Given the problem with PFAS contaminated water, we strived to eliminate PFOA (one of the most widespread PFAS) from water supplies.
Since we are working on optimizing a set of dehalogenases, we need a system that can allow us to select the most efficient bacteria. Our part BBa_K4868001 is a composite part consisting of two parts:
A fluoride-sensitive riboswitch, associated with a green fluorescent protein.
An insertion site, where the dehalogenase genes can be inserted in a modular way.
When the dehalogenases degrade PFOA and cleave the C-F bonds, fluoride is released. This will activate the riboswitch, resulting in the expression of the green fluorescent protein. The more effective the dehalogenases are at degrading PFOA, the more fluoride will be released, and the more the cells will fluoresce. The principle of the selection system can be seen on figure 1, and the basic mechanism of a riboswitch can be seen on figure 2.
To insert the dehalogenases after the DNA construct has been assembled in a backbone, we use the highly efficient Golden Gate Cloning method. The site where the dehalogenase will be inserted is occupied by a red fluorescent protein gene, which is replaced during the Golden Gate Cloning. Colonies that do not have the dehalogenase gene inserted will fluoresce red and can therefore be easily detected.
Fig. 1: Schematic of the mechanism of our system. IPTG induces expression of DeHa. DeHa will cleave C-F bond in PFOA, which releases free fluoride. The fluoride will active the riboswitch which induces expression of the marker gene, GFP. GFP is then measured to quantify the effectiveness of the given DeHa.
Fig. 2: Schematic of the basic principle of a riboswitch. Notice how mRNA is transcribed without the presence of fluoride. It forms a hairpin structure, blocking translation. Upon addition of fluoride, a conformational change occurs, which opens the hairpin to a looser structure, allowing translation.
Methods Used in the Laboratory
The design we chose shaped the methods we used in the project. Here are short descriptions of what methods we used and why. Detailed descriptions of these methods can be found in the dropdown “Method Details” at the bottom of the page.
Construction of plasmid
Plasmid assembly: To construct a plasmid for transformation, we used Gibson Assembly. Due to problems with synthesizing our insertion piece, we had to have it synthesized in two parts. Gibson Assembly is useful for inserting multiple fragments at once into a backbone.
Insertion of dehalogenases: The dehalogenase genes needed to be inserted into the plasmid after assembly, in an efficient manner. For this, we chose the Golden Gate Cloning technique, where we utilized the PaqCI type IIS restriction enzyme.
Creation of mutants
Mutagenesis: To create a huge library of mutants, as a means of creating better dehalogenases, we opted for untargeted mutagenesis with Error-prone PCR.
Selection of optimized enzymes: After creating an array of mutants, we needed a way to select the most efficient bacteria. Given the correlation between fluorescence and degradation efficiency, we decided to use FACS to sort the most fluorescent cells.
Extraction and evaluation of the dehalogenases
Protein purification: Since we wanted our final product to be purified enzymes, we used the HisCube - Ni-INDIGO His-Tag Protein Purification MINI Kit. The purification was verified with SDS-Page.
Measuring efficiency: It was essential to evaluate the efficiency and kinetics of our purified enzymes, and for this we used F-NMR and a fluoride probe, to measure the release of fluoride during the degradation.
Creating fluoride tolerant E. coli
Initially in our project, we wanted to create a strain of E. coli with an increased tolerance to fluoride. This would be beneficial in the experimental procedures generating more efficient dehalogenases, where the bacteria could be exposed to high concentrations of fluoride from the degradation process. Though we did succeed, we later learned that the fluoride tolerance would not be beneficial in our project, as described on the Engineering page.
To create this tolerant strain, we used Assisted Laboratory Evolution (ALE), where a selection pressure of increasing fluoride concentrations, and a mutagen, are applied to accelerate adaptation towards higher tolerance.
Plasmid construction using Gibson assembly
We used Gibson assembly to insert the two parts of our insertion piece into our plasmid backbone, which, before the assembly, was cut and linearized with the restriction enzymes SgrAI and HindIII.
For the Gibson assembly to be successful, we added 30 base pair overhangs to both sides of both parts of the insertion piece. The added overhangs are identical to the DNA parts where the assembly will be taking place. This way the 5' exonuclease will create single-stranded overhangs that will be complementary and seamlessly assemble our insertion piece into our plasmid backbone.
Fig. 3: Overview of Gibson assembly used for plasmid construction. The single-strand DNA overhangs created by SgrAI and HindIII are not complementary, thus preventing the plasmid backbone from reassembling. The overhangs in the backbone do, however, match those in the insertion piece, allowing the integration of these DNA-sequences into the backbone.
