and various erythromycin concentrations (0 µg/ml, 1 µg/ml, 100 µg/ml). TOP10 cells grown in LB with
50 µg/ml kanamycin were a positive control. Negative controls comprised TOP10 cells grown in LB with
no antibiotic and GKCW104 transformed with the constitutive optical device grown with LB, with no
erythromycin and arabinose added. Optical density and fluorescence were measured every 15 minutes
over 15 h. Experiment was performed at 37°C. The GFP intensity was normalized using OD600
measurements. Protocol: Plate-reader experiment with GKCW104.
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