This method can help to produce large amounts of recombinant RNAs of interest for application in research or industry. The main advantage is that recombinant RNA is produced in Escherichia coli in a cheap process that can be easily scaled up. Importantly, the RNA is produced on a circular RNA scaffold, which facilitates purification to homogeneity.
In contrast to DNA or proteins, RNAs of interest are not easily produced in large amounts in biofactory systems, such an Escherichia coli culture. Our method is based on co-expression of the RNA of interest inserted into a highly stable circular scaffold and applying a ligase that mediates RNA circularization. The circular RNA scaffold derives from a patented viroid.
Viroids are relatively small, non-coiling, highly base-paired circular RNAs that are infectious to some higher plants. We can produce tens of milligrams of the recombinant RNA per liter of bacterial culture in regular laboratory conditions. To begin this procedure, amplify the cDNA by PCR as outlined in the text protocol.
Next, add 100 nanograms of the plasmid pLELVd-BZB to a 0.5 milliliter tube. Add 10 U of the type IIS restriction enzyme BpiI and enough of buffer G to create a 20 microliter reaction. Incubate at 37 degrees Celsius for one hour to digest the plasmid.
After this, separate the PCR and digestion products by electrophoresis in a one percent agarose gel in TAE buffer. Stain the gel by shaking it in 200 milliliters ethidium bromide at a concentration of 0.5 micrograms per milliliter. Using a UV transilluminator, visualize the DNA.
Use a scalpel to cut out the bands corresponding to the amplified cDNA and the BpiI digested plasmid. Using silica gel columns, elute the DNAs from the gel fragments. Quantify the concentration of the DNA by spectrophotometric analysis.
Set up a Gibson Assembly reaction using the amplified cDNA and the digested plasmid. Incubate at 50 degrees Celsius for one hour. Then, use a silica gel column to purify the reaction.
After electroporating competent E.coli DH5-Alpha Cells, pick several white colonies and transfer them to liquid LB medium. Grow the colonies overnight at 37 degrees Celsius. Next, use a miniprep kit to purify the plasmids, and analyze their sizes by electrophoresis in a one percent agarose gel in TAE buffer.
First, co-electroporate the selected E.coli strain with both the pLELVd-BZB derivative that contains the cDNA corresponding to the RNA of interest and the plasmid P15LTRNISM to co-express the eggplant tRNA ligase. Transfer SOC liquid medium into the electroporation cuvette to recover the cells, and incubate at 37 degrees Celsius for one hour. Then, plate the bacteria on LB solid medium containing 50 micrograms per milliliter ampicillin, and 34 micrograms per milliliter chloramphenicol.
Incubate at 37 degrees Celsius overnight. The next day, add 250 milliliters of liquid TB medium, containing 50 micrograms per milliliter ampicillin, and 34 micrograms per milliliter chloramphenicol, to a one liter baffled Erlenmeyer flask. Retrieve the incubated E.Coli, and then pick a colony and inoculate the medium in the flask.
Incubate at 37 degrees Celsius with vigorous shaking at 180 RPM for 12 to 16 hours before harvesting the bacteria. Pour the harvested E.Coli culture into a 250 milliliter centrifuge bottle and spin down the cells at 14, 000 G for 10 minutes. Discard the supernatant and resuspend the cells in 30 milliliters of water.
Transfer this suspension to a centrifuge tube, and spin down the cells again using the previous conditions. Discard the supernatant and add 10 milliliters of chromatography buffer to the cell pellet. Vortex to resuspend the cells in the buffer.
Add one volume of phenol:chloroform and vortex vigorously to break the cells. Then, centrifuge at 12, 000 G for 10 minutes. Recover the aqueous phase, add one volume of chloroform, and vortex vigorously.
Centrifuge at 12, 000 G for 10 minutes. After this, filter the RNA preparation through a 45 micrometer syringe filter. Purify the RNA using a one milliliter diethyl ethanol amine column connected to a liquid chromatography system.
Adjust the flow rate to one milliliter per minute, and equilibrate the column with 10 milliliters of chromatography buffer. Then, load the sample and wash the column with 10 milliliters of chromatography buffer. Elute the RNA with 20 milliliters of elution buffer, and collect one milliliter aliquots.
Using two-dimensional polyacrylamide gel electrophoresis, separate the circular RNAs from their linear counterparts. First, prepare a five percent polyacrylamide gel in TBE buffer and containing eight molar urea as outlined in the text protocol. Mix 20 microliters of the RNA preparations with one volume of loading buffer.
Incubate at 95 degrees Celsius in a heating block for 1.5 minutes, and then snap-cool on ice. Load the samples in the polyacrylamide gel and run the electrophoresis at the appropriate conditions for the gel dimension. After this, stain the gel in 0.5 microgram per milliliter ethidium bromide for 15 minutes.
Wash the stained gel with water, and then visualize the RNA under UV light. Electrophoretic analysis of several recombinant plasmids in which different cDNAs are inserted show different migrations when compared to pLELVd-BZB. Note that pLELVd-BZB contains the lacZ marker, which is replaced by the cDNA corresponding to the RNA of interest, so while the migration does depend on the size of the inserted cDNA, the recombinant plasmids will usually migrate faster than the control plasmid.
Production of the recombinant RNA in co-electroporated bacterial cultures is monitored by breaking the cells and analyzing the RNA by denaturing PAGE. Strong bands can be seen, which correspond to empty ELVd and chimeric ELVd forms, in which different RNAs of interest were inserted. Interestingly, a major fraction of the recombinant RNA is seen as a circular form.
The circularity of the main fraction is observed by using a combination of two PAGEs under denaturing conditions at high and low ionic strength. The RNA preparation can be further purified by anion exchange chromatography. As seen here, the E.Coli RNA is efficiently retained at low ionic strength, and subsequently eluted at high ionic strength, with most of the RNA being collected in fractions two and three.
Recombinant RNA can be further purified to homogeneity by 2-D electrophoresis. This protocol allows the easy production of large amounts of recombinant RNA in Escherichia coli and purification to homogeneity thanks to the circularity of the final product. Remember that the RNA of interest results embedded into a circular RNA scaffold derived from a plant viroid.
If you wish to separate both moieties, you need to use a standard strategy, such as ribozymes, DNAzymes, or RNase H.The yield of this protocol depends on the particular RNA of interest, as small RNAs are likely to be produced in higher amounts than larger ones. For larger scale expressions, take into consideration that optimum time to harvest bacteria depends on many factors including the E.Coli strain, culture medium, and growing conditions. We recommend a preliminary time course assay to find the optimal production window in your particular conditions.
Remember that the recombinant RNAs accumulate in bacterial cells transiently, and that they completely disappear at the late-growing phase.