Insertion of the dehalogenases with Golden Gate Cloning
Fig. 4: Principle of Golden Gate cloning. Golden Gate cloning includes, firstly, the cutting and removal of RFP out of the insertion piece, residing in the backbone. Like in Gibson assembly, the cut plasmid is unable to re-assemble. Next, the ligation step occurs, which integrates the DNA-sequence of the dehalogenase of interest into the insertion piece.
Golden Gate Cloning is a method for assembling DNA fragments. The method utilizes the IIS-restriction enzymes, where we specifically used PaqCI. Two or more DNA fragments flanked with compatible recognition sites can then be digested and seamlessly ligated. Because the product will no longer contain the original recognition site, there will be no re-digestion, driving the process towards 100% completion.
Golden gate cloning was chosen since the design would allow the red fluorescent protein to be cut out, and the DeHa enzymes to be inserted in its place. Our Golden Gate insertion site is controlled by the T7 promoter, which means that under the presence of IPTG, the colonies will either turn red or white depending on whether the cloning was successful. In this way, we also ensured that the colonies we selected for further analysis had received the DeHa, since it is not possible for the plasmid to re-ligate itself without DeHa, because of the overhangs we designed1. Figure 4 gives an overview of the RFP-dehalogenase exchange that happens in Golden Gate cloning.
Mutagenesis with Error-prone PCR
Since there were several unknown factors about the enzymes, it would be difficult to make targeted modifications. This is why we are using random mutagenesis instead. Error-prone PCR was chosen to create DeHa mutant libraries.
This approach was chosen because, unlike using in vivo mutation of the DeHa gene to generate diverse isotypes, this approach prevents mutations in the host metabolism and the riboswitch-selection system. This means we can be sure that improved PFOA degradation will be caused by a more efficient dehalogenase and not be a mutation in a different gene. This is important because the enzymes were purified following the selection process and any mutations outside the DeHa sequence would not improve the PFOA degradation in vitro.
The error-prone PCR kit from Jena Bioscience was used, which included PCR components that increase the mutation frequency of the DNA polymerase by 0.6-2.0%2. These PCR components included enhanced concentrations of magnesium ions and unbalanced levels of dNTPs. Using a Taq-polymerase, which does not proofread, increased the error rate.
Performing Error-prone PCR on the DeHa sequences isolated will ensure that there are several different sequences produced which all can be transcribed and translated to different enzymes. A schematic of an error-prone PCR reaction can be seen on figure 5.
Fig. 5: Simplified schematic of an error-prone PCR reaction. As indicated in the figure, an error-prone PCR reaction works the same way as a normal PCR reaction, amplifying the desired DNA-product. Using the specified kit mentioned in the text above, the mutation rate is 1/10,000.
Selection of optimized enzymes with FACS
Fluorescence-activated Cell Sorting (FACS) is a type of flow cytometry, which uses the scattering of light or fluorescence activity to sort cells. An illustration of this principle can be seen in figure 6.
Fig. 6: Schematic overview of fluorescence-activated cell sorting (FACS). Bacteria are sorted by measuring the fluorescence of one single cell at a time. The machine can then sort the cells into different populations, e.g., low-fluorescence cells (orange) and high-fluorescence cells (green).
FACS was chosen as it is a high-throughput way to screen the DeHa libraries, which can easily contain several thousands of different enzyme variants.
After sorting the most fluorescent cells into individual wells on 96-well plates, the cells were incubated to initiate cell division. After 24 hours of incubation, the OD600 and fluorescence were measured which made it possible to select the most fluorescent bacterial lines among the variants. From the selected bacterial lines, the plasmid containing the improved dehalogenase enzyme was purified and submitted for sequencing.
After sequencing results had been assessed to make sure that the sequences differed from the original sequence and each other, the bacterial strains containing the plasmids were cultivated at a larger scale and the proteins were purified from them and tested.
Protein purification with the HisCube - Ni-INDIGO His-Tag Protein Purification MINI Kit and SDS-Page
The resin used in this kit consisted of cross-linked agarose beads and had a protein binding capacity of up to 100 mg/mL3. The used kit has the article no. 80101.
Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-page) is a method used to separate complex mixtures of protein. The first step is the denaturation of the proteins with an anionic detergent, which also binds to the protein, giving them a negative charge proportional to the protein's molecular mass. This is followed by electrophoresis, where the porous acryl gel matrix separates the proteins according to their size. The predicted result of an SDS-page resulting from the purified dehalogenases DeHa 1, 2 4, and 5 can be seen on figure 7.
