Editing In vitro translation (section)

=== Representative Results ===
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We have presented a sonication-based ''E. coli ''extract preparation protocol that can be completed over a four-day span, with '''Figure 1''' demonstrating the procedural breakdown over each day. There is malleability to the steps that can be completed in each day with various pausing points, but we have found this workflow to be the most effective to execute. Additionally, both the cell pellets (step 1.3.18) and fully prepared extract (step 2.10) are stable at -80 °C for at least a year, allowing the user to create larger stocks of each to save for use at a later time'''<sup>17</sup>'''. Not only is the extract stable over long time periods, but the extract can also undergo at least five freeze thaw cycles without a significant loss of productivity ('''Figure 4'''). This allows for larger aliquots of extract to be stored for multiple uses if freezer storage space is limited. However, we recommend multiple smaller aliquots (~100 μL) of extract whenever possible.

With every new extract preparation, we recommend that the user performs a magnesium titration in order to determine the optimal amount of magnesium for that batch of extract. Users can quantify batch-to-batch variability in total protein concentration of the cell extract by Bradford assay. For higher performing extracts, we typically see total protein concentrations of 30-50 mg/mL, and within this range there is no obvious correlation between total protein concentrations and cell extract performance. Therefore, we recommend that users tune magnesium concentrations accordingly to ensure that protein and nucleic acid functionality are maximized for each extract batch. Magnesium levels are important for proper DNA replication, transcription and translation, but excessive levels can be detrimental to these processes'''<sup>53</sup>'''. In order to demonstrate this dependency, we have performed a co-titration of magnesium and extract volume to determine the optimal combination that minimizes the amount of extract necessary, while maintaining a productive reaction ('''Figure 5'''). From this experiment, we recommend using 5 µL of extract and 10 mM Mg<sup>2+</sup> for extract with a total protein content of 30 mg/mL, in order to obtain over 1,000 μg/mL of sfGFP.

Our experience with CFPS has also allowed us to determine steps within the protocol that can be varied without detriment to the overall productivity of the system, and others that are integral for a high performing CFPS system. Most notably, the final OD<sub>600</sub> of cell harvest does not significantly affect the final output of the CFPS reaction, and cells can feasibly be harvested anywhere from 2.7 - 4.0 OD<sub>600</sub>. This represents the early exponential phase of growth where ribosome concentration per cell is the highest and the translational machinery is the most active to support rapid growth. This observation allows users flexibility to optimize their own procedures. We recommend harvesting at approximately 3.0 OD<sub>600</sub> in order to capture the cells at an OD<sub>600</sub> closer to 3.3 by the time harvesting is complete ('''Figure 2A'''). Variables that do impact CFPS yields include template DNA quality, reaction vessel size, and the relative quantities of cell extract and magnesium ion present in the reaction. We have found DNA quality to have notable batch-to-batch variation. In order to resolve this, we recommend that users purify DNA via a midi or maxi prep, followed by an additional DNA cleanup step either on the DNA purification column used in the maxiprep, or post-purification using an additional DNA cleanup kit. This improves the reproducibility in DNA quality for CFPS reactions and results in more robust protein production ('''Figure 2B'''). The reaction vessel also impacts volumetric yields, such that the protein production of identical reaction setups in varying vessel volumes can differ up to 40%. It has been theorized that the boost in in volumetric yield observed in larger vessels is due to an increased surface area of the reaction mixture, allowing for better oxygen exchange ('''Figure 2C'''), and others have further boosted volumetric yields by running CFPS reactions in large flat-bottom plates, which we recommend for reactions over 100 µL'''<sup>17</sup>'''<sup>,</sup>'''<sup>31</sup>'''<sup>,</sup>'''<sup>37</sup>'''<sup>,</sup>'''<sup>52</sup>'''.

http://cloudflare2.jove.com/files/ftp_upload/58882/58882fig1.jpg<br/>'''Figure 1'''''': Timeline for culture growth, production of cell extract, setup and quantification of CFPS reactions. '''The user can implement the CFPS platform for their research applications through this four-day workflow. Reagent preparation represents the primary time and cost investment for the first round of this experiment and diminishes substantially after reagents stocks are established. Additionally, cell pellets and prepared cell extract can be stored for over a year at -80 °C, allowing the user to begin the timeline at various steps for faster results. The user can also pause at various steps to modify the timeline of this workflow.&nbsp;

