Introduction
We want to build a plasmid system to produce a hydrolytic enzyme to degrade plastic. To achieve this goal, we have designed multiple expression elements and techniques. During the component-building process, we tried various approaches, learned from our experiences, learned from our mistakes, and finally achieved some results. This year's exploration and work also provided valuable experience for the follow-up team.
Cycle 1: Constructed by homologous recombination
Several homologous segments were designed to link multiple genes simultaneously, using seamless cloning to construct a fully functional system. Significant difficulties arose while there were some successes during construction, such as the successful replacement of the promoter. Lessons learned, and solutions designed to overcome the present experimental problems led to the successful construction of the expression system.
Design
A multifunctional system can achieve a more convenient effect by enabling a plasmid with multiple devices to perform tasks individually that require multivariate single-functional plasmids. According to this idea, we connect devices with independent functions to form systems with complete functions.
The basis for forming the final system was laid during the synthesis of each device. The promoter sequence was ligated to the expression vector by seamless cloning.
When constructing the whole system, different gene segments were added using different PCR primers, which, in principle, ensured the feasibility of the experiment.
Build
The homologous recombinase construction pathway was selected using the most efficient fusion cloning principle. The primers were designed and operated per the manufacturer's instructions: the gene fragment was amplified by polymerase chain reaction and gel electrophoresis to validate the vector and gel extraction for purification. Finally, the high-purity vector and fragment were ligated with homologous recombinase to construct the system, and the ligated vector was transformed into Escherichia coli for verification. Clonal hosts were inoculated into LB plates containing Kan 100 mg/ml for selection. After the growth of a colony, the colony was selected for PCR validation. If a target band was observed, the bacterial solution was mixed with a 1:1 mixture of glycerol and water, and the seeds were stored at − 80 ° C.
Test
After many attempts, we have made some achievements, and it is gratified that our expression vector was constructed and operated successfully.
Fig.1 DNA electropherograms of the EG promoter and the p7756 promoter. (A) Lane 1 is the marker; Lane 2 is the EG promoter; (B) Lane 1 is the marker, and Lane 2 is the p7756 promoter. the promoter is around 270bp.
Learn
After multiple attempts and comparing successful and failed examples, we determined that the lack of successful results may stem from two factors: 1) the discrepancy in the selected linkage system during seamless cloning and 2) an insufficient amount of single colonies chosen for validation, resulting in an increased incidence of false-positive outcomes. Following the optimisation above, we effectively created and validated the expression vector.
Cycle 2: Promoter strength test.
To choose a more effective promoter for PETase expression and secretion, we linked the fluorescent protein mcherry to each promoter and gauged the mcherry fluorescence intensity using a fluorescence spectrophotometer. This allowed us to identify the most suitable promoter.
Design
Initially, we chose the inducible promoter p7756 and the constitutive promoter EG from Rhodococcus opacus. Then, we designed the regulatory genes upstream and connected the fluorescent protein mcherry downstream. The constructed plasmid was electrotransferred into Rhodococcus opacus. Subsequently, the optimal promoter was determined by conducting carbon gradient experiments under controlled conditions and measuring the fluorescence intensity of mcherry using a fluorescence spectrophotometer. The optimal promoter was identified by designing a carbon gradient experiment alongside varying control conditions.
Build
Initially, we chose the inducible promoter p7756 and the constitutive promoter EG from Rhodococcus opacus. We then designed the regulatory genes upstream and ligated the fluorescent protein mCherry downstream. Next, we amplified the target genes through PCR and seamlessly cloned them before screening the constructed plasmids in an LB medium containing Kan resistance. Finally, we electrotransformed the constructed plasmids into Rhodococcus opacus while designing experiments using a carbon source gradient and various control conditions. The plasmid was electrotransferred into Rhodococcus opacus. The best promoter was identified by conducting a carbon gradient experiment and varying control conditions. The fluorescence intensity of mcherry was measured using a fluorescence spectrophotometer. The most influential promoter was then selected.