Fig. 7: Schematic representation of an SDS-page. The illustration represents the expected SDS-page resulting from purified protein samples of DeHa 1, 2, 4, and 5, respectively. The DeHa 1, 2 and 5 are expected to show bands at 25 kDa, whereas DeHa 4 has a size of approximately 35 kDa. The protein ladder used for size reference, also used in the laboratory experiments, is the PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa (Cat. No. 26619).
Measuring efficiency with F-NMR and a fluoride probe
We chose to use F-NMR since fluoride has a spin. It is possible to measure how the PFOA molecules are cut by the enzyme without the enzyme interfering with the measurements (because the enzymes do not contain Fluorine). F-NMR was used to determine if and how fast the enzymes work.
Fig. 8: 19F nuclear magnetic resonance spectroscopy image of PFOA.
Firstly, F-NMR is used to determine whether the enzyme cleaved the F-C and C-C bonds. This can be seen if the peaks in the spectra have shifted or disappeared. Afterward, F-NMR was used to study the kinetics of the enzymes, by taking an F-NMR spectra at regular intervals and comparing the spectra and see how the peaks “move around” in the spectra.
Fluorine-19 nuclear magnetic resonance spectroscopy (F-NMR) is a method typically used for structural analysis of molecules. This method takes advantage of the fact that certain nuclei have a spin. A Spin depends on the isotope of the element. When applying an external magnetic field, the nuclei will align with or against the field. Then a radio frequency is applied that makes the nuclei temporarily shift alignment. When the radio frequency is turned off, the nuclei emit energy which is detected, and results in an interpretable spectrum4. An example of such a spectrum can be seen in figure 8.
One of the final planned experiments of this project was to measure the enzymatic activity of the variant dehalogenases and compare it to the original dehalogenases, using an ion-sensitive electrode. Unfortunately, due to delivery issues, it was not possible to obtain the electrode before The Grand Jamboree. It would have been a valuable tool for determining the enzymatic activity of both the original and variant dehalogenases. An ion-sensitive electrode measures the electric potential of a solution, which results from the presence of ions. By measuring the electric potential of stock solutions with known fluoride concentrations, a reference curve could be created. With this reference curve, the electric potential of the solution can be converted to the concentration of fluoride.
Fig. 9: Illustration of an ion-sensitive electrode.
In essence, the planned experiment involved creating several PFOA solutions corresponding to the number of dehalogenases to be tested. All would have the same PFOA concentration. Next, the desired enzyme would be added to its respective beaker. PFOA degradation using the dehalogenases of interest yields free fluoride ions, which would affect the electric potential of the solution. The electrode would then be used to measure the electric potential of the solutions over time, at set intervals. Using the reference curve, the data points could be converted to the fluoride concentration of the respective solutions at a given time. Thereby, the change in fluoride concentration over time could be calculated, and from this, enzymatic activity would be derived.
In conclusion, an ion-sensitive electrode would allow for the measurement of the enzymatic activity of both the original and variant dehalogenases. This would reveal whether optimization of dehalogenases using error-prone PCR is feasible.
For further information on how this experiment would have been conducted, please refer to SOP 30.
The DNA parts include the pET-51b plasmid and an insertion piece with a fluoride-activated riboswitch which is coupled to GFP. GFP will thereby be the selection tool for the most effective PFOA-degrading enzymes. The insertion piece also includes an insertion site for the dehalogenases. The insertion site has RFP as a placeholder and selection marker. It also contains PaqCI sites that match the PaqCI sites designed on the genes encoding the dehalogenases. This way, each respective dehalogenase can replace RFP, using Golden Gate cloning. This means that RFP can function as a second selection marker for successful Golden Gate cloning. Because the insertion piece is too long to synthesize, it had to be ordered in two separate parts. This made Gibson assembly an obvious choice for plasmid assembly. The final plasmid design has the part number BBa_K4868001. For a visual representation of our insertion piece, please visit our project description.
Gibson Assembly
Gibson assembly is a method that allows two separate pieces of DNA to be ligated and inserted into a plasmid. Therefore, it was used to couple the two segments of the insertion piece to the pET-51b backbone. The backbone had previously been cut with the restriction enzymes HindIII and SgrAI, giving it overhangs, which matched those on the insertion piece.
For more details on Gibson assembly, please see SOP 9.
