Research Overview
We followed two parallel research lines. The first aimed to integrate our designed construct LoxPort into the genome of Rhodococcus opacus PD630. The second focused on characterizing a set of constitutive promoters in R. opacus to evaluate their strength and suitability for gene expression.
Below, you can find the common protocols we used for both research lines.
Whether you have never held a pipette in your life or you already know these techniques inside out, we encourage you to take a look!
Producing pKSAC45 + the construct
The designed construct, synthesized in a medium-copy plasmid, was introduced into E. coli cells using the standard heat-shock transformation protocol. Transformed cells were plated on selective agar plates containing ampicillin and IPTG. After colony growth, a single colony was picked and cultured.
Plasmid DNA was purified from this culture using a MidiPrep kit from Macherey-Nagel. An additional wash step was performed to reduce salt content and improve the final DNA concentration.
Next, the construct was ligated into the suicide plasmid pKSAC45 using the SphI and EcoRI restriction enzymes. The construct, carrying the required homology arms, enables double-crossover homologous recombination once delivered into R. opacus. The pKSAC45 backbone serves as the non-replicative vector for integration.
Cells were plated on kanamycin-selective agar plates, and plasmids were subsequently isolated using a MiniPrep kit from Macherey-Nagel.
Promoter Characterization Study
A set of constitutive promoters was selected to evaluate their relative expression strengths: p2, pB2, pB3, and pLac (see more in the Design page).
Promoter pLac was amplified by PCR from a vector containing an IPTG-inducible selection system. Promoters p2, pB2, and pB3 were amplified by PCR from the Rhodococcus jostii RHA1 genome; in the case of pB2 and pB3, partial fragments of the native promoter were used. All promoters were cloned with restriction sites for NcoI and BsrGI.
The sfGFP coding sequence was also amplified by PCR, introducing mutations to remove undesired restriction sites (BsrGI and SapI) and adding NcoI and NotI recognition sites. To construct the reporter system, the pTip plasmid was digested, and its original promoter was replaced with one of the selected promoters driving sfGFP expression through restriction cloning.
The recombinant plasmids were first introduced into E. coli via the standard heat-shock transformation protocol. Transformed cells were plated on ampicillin-containing agar plates, and plasmid DNA was subsequently purified using a MiniPrep kit from Macherey-Nagel.
Validated constructs were then electroporated into Rhodococcus and plated on tetracycline-selective agar plates. After colony growth, approximately 100 colonies were picked, re-streaked, and re-cultured to ensure clonal stability. From each promoter construct, four colonies were randomly selected and grown in liquid medium until reaching an OD₆₀₀ of 0.5.
Finally, the fluorescence of these cultures was measured, providing a comparative assessment of promoter strength across the different constitutive elements.
Determination of enzyme kinetic parameters through spectrophotometric analysis
The first step in the construction of a biosensor is the study of the behavior of the enzyme we are working with, which in our case is Porcine Pancreatic Lipase. To design a functional and optimized sensor, it is essential to determine the enzyme’s activity with respect to a standard substrate, pNPP. This is achieved through a lipase assay test as seen in the protocol below.
Once we have an idea of the minimum enzyme concentration required to obtain a significatively different signal from the control, we can consider using a substrate more similar to our target analyte. In this case, the chosen substrate is palmitic acid, which is degraded into fatty acids by lipase. However, since we are the one chosing the concentration of the substrate, we can use this knowledge to assess the accuracy of the second assay.
This is the goal of the next protocol, currently under development, where, unlike in the first experiment, we will prepare solutions with varying substrate concentrations instead of varying enzyme concentrations. This will allow us to calculate the Michaelis-Menten constant and other useful kinetic parameters.
Once this phase is completed, we can move on to the next step in the biosensor workflow: optimizing the method in liquid phase
PROTOCOLS
Each protocol section is expandable and contains three main sections: Description, Quick Protocol, and Complete Protocol PDF.
High-fidelity PCR with Q5 polymerase
Used to amplify DNA fragments with high accuracy, minimizing mutations during amplification.
Quick protocol:
- Prepare reaction mixes in PCR tubes as follows on ice:
- Template DNA quantity is described here:
- Set the thermocycler with the following protocol:
Agarose gel electrophoresis
Separates DNA fragments by size to verify PCR products or digestion results.
Quick protocol:
- Gel casting
- Melt 0.35g in 50 ml TAE buffer, in a 250 ml Erlenmeyer flask.
- Cool down till touchable hot.
- Add 2.5 μl EtBr to reach 0.5μg/ml final concentration.
- Cast the gel in a sealed tray, and place the desired well comb.
- Cool down for 20-30 minutes at RT.
- [optional]: cool down at 4°C for 10 minutes.
- Sample preparation
- Add 6x loading dye to correctly concentrated DNA (for PCR verification, use 2 μl of product and 8 μl of bi-distilled water).
- Load the whole sample mix in a well.
- Load MW marker in an external well.
- Run Conditions
- For PCR or digestion verification: constant 100 mV, roughly 35 min.
- For gel extraction: constant 80 mV, roughly 50 min
NucleoSpin Gel and PCR Clean-Up
Purifies DNA fragments from PCR reactions or agarose gels for downstream applications.
During our experiments, we utilized Macherey-Nagel’s kit. The protocol on the company’s website can be found here.