Test
Mcherry's fluorescent intensity was detected at an excitation wavelength of 610 nm. The fluorescence intensity was higher when the guaiacol inducer was added. The constitutive promoter, EG, had a higher response than the induced promoter, p7756. The EG promoter remained enhanced under the tested carbon gradient, suggesting that increasing glucose concentration in future experiments is appropriate.
Fig.2 The promoter strength was detected by fluorescence spectrophotometry. the EG promoter with or without the addition of the inducer guaiacol and the p7756 promoter with or without the addition of the inducer guaiacol, and the strength of promoter fluorescence was expressed with increasing glucose concentration for all four sets of conditions.
Learn
After many attempts and comparing the success and failure examples, we concluded that the RM medium containing high sugar is favourable to the corresponding EG promoter, and we designed regulatory genes upstream of the EG promoter. The addition of the inducer guaiacol was able to promote the expression response of the constitutive promoter EG, which inspired us that the expression of PETase in Rhodococcus opacus can be cultivated using the RM medium with high sugar and the addition of guaiacol.
Cycle3: Extracellular expression part
After realising that extracellular expression of specific proteins might not be possible with the existing system, we decided to redesign the plasmid. We decided to redesign the plasmid, remove the signal peptide and try to express it in cells. We amplified the PETase gene sequence by PCR and inserted it behind the EG promoter to construct a plasmid without the signal peptide. After electrotransformation, we extracted the total protein of Rhodococcus opacus, purified it on a nickel column and detected it by SDS-PAGE. This time, we successfully detected the rest of our protein and achieved the synthesis of PETase.
Design
Having established that specific proteins could not be expressed extracellularly by existing systems, we found that the intracellular expression level was much higher than extracellular expression. Intracellular proteins are more stable than extracellular proteins, avoiding the problem of signal peptide mismatches that can lead to undetectable expressed proteins. Although it is challenging to detect proteins in cells, considering various factors, we decided to reconstitute the plasmid, remove the signal peptide and try to express proteins in cells.
Build
We designed a new PCR primer based on the original sequence, ligated PETase into the vector containing the EG promoter and His tag, electrotransferred the correctly validated vector into Rhodococcus opacus, and induced the culture by the pre-gradient experiment.
To ensure the successful construction of the target plasmid, we first transformed the enzyme conjugate product into E.coli for detection and amplification of E. coli. After determining the acquisition of the target plasmid, we also used Rhodococcus opacus for electro-transfection and expression.
Test
To confirm the successful construction of the target plasmid, we first transformed PETase into E.coli and then into Kan-resistant LB plates. Only E. coli successfully transformed with the plasmid were able to grow normally. Colony PCR was used to determine whether or not the transferred plasmid was correct, and further sequence validation was carried out to ensure that the plasmid was successfully constructed without mutation.
The promoter did not need to be induced for this construction, but adding the inducer guaiacol could enhance expression as we added regulatory genes upstream of the promoter during construction. We then centrifuged to collect the precipitate and centrifuged to collect the supernatant after breaking the cells of the precipitate and separated the target protein from a large number and different types of total proteins by applying SDS-PAGE to determine the presence of the target protein.
The target protein was located at approximately 29 kDa, which was in better agreement with the theoretical results, thus successfully confirming the expression of PETase.
Fig.3. Intracellular production of IsPETase. (A) DNA electropherograms of the IsPETase. (B) SDS-PAGE gel of 10-fold concentrated media from secretion systems. M indicates protein molecular weight marker. Lane 1 is the EG promoter and IsPETase. Lane 2 is the p7756 promoter and IsPETase. IsPETase size is indicated by a triangle and label.
Learn
Through intracellular experiments, we identified the problem of matching the signal peptide to the spatial structure of the protein, i.e. even the standard signal peptide on the most popular engineered plasmids could not match the protein to any spatial structure. This part of the experiment rescues our results by expressing enzymes not detectable by extracellular expression and allows the synthesis of mainly degradative enzymes.