Transformation into competent cells from Gibson assembly kit
In this step, the product from the Gibson assembly is transformed into NEB(®) 5-alpha Competent E. Coli cells (Cat. No. NEB #C2987), referred to as NEB C2987. This was done with a heat-shock transformation, before being plated onto selection plates with 100 μg/mL ampicillin. The NEB competent cells do not encode ampicillin resistance on their genome, but pET-51b does. Therefore, ampicillin was used as a negative selection marker.
For more details on the used heat-shock protocol, please see SOP 13.
Colony PCR
Colony-PCR is a method used to verify that the plasmid is assembled correctly by using primers that bind to short sequences in the backbone which flank the insertion piece. Therefore, a colony PCR from a successful Gibson assembly will reveal a band with the same length as the insertion piece, when run on a gel, which is roughly 2000 bp. Colony PCR was usually done on 8 colonies per transformation, followed by a gel electrophoresis. Two of the colonies that contained the insertion piece were selected, cultured, and saved in a freeze stock. An overview of a colony-PCR workflow can be seen on figure 9.
For a more detailed description of how colony-PCR was run, please see SOP 15.
Fig. 9: Overview of colony-PCR workflow. From a plate of successful mutants, e.g., 8 colonies are picked up and streak plated. Then, a PCR reaction is set up with genetic material from each colony. A gel-electrophoresis is then run to investigate the size of the genetic material. This reveals whether the bacteria contain the desired plasmid.
Plasmid purification of product from Gibson assembly
In this step, the plasmids from a successful Gibson assembly reaction were purified using the HisCube - Ni-INDIGO His-Tag Protein Purification MINI Kit (Article no. 80101), from Cube Biotech. After purification, the DNA concentration was measured and the purified plasmids were then stored in the freezer (−20℃), ready for Golden Gate cloning.
For more details on how plasmid purification was performed, please see SOP 14.
Golden Gate cloning to replace RFP with a dehalogenase
In this step, the placeholder RFP was replaced with one of the dehalogenases using Golden Gate cloning. One of the advantages of Golden Gate cloning is that the DNA cutting and ligation steps can be combined in a single reaction. The plasmid cannot re-assemble without a dehalogenase gene. This is because the overhangs created by the PaqCI in the backbone are not complementary.
For more details on Golden Gate cloning, including the program used in the PCR-machine, please see SOP 11.
Transformation into NEB C3040I cells
In this step, the Golden Gate assembly is transformed into NEB® Stable Competent E. coli (High Efficiency) (Cat. No. C3040I), referred to as NEB C3040I. 2 µL of the Golden Gate mixture was added to the cells, which were then transformed by following a heat-shock protocol. Then, they were plated out on selection plates with 100 μg/mL ampicillin. NEB C3040I cells do not encode ampicillin resistance on their genome, but pET-51b does. Therefore, ampicillin was used as a negative selection marker.
For more details on the used heat-shock protocol, please see SOP 13.
Colony PCR
Colony-PCR was used to verify whether the plasmid was assembled correctly after Golden Gate cloning. The dehalogenases are approximately the same length as the placeholder RFP. This means that, when amplifying the entire insertion piece, it was nearly impossible to see if the respective dehalogenase had successfully replaced RFP. Therefore, a new set of primers was designed. The forward primer bound to the dehalogenase gene but was unable to bind to RFP. The reverse primer bound to the plasmid backbone. Therefore, the DNA-product was only amplified if RFP had been successfully replaced by a dehalogenase.
Colony PCR was done on a total of 8 colonies per transformation. After gel electrophoresis, two of the colonies that contain the plasmid with the respective dehalogenase are saved as a freeze stock. For an overview of colony-PCR workflow, please see figure 11.
For a more detailed description of how colony-PCR was run, please see SOP 15.
Fig. 11: Overview of colony-PCR workflow. From a plate of successful mutants, e.g., 8 colonies are picked up and streak plated. Then, a PCR reaction is set up with genetic material from each colony. A gel-electrophoresis is then run to investigate the size of the genetic material. This reveals whether the bacteria contain the desired plasmid.
Plasmid purification of product from Golden Gate cloning
Purification of the Golden Gate assembled plasmid was necessary at this time of the project. This is because NEB C3040I lacks a T7 promotor which is necessary for the expression of the dehalogenases and thereby the selection of the most efficient variant dehalogenases. After purification, the plasmid was transformed into the T7 Express Competent E. coli strain (High efficiency) (Cat. No. C2566I)
An important note: This Golden Gate cloning was performed before NEB C2566I was used. NEB C2566I could accept the direct product of Golden Gate cloning.