Ligation
Joins DNA fragments together, typically inserting an amplified construct into a plasmid backbone.
Quick protocol:
- Assemble the reaction on ice following the table
- Incubate for 16h overnight or 10 minutes at room temperature, we assessed that also ligation incubated at 4°C over-weekend performed well.
- OPTIONAL: inactivate enzymes at 65°C for 10 minutes [1].
- Store at -20°C for further use.
Bacterial transformation
Introduces recombinant plasmids into E. coli or other bacteria for propagation and expression.
Quick protocol:
- First incubation
- Defrost an aliquot of competent cells
- Add 400pg to 100ng of plasmid
- Incubate on ice for 30’
- Heat Shock
- Transfer for 2’ at 42°C
- Place back on ice for 2’
- Cell Recovery
- Add 1 ml sterile LB
- Incubate 45’ at 37°C with shaking
- Plating
- [OPTIONAL] Plate 100μl onto selective plates
- Pellet the remaining suspension
- Remove all supernatant but ~100μl
- Resuspend the pellet
- Spread resuspended pellet onto a selective plate
- In a sterile environment:
- In a sterile environment:
Miniprep plasmid extraction
Isolates plasmids amplified in E. coli cells
During our experiments, we utilized Macherey-Nagel’s kit. The protocol on the company’s website can be found here.
PCR with ExTaq polymerase
Performs routine DNA amplification where high fidelity is not essential, e.g., for screening colonies.
Quick protocol:
- Prepare reaction mixes in PCR tubes as follows on ice:
- Template DNA quantity is described here:
- Set the thermocycler with the following protocol:
Agarose plate preparation
Provides a selective growth medium for bacterial cultures, often containing antibiotics.
Quick protocol:
- Melt sterile LB agar inside the sterile bottle
- Cool down to touchable hot
- In a sterile environment, add antibiotic to reach the desired concentration
- In a sterile environment, rapidly pour roughly 15-20 ml of LB agar in each petri dish
- Make plates cool down in a sterile environment with the lid open, to avoid condensation formation
- Once solid, store upside down at 4°C
Primer design
Learn how to create short DNA sequences that define the start and end points of PCR amplification
A-tailing
Adds a single adenine overhang to PCR-amplified DNA fragments, preparing them for cloning into T-overhang vectors.
During our experiments, we utilized NEB’s Klenow fragment (3’->5’ exo-) protocol. The protocol on the company’s website can be found here.
sfGFP fluorescence measurement
Quantifies reporter gene expression by measuring the fluorescence of superfolder GFP in cells or cultures.
Quick protocol:
- Day 1
- Under a sterile hood, plate the colonies to be studied onto selective plates (if needed)
- Grow 2 to 4 days at 30°C with orbital shaking at 150rpm
- Day 2
- Under a sterile hood, pick biomass to fill half of a 10 μl microbiology loop
- Transfer the biomass in 600 μl LB and vortex thoroughly
- Sediment for 2 mins
- Inoculate 300 μl of the supernatant into the pre-inoculum of 10 ml LB (with selection) in a sterile Erlenmeyer flask.
- Grow for roughly 18 hours at 30°C with orbital shaking at 150rpm
- Day 3
- Measure OD600 with a spectrophotometer
- Inoculate a new sterile Erlenmeyer flask with 10 ml LB (with selection) with bacteria from the pre-inoculum to reach OD600 = 0.05.
- Grow at 30°C with orbital shaking at 150rpm till OD600 = 0.5
- Control OD600 with the spectrophotometer
- Once OD600 = 0.5 is reached, measure fluorescence and 600nm absorbance on 200 μl of culture using a plate reader
Electroporation transformation
Introduces plasmid DNA into bacterial cells by applying an electrical pulse that temporarily permeabilizes the cell membrane.
Quick protocol:
- Pre-inoculum
-
In a sterile environment:
- Plate Rhodococcus opacus PD630 on an LB agar plate and wait 2 days to have biomass
- Inoculate in a flask with 20mL of LB + Gly(0.85%) + Sucrose (1%), O/N at 30°C
- Inoculum
-
In a sterile environment:
- Inoculate in the evening the right amount of cells in order to have a starting OD600 equal to 0.02 in a flask with 50mL of LB + Gly(0.85%) + Sucrose(1%)
- The next morning wash 3 times the cells with H2O, resuspend in 2.5mL and split into aliquots of 200 μl
- Electroporation
- Electroporate the cells with set parameters (25 μF, 200 ohm, 2.5 kV)
-
In a sterile environment:
- Add 600 μl of fresh LB to cells and incubate O/N for recovering
- Plating
-
In a sterile environment:
- Centrifuge the colture and leave 300 μl of supernatant, resuspend
- Plate around 150μl onto 2 selective plates
- Incubate for 2-3 days at 30°C
Restriction Enzymes Digestion
Cuts DNA on a specific target sequence, used mainly for cloning and construct verification.
If you don’t know how much DNA you should cut, please refer to the complete PDF file.
Quick protocol:
- Assemble the reaction on ice following the table:
- Incubate for times varying from 2 hours to overnight at 37°C (see optimization section).
- OPTIONAL: inactivate enzymes (enzyme specific process, see documentation from the producer if you intend to do so) [1].
- Store at -20°C for further use.
