The overall goal of this experiment is to describe how bacteriophages are synthesized from their genome using an all E.coli cell-free transcription translation system. This method can help answer key questions in the synthetic biology field, such as elucidating the molecular self-assembly mechanisms in living systems. The main advantage of this technique is the versatility of the platform.

Many biological systems can be fully recapitulated and finely tuned for experimental analysis. Begin this procedure with preparation of phage plaques as described in the text protocol. Use the end of a sterile Pasteur pipette to core and remove a single plaque.

Blow the plaque into the diluted cells. Then incubate at 37 degrees Celsius for two hours. To test for full infection, take a 500 microliter sample into a 1.5 milliliter tube and add 10 microliters of chloroform.

Immediately vortex the mixture at high speed and observe if the solution clarifies, which indicates phage infection. Once the cells are fully infected, incubate for another two hours. Next, add DNase before centrifuging the cells at 8, 000 times g and four degrees Celsius.

Discard the supernatant, then re-suspend the pellet in 10 milliliters of 1X Tris magnesium chloride. Add 500 microliters of chloroform and vortex at high speed to lyse the cells. Clarify by centrifugation at 12, 000 times g for 10 minutes at four degrees Celsius.

Then decamp the supernatant into a 15 milliliter conical tube and store at four degrees Celsius. Prepare four five to 45%sucrose gradients by pipetting 2.5 milliliters of 45%sucrose into four ultracentrifuge tubes. Top the layer with approximately 2.6 milliliters of 5%sucrose, stopping when the liquid reaches two millimeters from the rim of the tube.

Mix the gradients by tilt tube rotation using a gradient forming instrument for 43 seconds at 86 degrees and 23 RPM. Using a one milliliter pipette, add 500 microliters of the pooled phage suspension by bringing the tip into contact with the liquid surface and very slowly dispensing the volume onto the top of the sucrose gradient, being careful to not mix through rapid pipetting. Centrifuge at 70, 000 times g for 20 minutes at four degrees Celsius.

Submerge a blunt canula into the solution until the tip is centered on the very upper edge of the phage band. Remove the band by drawing on the syringe plunger until the majority of the phage band has been removed. Then add the sucrose volume with the suspended phage to three or four ultracentrifuge tubes.

Fill the tubes to between three and four millimeters from the top of the tube with cold 1XTM. Cover the tubes with paraffin film and invert to mix. Place the tubes back in an ultracentrifuge and spend at 145, 000 times g for one hour at four degrees Celsius.

Quickly pour off the supernatant and drain upside down on disposable wipes. Divide 200 to 400 microliters of cold 1XTM across all pellets and re-suspend overnight at four degrees Celsius. No shaking is required.

The day before making titers, prepare a culture of E.coli host cells in 10 milliliters of LB growth media and leave the culture in a shaking incubator set to 250 RPM and 37 degrees Celsius overnight. Next, prepare a master mix by pipetting 5.25 milliliters of top agar, 220 microliters of diluted phage samples, and 50 microliters of host bacterial cells to a 14 milliliter culture tube. Cap the culture tube and vortex at high speed.

Retrieve two culture plates from the 37 degree Celsius incubator. Then add 2.5 milliliters of master mix slowly to the center of the first plate. Gently rotate the plate by hand to distribute the master mix evenly so that it spans the entire culture plate.

Wait 20 minutes to ensure solidification of the top agar. Incubate the plates at 37 degrees Celsius for four to seven hours. Count the plaques and determine the phage concentration for each reaction sample.

Dilute a portion of the phage stock to 400 microliters with 1XTM buffer in a fresh 1.5 milliliter tube. Add an equal volume of Tris phenol chloroform to the dilution. Shake the mixture gently on a laboratory rocker or by hand for five minutes.

Then centrifuge the resultant emulsion at 17, 000 times g in a benchtop centrifuge for five to 10 minutes. Remove the upper aqueous phase to a new tube, taking care not to disturb the interphase boundary. Repeat these steps an additional two times for a total of three phenol extractions.

Transfer the aqueous phase to a fresh tube and add an equal volume of chloroform. Shake and centrifuge before transferring the aqueous DNA sample to a clean tube. Next, add 0.4 volumes of three molar sodium acetate and three volumes of 95%ethanol.

Place the tube in a minus 20 degree Celsius freezer for overnight precipitation. Centrifuge at 17, 000 times g in a benchtop centrifuge for 10 to 15 minutes. Remove and discard the supernatant, either by decanting or pipetting.

Then add 500 microliters of 70%ethanol and gently shake the tube until the pellet floats free from the bottom of the tube. Following centrifugation, remove and discard the ethanol, taking care to not disturb the pellet. After adding ethanol and centrifuging as before, remove and discard the ethanol a second time, once again taking care not to disturb the pellet.

Air dry the pellet on the benchtop for 30 to 60 minutes. Once dry, add 50 microliters of double-distilled water to the pellet and incubate for one hour at room temperature or overnight at four degrees Celsius to re-suspend. Determine the DNA concentration using absorption measurements at 280 nanometers.

To perform the cell-free reaction, aliquot the indicated volume of crude extract, 33%of the final reaction volume, into a microcentrifuge tube. Prepare the master mix as listed in the text protocol. Add the appropriate volume of each component to the crude extract.

After adding the last component, homogenize the reaction by vortexing. Then split the master mix into n microcentrifuge tubes. Next, add the indicated volumes of the variable components to the master mix array.

Add water to each reaction to reach the desired final reaction volume before vortexing. Then incubate the microcentrifuge tubes at 29 degrees Celsius for at least eight hours or overnight. After preparing the cells for phage titer as described in the text protocol, titer the final cell-free reaction LB solution as demonstrated earlier in this video.

Shown here are representative results of a cell-free reaction synthesizing phi X 174 phage. This phage plaque shows a successful phage reaction that was titered at an appropriate dilution, evident by the countable number of plaques. However, a successful reaction can show up poorly if the dilution factor is too low for the phage yield of the reaction.

This plate shows an uncountable number of plaques. Shown here is a plate with inhomogeneous distribution of phage plaques due to insufficient pre-equilibration of culture plate temperature. During the phage titer, the culture plate solidified the reaction in the lower right portion of the plate.

Molecular crowding conditions have a strong effect on phage synthesis for phi X 174 and T7.In particular, phage yield for T7 drops by three orders of magnitude when PEG concentrations drop from four to 2%weight by volume. Once mastered, phage purification and DNA extraction can be done in two days if it is performed properly. The cell-free reaction takes just hours when there are no issues.

After watching this video, you should have a good understanding of how to grow phage, extract and purify their DNA, and use the result to reconstitute fully infectious phage using the cell-free reaction system. Working with phenol can be extremely hazardous, and precautions such as lab coat, eye protection, and gloves should be worn while performing this procedure. All work with open containers of phenol should be performed in a fume hood.