For more details on how plasmid purification was performed, please see SOP 14.
Transformation of BBa_K4868001 into NEB Express Competent Cells
In this step, the successful Golden Gate product was transformed into NEB C2566I, using a heat-shock protocol. The success of the transformation was tested by being plated out on selection plates with 100 μg/mL ampicillin and 0.4 mM IPTG. Ampicillin was used as a positive selection marker, and IPTG was used as a negative selection marker. This is because, if Golden Gate had failed, the cells would still express RFP. The NEB C2566I cells do not encode ampicillin resistance on their genome, whereas the plasmid does. Therefore, ampicillin was used as a negative selection marker for the transformation. IPTG was used as a negative selection marker for the Golden Gate cloning. This is because NEB C2566I contains a genomic T7 polymerase, which is IPTG-inducible. Had RFP not been replaced by a dehalogenase, cell cultures would therefore gain a red color in IPTG-rich media.
For more details on the heat-shock protocol, please see SOP 13.
Riboswitch tests
With the plasmid successfully transformed into NEB C2566I, the functionality of the riboswitch, and thereby the proposed selection system was tested. The sensitivity of the riboswitch was tested by growing bacterial cultures in varying amounts of NaF. This was to prove that the fluorescence of the cells was correlated to the concentration of fluoride. The fluorescence of the cells was investigated in several ways; please read more about the different methods below, see figure 12.
Fig. 12: Overview of riboswitch-tests performed on NEB C2566I. Firstly, day-cultures of ALE1 containing the plasmid of interest were set up in different NaF-concentrations. These were then investigated using fluorescence miscroscopy, in 96-well plates on a plate-reader and on agar-plates under a blue-light transilluminator.
Fluorescence microscopy to test riboswitch sensitivity in NEB C2566I
In preparation for fluorescence microscopy, day-cultures with varying NaF concentrations were made and induced with 0.4 mM IPTG. The fluorescence of the cells was then analyzed under the microscope. This was useful for both investigating whether GFP-expression increased with the NaF-concentration and if there was any remaining RFP-induction after the Golden Gate cloning.
For more details on the experimental setup for fluorescence microscopy imaging, please visit SOP 17.
Assessing the fluorescence of bacterial cultures using a plate reader
Aside from assessing cell fluorescence under the microscope, quantitative data was needed to confirm the observations from the fluorescence microscope. Thus, a 96-well plate with a range of NaF concentrations was prepared. Both OD600 and fluorescence was measured on a plate reader. The collected data confirmed that the fluorescence of cells containing the plasmid did increase with the NaF-concentration.
Analyzing fluorescence of single bacterial colonies on agar plates
It was tested whether it was possible to see an increase in fluorescence on agar plates with increasing NaF concentrations. The fluorescence was measured with a blue light transilluminator. Although not a quantitative method, this approach might be useful to preselect the most fluorescent colonies when selecting the most improved enzymes.
FACS
Even though each of the methods so far confirmed the activity of the riboswitch, none of them were ideal for the selection of the improved dehalogenases. Using FACS made it possible to collect valuable fluorescence data for a more detailed analysis of the bacterial cultures.
For a more detailed description of how FACS was conducted, please visit SOP 22.
Error-prone PCR of dehalogenase genes
To optimize the dehalogenases, mutations were introduced in the genes encoding the dehalogenases, generating multiple variants. For this purpose, the JBS Error-Prone Kit (Cat. No. JBS PP-102) was used. This kit includes a Taq DNA-polymerase that lacks proofreading abilities, a special dNTP mix, and other components that contribute to a mutagenesis rate of 19%. By running the error-prone PCR-program on the dehalogenase genes before Golden Gate cloning, no mutations were introduced to the plasmid backbone. Therefore, BBa_K4868001 retains its functionality, except for the dehalogenase introduced with Golden Gate cloning. This means that any increase in fluorescence of cells containing a variant dehalogenase comes from an improved enzymatic activity and not from elsewhere.
Golden Gate cloning with variant dehalogenases
In this step, the placeholder RFP is replaced with one of the variant dehalogenases, using Golden Gate cloning. One of the advantages of Golden Gate cloning is that the DNA digestion and ligation steps can be combined in a single reaction. The plasmid cannot re-assemble without a dehalogenase gene. This is because the overhangs created by the HindIII and SgrAI in the backbone are not complementary.
For more details on Golden Gate cloning, including the program used in the PCR machine, please see SOP 11.
