Editing In vitro translation (section)

=== Protocol ===
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1. Media Preparation and Cell Growth

#'''Day 1'''
##Streak ''E. coli ''BL21*(DE3) cells from a glycerol stock onto an LB agar plate and incubate for at least 18 h at 37 °C.
##Prepare 50 mL of LB media and autoclave the solution on a liquid cycle for 30 min at 121 °C. Store at room temperature.
#'''Day 2'''

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*Prepare 750 mL of 2x YTP media and 250 mL of 0.4 M D-Glucose solution as described in the supplemental information.
*Pour the 2x YTP media into an autoclaved 2.5 L baffled flask and the D-Glucose solution into an autoclaved 500 mL glass bottle. Autoclave both solutions on a liquid cycle for 30 min at 121 °C.
*Ensure that both sterile solutions are stored at 37 °C if cell growth is being performed on the next day, to maximize growth rates upon inoculation. Do not combine solutions until inoculation.<br/>NOTE: Solutions can be stored at 4 °C for 1-2 d if needed, though the 2x YTP media is highly prone to contamination.
*Start an overnight culture of BL21(DE3) by inoculating 50 mL of LB media with a single colony of BL21(DE3) using a sterilized loop and sterile technique to avoid contamination.
*Place the 50 mL of BL21*(DE3) LB culture into a 37 °C 250 rpm shaking incubator and grow overnight for 15-18 h.
*Prepare and sterilize all materials required for days 3 and 4, including: two 1 L centrifuge bottles, 4x cold 50 mL conical tubes (weigh and record masses of three), and many 1.5 mL microfuge tubes.
*'''Day 3'''
*#Remove the 50 mL overnight culture of BL21*(DE3) in LB from the shaking incubator and measure the OD<sub>600</sub> on a spectrophotometer using a 1:10 dilution with LB media. Calculate the volume of overnight culture necessary to add to 1 L of media for a starting OD<sub>600</sub> of 0.1 (For example, if an OD<sub>600</sub> of a 1:10 dilution is read as 0.4, inoculate 25 mL of the undiluted OD<sub>600 </sub>= 4.0 overnight culture into 1 L of 2x YTPG).
*#Remove the warmed 2x YTP media and D-Glucose solutions from the 37 °C incubator along with the 50 mL of LB culture. Using sterile technique, carefully pour the D-Glucose solution into the 2x YTP media (avoiding the sides of the baffled flask).<br/>NOTE: Addition of D-Glucose completes the recipe for 1 L of 2x YTPG.
*#Maintaining sterile technique, inoculate the 1 L of 2x YTPG solution with the appropriate amount of the 50 mL culture to begin the 1 L culture at a 0.1 OD<sub>600</sub>. Immediately place the inoculated 1 L culture into a 37 °C shaking incubator at 200 rpm.
*#Take the first OD<sub>600 </sub>reading after the first hour of growth (lag phase typical takes 1 h). Do not dilute the culture. Continue taking OD<sub>600</sub> measurements approximately every 20-30 min until OD<sub>600</sub> reaches 0.6.
*#Upon reaching OD<sub>600</sub> = 0.6, add 1 mL of 1M IPTG (final concentration in 1 L culture = 1 mM) to the 2x YTPG culture.<br/>NOTE: Ideal induction OD<sub>600</sub> is 0.6; however, a range of 0.6-0.8 is acceptable. Induction by IPTG is for endogenous production of T7 RNA Polymerase (T7RNAP).
*#After induction, measure the OD<sub>600</sub> approximately every 20-30 min until it reaches 3.0.<br/>NOTE: Cool down the centrifuge to 4 °C during this time. Prepare cold S30 buffer as detailed in the Supplementary Information. If the S30 buffer is prepared in advance, ensure that DTT is not added until the day of use.
*#Once the OD<sub>600</sub> reaches 3.0 ('''Figure 2A'''), pour the culture into a cold 1 L centrifuge bottle in an ice-water bath. Prepare a water-filled 1 L centrifuge bottle of equal weight to be used as a balance in the centrifuge.<br/>NOTE: Absorbance values vary from instrument-to-instrument. While the OD<sub>600</sub> of harvest of BL21(DE3) is not a sensitive variable, it is recommended that the user evaluate and optimize this variable as a troubleshooting measure. Larger spectrophotometers may result in relatively lower OD<sub>600 </sub>readings compared to smaller cuvette-based spectrophotometers.
*#Centrifuge the 1 L bottles for 10 min at 5,000 x g and 10 °C to pellet cells.
*#Slowly pour off the supernatant and dispose of it according to the institution's biological waste procedures. Place the pellet on ice.
*#Using a sterile spatula, scrape the cell pellet from the centrifuge bottle and transfer it to a cold 50 mL conical tube.
*#Add 30 mL of cold S30 buffer to the conical tube and resuspend the cell pellet by vortexing with short bursts (20 - 30 s) and rest periods (1 min) on ice until fully resuspended with no chunks.
*#Once the pellet is fully resuspended, use another 50 mL conical tube with water as a balance and centrifuge for 10 min at 5000 x g and 10 °C (pre-cooled to 4 °C).<br/>NOTE: This completes the 1<sup>st</sup> of 3 washes required when harvesting the cells.
*#Pour out the supernatant and dispose of it according to the institution's biological waste procedures. Resuspend the pellet with 20-25 mL of cold S30 buffer and centrifuge for 10 min at 5000 x g and 10 °C (pre-cooled to 4 °C).<br/>NOTE: This completes the 2<sup>nd</sup> of 3 washes.
*#Again, pour out the supernatant and dispose of it according to the institution's biological waste procedures. Add exactly 30 mL of S30 buffer and vortex again to resuspend the pellet.
*#Using the 3 pre-weighed, cold 50 mL conical tubes and a serological pipette filler with a sterile pipette, aliquot 10 mL of resuspended pellet/S30 buffer mixture into each of the 3 conical tubes.<br/>NOTE: Splitting the cells into 3 tubes is not required, but this step results in smaller cell pellets (~ 1 g) for increased convenience at later steps.
*#Centrifuge all tubes, using appropriate balances as needed, for 10 min at 5000 x g and 10 °C (pre-cooled to 4 °C).<br/>NOTE: This completes the final wash step.
*#Pour out the supernatant and dispose of it according to the institution's biological waste procedures. Remove the excess S30 buffer by carefully wiping the inside of the conical tube and cap with a clean tissue; avoid touching the pellet.
*#Reweigh the tubes on an analytical balance and record the final pellet weight on each tube.<br/>NOTE: The protocol can be paused at this point. The pellets can be flash frozen in liquid nitrogen and stored at -80 °C for up to a year until needed for extract preparation.

