Our protocol provides a simplified workflow for Streptomyces venezuelae cell-free transcription-translation. Our system harnesses the metabolic enzymes and pathways from this non-model organism. The main advantages are the heterologous expression of genes with high GC content, the provision of a favorable protein forwarding environment, and an open system for fine-tuning of biosynthetic pathways.
Our method will open new opportunities into studying Streptomyces and related high GC bacteria. It allows to study enzymes and gene expression in these bacteria. This technique has been developed for ease of implementation by new users.
The only step that requires prior experience or familiarization is sonication to produce highly active crude extracts. To begin harvesting the pre-cultured cells on the third day, dilute the overnight culture at the ratio of one-to-10 with a fresh GYN medium for the optical density or OD measurement at 600 nanometers. If the OD of the culture at 600 nanometers is less than 0.3, increase the shaking speed to 250 to 300 rotations per minute and grow until an OD reaches 0.3.
Grow for no longer than an additional two hours. If OD is greater than 0.3, transfer the cultures to centrifugation containers and rapidly cool on wet ice for 30 minutes. While waiting for the cell culture to cool, prepare four milliliters of fresh one molar dithiothreitol, S30A and S30B buffers and keep them on ice.
After weighing an empty 50 milliliter centrifuge tube, pre-chill it at minus 20 degrees Celsius. Add two milliliters of one molar dithiothreitol to one liter of S30A buffer on ice and mix well. Centrifuge the cells at 6, 000 times G for 10 minutes at four degrees Celsius.
After discarding the supernatant in a quick motion, add 250 milliliters of S30A buffer and resuspend the cells by shaking the centrifugation bottles vigorously until the cell clumps are homogeneously dispersed. Then centrifuge the cells again for six minutes. After centrifugation, discard the supernatant and repeat the addition of S30A buffer, followed by centrifugation as demonstrated.
Next, add two milliliters of one molar DTT to one liter of S30B buffer on ice and mix well. Add 250 milliliters of S30B buffer to the cells and repeat centrifugation. Resuspend the cell pellet in 10 milliliters of S30B buffer and transfer the cell suspension to the pre-weighed pre-chilled 50 milliliter centrifuge tube.
If required, transfer the residual cells with an additional five to 10 milliliters of S30B buffer and fill the tube to 50 milliliters volume with S30B. Centrifuge the cells for 10 minutes. Discard the supernatant and repeat the centrifugation at previous settings as demonstrated.
After discarding the supernatant, aspirate the remaining S30B supernatant with a 100 to 200 microliter pipette and weigh the wet cell pellet. For every one gram of wet cells, add 0.9 milliliters of S30B buffer. Resuspend the cells using either a Pasteur pipette or vortex.
Centrifuge briefly for about 10 seconds up to 500 G to sediment the cells. Lower the sonicator tip into the cell suspension until the tip is about one centimeter below the liquid surface. Input different parameters in the sonicator settings.
Set frequency to 20 kilohertz, amplitude to 65%pulse on and off time to 10 seconds, and total sonication time to one minute. Begin sonication and move the tube up or down and sideways during first two resting cycles to ensure the cells are evenly sonicated. If the cells are not fully lysed, invert the tube two to three times and repeat the sonication for an additional one or two 10-second cycles with frequent mixing until the cells are fully disrupted.
Centrifuge the lysed cells to remove the cell debris, then transfer the supernatant into a 1.5 milliliter microcentrifuge tube as one milliliter aliquot. For the runoff reaction of the cell extracts, incubate the cell containing the cell extract at 30 degrees Celsius for 60 minutes on a heat block or in the incubator without a shaker. For the transcription-translation reaction, thaw the cell extract SMM or MES solution and plasmid DNA on ice.
Pre-chill a 384-well plate at minus 20 degrees Celsius. Set up the transcription-translation reaction on the ice where 25%of the volume is plasmid DNA, 33.33%is cell extract and 41.67%is SMM solution. Gently vortex the mixture for about five seconds at a low speed setting to ensure the solution is homogenous while avoiding foaming or bubble formation.
Transfer three aliquots of 10 microliters into three wells of a 384-well plate as a technical triplicate without introducing air bubbles. After sealing the plate with a transparent cover, spin the plate at 400 times G for five seconds and incubate the reaction at 28 degrees Celsius either in an incubator or a plate reader without shaking. Streptomyces venezuelae cell-free transcription-translation was optimized for high yield expression of fluorescent proteins from the pTU1-A-SP44 standard plasmid, as well as biosynthetic enzymes detected by gel electrophoresis.
The expression of the pTU1-A-SP44-mScarlet-I standard plasmid was measured using several methods. The real-time fluorescence measurements of mRNA using the dBroccoli RNA aptamer, immature mScarlet-I protein using the FlAsH tag system, and mature mScarlet-I protein were carried out. The in-gel fluorescence staining of mScarlet-I using FlAsH tag was conducted in addition to the Coomassie Blue staining of the total cell-free protein.
Following this procedure, activity assays can be performed to study enzymes and pathways that are challenging using standard methods. It also extends towards scaled up biosynthesis and high throughput gene expression. We are developing this technique to study metabolic enzymes from high GC bacteria.
We have shown the potential of our system to study scaled up biosynthesis in cell-free reactions.