Transformation into competent expression strain
The Golden Gate product was transformed into NEB C2566I Express competent E. coli cells, by following a heat-shock protocol. Whether transformation was successful was tested by plating out the bacteria on selection plates with 100 μg/mL ampicillin, 0.4 mM IPTG, and 1.2 mM PFOA. Ampicillin was used as a positive selection marker, and IPTG was used as a negative selection marker. This is because, if Golden Gate had failed, the plasmid would still contain RFP. This means that any bacterial culture grown on IPTG would be red.
From previous experimentation, it was concluded that the PFOA concentration at its maximum solubility, 1.2 mM, is not enough to fully activate the riboswitch. Therefore, there was added 88 µL pr. cm2 of a 1.2 mM PFOA solution in water on top of the dried agar plates. Once dry, an even layer of PFOA was left on top of the plates.
For more details on the used heat-shock protocol, please see SOP 13.
FACS selection
Because all colonies from the transformation were on plates, they were all washed off, resuspended, and diluted, so the FACS machine could measure fluorescence efficiently. The strategy was to sort the 0.1% most fluorescent single cells into wells on a 96-well plate. This was repeated for each dehalogenase. The 96-well plates were then incubated and streaked onto agar plates.
For a more detailed description on how FACS was conducted, please visit SOP 22.
Purification and sequencing of plasmids containing variant dehalogenases
A total of 40 plasmids were purified and sent for sequencing. After receiving sequencing results, both the unmodified and variant dehalogenases were compared, to see which mutations had occurred.
For more details on how plasmid purification was performed, please see SOP 14.
Plate reader selection
The variant dehalogenases were tested, by being plated onto agar plates with 1.2 mM PFOA, followed by overnight incubation at 37℃, allowing the bacteria to grow. Next, they were transferred to 27℃, being the optimal temperature of the enzymes. After tracking the fluorescence for 24 hours, the bacteria were washed off the plates, normalized, and had both OD600 and fluorescence measured. Then, wells that had the highest fluorescence compared to the OD600 were chosen for further experimentation.
Protein purification
This process started with making overnight cultures of bacteria with the desired enzymes, and then large day cultures. Once they had reached the exponential phase, they were induced with 0.4 mM IPTG. Inducing the cells in their exponential phase gives the greatest protein yield. The cells were lysed using a French press and the proteins were then purified using the HisCube - Ni-INDIGO His-Tag Protein Purification MINI Kit (Article No. 80101).
For more details on protein purification using the above-mentioned purification kit, please refer to SOP 25.
Protein purification
We intended to do the following but unfortunately we were unable to before the deadline:
This process started with making overnight cultures of bacteria with the desired enzymes, and then large day cultures. Once they had reached the exponential phase, they were induced with 0.4 mM IPTG. Inducing the cells in their exponential phase gives the greatest protein yield. The cells were lysed using a French press and the proteins were then purified using the HisCube - Ni-INDIGO His-Tag Protein Purification MINI Kit (Article No. 80101).
For more details on protein purification using the above-mentioned purification kit, please refer to SOP 25.
SDS-page
To check the expression of the dehalogenases, an SDS page was performed. Both induced and uninduced samples were compared to confirm the presence of the respective dehalogenases.
For a thorough explanation of how to perform an SDS page using a 16% Tris-Glycine gel, please refer to SOP 21.
SDS-page
We intended to do the following but unfortunately we were unable to before the deadline:
To check the expression of the dehalogenases, an SDS page was performed. Both induced and uninduced samples were compared to confirm the presence of the respective dehalogenases.
For a thorough explanation of how to perform an SDS page using a 16% Tris-Glycine gel, please refer to SOP 21.
F-NMR
F-NMR was another approach that could be used to test the efficiency of the variant dehalogenases. This method also allowed the investigation of all the byproducts of the PFOA degradation. Samples were made so the enzymes had 71 hours, 59 hours, and 47 hours to degrade PFOA. By comparing the spectra from each sample, the efficiency of the enzyme could be determined.
For more details on protein F-NMR, please refer to SOP 29.
F-NMR
We intended to do the following but unfortunately we were unable to before the deadline:
F-NMR was another approach that could be used to test the efficiency of the variant dehalogenases. This method also allowed the investigation of all the byproducts of the PFOA degradation. Samples were made so the enzymes had 71 hours, 59 hours, and 47 hours to degrade PFOA. By comparing the spectra from each sample, the efficiency of the enzyme could be determined.
For more details on protein F-NMR, please refer to SOP 29.