2. Crude Cell Extract Preparation - Day 4

#For extract preparation, keep cells cold on ice during each step. Add 1 mL of cold S30 buffer per 1 g of cell mass of the pellet. Ensure that dithiothreitol (DTT) has been supplemented to the S30 buffer to a final concentration of 2 mM.<br/>NOTE: Cool down the microcentrifuge to 4 °C during this time.
#Resuspend the cell pellet by vortexing with short bursts (20 - 30 s) and rest periods (1 min) on ice until fully resuspended. If resuspension is difficult, leave the pellets on ice for 30 min to defrost.
#Transfer 1.4 mL of resuspended cells into a 1.5 mL microfuge tube.
#Place one 1.5 mL tube containing 1.4 mL of resuspended cells into an ice water bath in a beaker. Sonicate for 45 s on followed by 59 s off for 3 total cycles, with amplitude set at 50%. Close and invert the tubes to gently mix during the off periods. In total, deliver 800-900 J of energy to each 1.5 mL microfuge tube containing 1.4 mL of resuspended cells ('''Figure 3A''' '''& 3B''').<br/>NOTE: This step is sensitive to the sonicator type and model used and should be optimized if equipment is different than listed for this procedure. Two complementary approaches can be used to scale-up the amount of extract prepared during this step: 1) multiple 1.5 mL microfuge tubes can be sonicated in parallel, and/or 2) larger volumes can be sonicated in conical tubes (up to 15 mL of cell resuspension per tube), scaling the amount of energy delivered as previously described <sup>29</sup><sup>,</sup><sup>45</sup>.
#Immediately after sonication is complete, add 4.5&nbsp;µL of 1 M DTT (supplementing an additional 2 mM DTT) into the 1.4 mL of lysate and invert several times to mix. Place the tube on ice. Repeat steps 2.4 and 2.5 for any additional tubes of resuspended cells before proceeding to centrifugation.
#Microcentrifuge samples at 18,000 x g and 4 °C for 10 min ('''Figure 3C''').
#Pipette the supernatant into a new 1.5 mL microfuge tube. Do not disturb the pellet; it is preferable to leave some supernatant behind to maintain purity than to disrupt the pellet in efforts to maximize yield.