Cycle4: Intracellular expression part
We first designed all enzymes and short peptides for extracellular expression to achieve our goal. Therefore, after electrotransformation of the plasmid into Rhodococcus opacus, we tried to detect the target proteins in the supernatant directly. After centrifugation, the supernatant was subjected to SDS-PAGE. However, only part of the target protein was detected after several attempts. After analysis, we suspected the target protein could not be detected because of low and unstable extracellular expression or the spatial structure mismatch between the signal peptide and the protein. Therefore, in the future, we would like to try expression in the cell.
Design
To facilitate the synthesis of hydrolases directly involved in plastic degradation, we initially chose to ectopically express all the enzymes and signal peptides to synthesise hydrolases using the relevant enzymes in the culture medium. Therefore, we added a signal peptide before all target genes to secrete all proteins. Meanwhile, to enhance expression, we chose the constitutive promoter EG to initiate and enhance the expression of downstream genes. To stabilise the target gene in Rhodococcus opacus and form a high copy for efficient expression, we selected a suitable restriction site, linearised the plasmid and integrated it into the yeast genome to ensure it remained stable after delivery. We aim to achieve a high copy by repeated integration. At the same time, we added its marker at the end of the gene for protein purification and identification.
Build
We first amplified the signal peptide and vector backbone by PCR, then ligated the signal peptide and vector by double enzyme cleavage, then ligated the PETase downstream of the signal peptide by seamless cloning, and finally, by validation, the plasmid with the correct validation was transferred into E. coli. The E. coli was expanded to obtain sufficient target plasmids. The purified plasmid was then electrotransferred into Rhodococcus opacus and induced in the RM medium. Finally, the supernatant was centrifuged and collected, concentrated and subjected to SDS-PAGE to validate the secretory expression of the extracellular enzyme.
Test
To confirm the successful construction of the target plasmid, we first transformed PETase into E. coli and then into LB plates containing Kan resistance. Only E. coli successfully transferred into the plasmid were able to grow normally. Colony PCR was used to determine whether or not the transferred plasmid was correct, and further sequence validation was carried out to ensure that the plasmid was successfully constructed without mutation.
The promoter did not need to be induced for this construction, but we added regulatory genes upstream of the promoter during construction to enhance expression by adding the inducer guaiacol. We then collected the supernatant for concentration and used SDS-PAGE to separate the target proteins from a large number and different types of total proteins to determine the presence of the target proteins.
The target protein was located around 29 kDa, and there was no band in the supernatant, whereas there was a band in the supernatant after cell disruption, suggesting that Rhodococcus opacus could successfully express PETase, but the effect of these two signal peptides was less pronounced.
Fig.4 DNA electropherograms of the signal peptide. (A) M is the marker. Lane 1 is the signal peptide 1; Lane 2 is the signal peptide 2; Lane 3 is the signal peptide 3. (B) M is the marker. Lane 1 is the vector framework.
Fig.5. SDS-PAGE gel of 50-fold concentrated media from secretion systems. M indicates protein molecular weight marker. Lane 1 is the signal peptide 1 and PETase. Lane 2 is the signal peptide 2 and PETase. Lane 3 is the empty carrier. Lane 4 is the subcellular signal peptide 1 and PETase. Lane 5 is the subcellular signal peptide 2 and PETase. IsPETase size is indicated by a triangle and label.
Learn
However, after several SDS-PAGE tests, we found that some proteins were still not detected in the bands, but the bacterial liquid PCR was verified to be correct, and the expression was detected in the supernatant of the ruptured cells. Through analysis, there are two reasons: the low extracellular expression of the proteins, which are unstable in the extracellular environment and easily degraded by proteases and other enzymes. The other is that the signal peptide we used is a universal signal peptide, which lacks the specific design for the target protein, and the two are not suitable for each other, so the protein cannot be successfully secreted to the outside of the cell.