Fluoride probe
ALE1 and ALE2 were developed to have more fluoride tolerance since the fluoride released from PFOA degradation will become toxic upon accumulating. The increased tolerance was achieved by Assisted Laboratory Evolution. The Escherichia coli Rosetta strain was selected for this experiment since it has a T7 promotor, which plays a crucial part in our selection system for improved enzymes. The basis of the system is that when the cells are induced with IPTG, the T7 polymerase will be expressed, which then will allow the expression of the respective dehalogenase encoded in our plasmid.
Fluoride probe
We intended to do the following but unfortunately we were unable to before the deadline:
ALE1 and ALE2 were developed to have more fluoride tolerance since the fluoride released from PFOA degradation will become toxic upon accumulating. The increased tolerance was achieved by Assisted Laboratory Evolution. The Escherichia coli Rosetta strain was selected for this experiment since it has a T7 promotor, which plays a crucial part in our selection system for improved enzymes. The basis of the system is that when the cells are induced with IPTG, the T7 polymerase will be expressed, which then will allow the expression of the respective dehalogenase encoded in our plasmid.
E. Coli Rosetta
ALE1 and ALE2 were developed to have more fluoride tolerance since the fluoride released from PFOA degradation will become toxic upon accumulating. The increased tolerance was achieved by Assisted Laboratory Evolution. The Escherichia coli Rosetta strain was selected for this experiment since it has a T7 promotor, which plays a crucial part in our selection system for improved enzymes. The basis of the system is that when the cells are induced with IPTG, the T7 polymerase will be expressed, which then will allow the expression of the respective dehalogenase encoded in our plasmid.
ALE1
Gradually, the bacteria were exposed to increased NaF concentrations in LB-media. Additionally, they were exposed to UV light for a short time to induce mutations, thereby speeding up the adaption process. An illustration of the experimental workflow can be seen on figure 13.
For further details on how Adaptive Laboratory Evolution was used to increase the fluoride tolerance of E. coli Rosetta in liquid media, please visit SOP 31.
Fig. 13: Illustration of how the ALE1 experiments were set up. The ALE1 strain was developed in liquid media on 96-well plates, with a dilution row of NaF, and then exposed to UV-light to speed up the evolution process.
Transformation of pET-51b containing BBa_K4868001 into ALE1
Both the purified plasmid from Gibson assembly and Golden Gate cloning plasmid were transformed into the ALE1-strain using a heat-shock transformation.
For more details on the heat-shock protocol, please visit SOP 13.
Riboswitch tests of ALE1
With the plasmid successfully transformed into ALE1, the functionality of the riboswitch, and thereby the selection system was tested. The sensitivity of the riboswitch was tested by growing the bacteria in different NaF concentrations. In theory, the activity of the riboswitch, and thereby fluorescence should therefore gradually increase. The activity of the riboswitch was tested in three different ways: with fluorescence microscopy, a plate reader, and with a blue light transilluminator. An overview of the methods can be seen in figure 14.
Fig. 14: Overview of riboswitch-tests performed on ALE1. Firstly, day-cultures of ALE1 containing the plasmid of interest were set up in different NaF-concentrations. These were then investigated using fluorescence miscroscopy, in 96-well plates on a plate-reader and on agar-plates under a blue-light transilluminator.
Microscopy
The experimental procedure was the same as described further up on this page. For comparison, this experiment was also run in NEB C3040I cells and the original Rosetta strain. Surprisingly, ALE1 showed much lower fluorescence intensity than NEB C3040I and Rosetta. It was theorized that the increased fluoride tolerance from ALE1 might come from optimized efflux pumps. Decreased fluorescence meant that it would be unfavorable to use the ALE1-strain to select for improved enzymes later.
For more information on the results from fluorescence microscopy, please visit our Results page.
Assessing the fluorescence of bacterial cultures using a plate reader
Aside from assessing cell fluorescence under the microscope, quantitative data was needed to confirm the observations from the fluorescence microscope. Thus, a 96-well plate with a range of NaF concentrations was prepared. Both OD600 and fluorescence was measured on a plate reader. However, the results were not what we had hoped for, and so, we had to consider what to do next.
For further details on the results from the plate-reader experiment, please visit our Results page.
Analyzing fluorescence of single bacterial colonies on agar plates
It was tested whether it was possible to see an increase in fluorescence on agar plates with increasing NaF concentrations. The fluorescence was measured with a blue light transilluminator. Although not a quantitative method, this approach might be useful to preselect the most fluorescent colonies when selecting the most improved enzymes.