*Incubate the supernatant from the previous step at 250 rpm and 37 °C for 60 min by taping the tubes to the shaking platform of the incubator (this is the runoff reaction).
*Microcentrifuge samples at 10,000 x g and 4 °C for 10 min.
*Remove the supernatant without disturbing the pellet and transfer it to a new tube. Create many 100 µL aliquots of extract for storage.<br/>NOTE: The protocol can be paused here, and the extract can be flash frozen in liquid nitrogen and stored at -80 °C for up to a year until needed for CFPS reactions. At least 5 freeze-thaw cycles can be undergone without detriment to extract productivity ('''Figure 4''').

3. Cell-Free Protein Synthesis Batch Format Reactions

#Thaw Solutions A and B, DNA template, BL21(DE3) extract (if frozen), T7RNAP, and an aliquot of molecular grade water.<br/>NOTE: CFPS reaction template can be found in the '''Supplementary Information'''. Solutions A and B recipes are provided in the '''Supplementary Information''' and correspond to specific concentrations for numerous reagents to support the PANOx-SP based energy system for CFPS. The role of each reagent and acceptable variation in these reagent concentrations that can support CFPS have been determined<sup>50</sup>. A T7RNAP purification protocol can be found in the Supplementary Information<sup>51</sup>. Supplemental T7RNAP can increase volumetric yields but is not necessary if T7RNAP is induced during cell growth. Plasmid DNA template (pJL1-sfGFP) can be prepared using a maxiprep kit with two washes using the wash buffer in the kit, followed by a post-processing DNA-cleanup using a PCR purification kit ('''Figure 2B'''). Linear DNA templates can also be used in CFPS reactions.
#Label the necessary amount of microfuge tubes needed for CFPS reactions.<br/>NOTE: Reactions can be performed in various vessel sizes, but a smaller vessel can decrease volumetric protein yields ('''Figure 2C'''). Scaling up a reaction in the same size vessel may also reduce volumetric yields, as a function of decreasing the oxygen exchange, due to a decrease in the surface area to volume ratio. When increasing reaction volume above 100 μL, it is recommended to use flat bottom well plates <sup>31</sup><sup>,</sup><sup>37</sup><sup>,</sup><sup>52</sup>.
#Add 2.2 µL of Solution A, 2.1 µL of Solution B, 5 µL of BL21*(DE3) extract, 0.24 μg of T7RNAP (16 μg/mL final concentration), 0.24 ng of DNA template (16 ng/mL final concentration), and water to bring the final volume to 15 µL.<br/>NOTE: Vortex Solutions A and B frequently during reaction setup to avoid sedimentation of components and ensure that each reaction receives a homogenous aliquot of each solution. Avoid vortexing the extract, instead invert the tube to mix.
#After all reagents have been added to the reaction, mix each tube by pipetting up and down or gently vortexing while ensuring that the final reaction mixture is combined into a single 15 µL bead at the bottom of the 1.5 mL microfuge tube.
#Place each reaction into the 37 °C incubator without shaking for 4 h, or 30 °C overnight.<br/>NOTE: Successful reactions can be qualitatively assessed visually based on the green color of the sfGFP product within the CFPS reaction mixture ('''Figure 3D'''). Expression of the protein of interest can also be confirmed by SDS-PAGE ('''Supplemental Figure 2''').

4. Quantification of the Reporter Protein, [sfGFP]

#Load 48 µL of 0.05 M HEPES, pH 8, into each well needed for quantification (usually performed in triplicate per reaction tube).
#Remove reactions from incubator. Pipette up and down to mix each reaction, then transfer 2 µL of reaction into the 48 µL of 0.05 M HEPES, pH 8. Pipet up and down again in the well to mix.
#Once all reactions are loaded and mixed, place the 96 well plate into the fluorometer and measure the sfGFP endpoint fluorescence.<br/>NOTE: Excitation and emission wavelengths for sfGFP fluorescence quantification are 485 nm and 510 nm, respectively.
#Using a previously generated standard curve, determine the [sfGFP] from the obtained fluorescence readings.<br/>NOTE: Instructions for generating a standard curve of sfGFP concentration versus fluorescence intensity are provided in Supplementary Information ('''Supplementary Figure 3'''). Users will need to establish a standard curve for their instrument since instrument sensitivity may vary.