ALE2
This strain was developed, by plating them on agar plates and exposing them to gradually increasing NaF-concentrations over time. It was assumed that this would make the bacteria develop a higher tolerance to fluoride. Additionally, the plates with the bacteria were exposed to UV light for 15 seconds to induce mutations, thus accelerating the evolution process. An illustration of the experimental setup can be seen on figure 15.
Performing the experiment on agar plates gave the advantage of more easily tailoring the NaF-concentration to the tolerance of the bacteria each day, compared to the used dilution row. The disadvantage was that plates had to be cast each day, allowing room for uncertainty of measurement.
For further details on how Adaptive Laboratory Evolution was used to increase the fluoride tolerance of E. coli Rosetta in solid media, please visit SOP 32.
Fig. 15: Illustration of how the ALE2 experiments were set up. The ALE2 strain was developed in solid media on agar plates, with a dilution row of NaF, and then exposed to UV-light to speed up the evolution process.
Transformation of BBa_K4868001 into ALE2
Both the purified plasmid from Gibson assembly and Golden Gate cloning plasmid were transformed into the ALE2-strain using a heat-shock transformation.
For more details on the heat-shock protocol, please visit SOP 13.
Riboswitch tests of ALE2
Since ALE2 was developed separately from ALE1, it was theorized that the riboswitch tests would yield different results. The sensitivity of the riboswitch in ALE2 was tested in the same ways as ALE1; under a fluorescence microscope, with a plate reader, and with a blue light transilluminator.
An overview of the methods can be seen in figure 16.
Fig. 16: Overview of riboswitch-tests performed on ALE2. Firstly, day-cultures of ALE1 containing the plasmid of interest were set up in different NaF-concentrations. These were then investigated using fluorescence miscroscopy, in 96-well plates on a plate-reader and on agar-plates under a blue-light transilluminator.
Microscopy
To test the fluorescence system, we made day-cultures with increasing concentrations of NaF, ranging from 0 to 200 mM NaF. For comparison, we ran the same experiment parallel in NEB stable competent cells. The results of this experiment showed promising results, compared to ALE1. For further details, please visit our Results page. However, the strain could not accept the product of Golden Gate cloning. Therefore, it was concluded that ALE2 was unfit for continued use in our experiments.
For more information on the results from fluorescence microscopy, please visit our Results page.
Assessing the fluorescence of bacterial cultures using a plate reader
Aside from assessing cell fluorescence under the microscope, quantitative data was needed to confirm the observations from the fluorescence microscope. Thus, a 96-well plate with a range of NaF concentrations was prepared. Both OD600 and fluorescence was measured on a plate reader. However, the results were not what we had hoped for, and so, we had to consider what to do next.
For further details on the results from the plate-reader experiment, please visit our Results page.
The Wall
It was official. We hit a wall. The working theory was that the increased fluoride tolerance came from optimized efflux pumps. This meant that both strains could maintain a very low intracellular concentration of fluoride. To verify this proposal, both ALE1 and ALE2 were sent to whole genome sequencing. Whole genome sequencing would also reveal why ALE2, but not ALE1 showed fluorescence. In any case, both ALE strains were unfit for the selection of optimized enzymes. So, the tale of ALE ended here. Not all hope was lost, though! Other strains still responded well to fluoride under the fluorescence microscope.
For further information, please visit our Results page.
Unfortunately, the whole-genome sequencing results of ALE1 and ALE2 arrived just before the wiki-freeze, leaving too little time for analysis. The analysis will be ready for The Grand Jamboree, so stay tuned!
Standard Operating Procedures (SOP)
Results
Engler, C., & Marillonnet, S. (2014). Golden Gate Cloning. In S. Valla & R. Lale (Eds.), DNA Cloning and Assembly Methods (pp. 119-131). Humana Press. https://doi.org/10.1007/978-1-62703-764-8_9
Bioscience, J. JBS Error-Prone Kit - Random Mutagenesis by Error-Prone PCR https://www.jenabioscience.com/molecular-biology/cloning-and-mutagenesis/random-mutagenesis-kits/pp-102-jbs-error-prone-kit
Biotech, C. HisCube Ni-INDIGO - His-tag Protein Purification MINI Kit. https://cube-biotech.com/media/97/e2/2f/1666611404/80101%20INDIGO-Ni%20%20MINI%20Kit.pdf
Southern University of Denmark, NMR-Spektroskopi. https://www.sdu.dk/da/om_sdu/institutter_centre/fysik_kemi_og_farmaci/samarbejde/bestil-analyser/nmr/teori