http://cloudflare2.jove.com/files/ftp_upload/58882/58882fig2.jpg<br/>'''Figure 2'''''':'''&nbsp;'''Modifiable conditions for CFPS and the effects on volumetric reaction yields.''''''A.''' Extract productivity comparison based upon harvesting BL21(DE3) cells at various OD<sub>600</sub> readings. Based on this plot, we recommend harvesting at an OD<sub>600</sub> of 3.3 to produce at least 1000 µg/mL of target protein. Reactions were performed at a 15 μL scale in 1.5 mL microfuge tubes. '''B.''' Comparison of two DNA maxiprep wash protocols with and without post-purification DNA-cleanups. pJL1-sfGFP plasmids underwent a maxiprep with one or two washes followed by a post-purification cleanup by PCR purification kit. To achieve ~900 µg/mL of protein expression, we suggest performing a post-purification DNA cleanup regardless of the number of maxiprep washes. Reactions were performed at a 15 μL scale in 1.5 mL microfuge tubes. '''C.''' 15 µL CFPS reactions performed in various vessels ranging from 2 mL to 0.6 mL microfuge tubes. "Neg" represents a negative control where no DNA template was added to the reaction. All error bars represent 1 standard deviation of three independent reactions for each condition, each of which was quantified in triplicate.&nbsp;&nbsp;https://www.jove.com/files/ftp_upload/58882/58882fig2large.jpg&nbsp;

http://cloudflare2.jove.com/files/ftp_upload/58882/58882fig3.jpg<br/>'''Figure 3'''''':'''&nbsp;'''Key procedural setups and outcomes for creating productive extract. A. '''Proper setup of sonication ice water bath to ensure cooling of sample while heat is generated during sonication. '''B. '''1.5 mL microfuge tube containing resuspended cell pellet pre (left) and post (right) sonication. The resulting lysate should display a darker hue compared to resuspended cell pellet. '''C.''' Proper separation of the supernatant and pellet of cell lysate after 18,000 x g centrifugation. '''D. '''CFPS reactions after 4 h of incubation at 37 °C. 1.5 mL microfuge tube on the right (successful reaction) shows visible fluorescence of the sfGFP reporter protein at ~900 μg/mL. The negative control tube on the left, lacking template DNA and simulating an unsuccessful reaction, displays a clear solution with no fluorescence.

http://cloudflare2.jove.com/files/ftp_upload/58882/58882fig4.jpg<br/>'''Figure 4'''''': Change in protein expression over 5 freeze-thaw cycles for CFPS extract.''' Extract prepared from the same growth underwent five freeze thaw cycles via liquid nitrogen flash freezing followed by thawing on ice. No significant changes in extract productivity for expressing sfGFP were seen over the five freeze-thaw cycles. Reactions were performed at a 15 μL scale in 1.5 mL microfuge tubes. "Neg" represents a negative control where no DNA template was added to the reaction. All error bars represent 1 standard deviation of three independent reactions for each condition, each of which was quantified in triplicate. 



http://cloudflare2.jove.com/files/ftp_upload/58882/58882fig5.jpg<br/>'''Figure 5'''''': CFPS for reactions with varying [Mg'''<sup>'''2''''''+'''</sup>'''] and extract volumes versus [sfGFP]'''. [Mg<sup>2+</sup>] ranged from 8 mM to 14 mM with 2 mM increments and extract volumes ranged from 3 µL to 7 µL with 1 µL increments. The color code represents [sfGFP] produced from high (red) to low (purple). To maximize reagent efficiency while maintaining high protein production, we recommend using 5 µL of extract and 10 mM Mg<sup>'''2'''+ </sup>for extracts that have a total protein content of ~30 mg/mL, as determined by Bradford assay. Original points to generate the contour plot were based off endpoint fluorescence of three independent reactions for each condition, each of which was measured in triplicate. Reactions were performed at a 15 μL scale in 1.5 mL microfuge tubes.&nbsp;

'''Supplementary '''Figure 1''':&nbsp;''''''Cost per microgram of protein produced and per microliter of reaction across six cell-free protein synthesis platforms.''' Our platform is compared to five different cell free protein synthesis kits/platforms with varying productivity and pricing. Our sonication based CFPS platform is more cost-effective in both $/µg of protein and $/µL of reaction than most commercial kits and provides the ease of a kit for reaction setup, while remaining cost comparable to other academic CFPS platforms. https://www.jove.com/files/ftp_upload/58882/supplemental_figure_1.jpg

'''Supplementary '''Figure 2''':&nbsp;''''''SDS-PAGE of sfGFP expression in CFPS.''' Cell-free protein synthesis reactions with (+ DNA) and without (- DNA) DNA template for sfGFP were run on a 12% SDSPAGE gel to demonstrate the expression of sfGFP observed at 27 kDa (black arrow). Traditional SDS-PAGE techniques were used. Each sample loaded onto the gel included 18 µg of total protein based on Bradford assay quantification of total protein in the cell extract. Based on fluorescence intensity measurements and our standard curve, we estimate that the "+ DNA" lane contains 0.42 µg of sfGFP. In order to obtain these samples, CFPS reactions were run at a 15 µL scale in 1.5 mL microfuge tubes producing volumetric yields consistent with '''Figure 3C'''. https://www.jove.com/files/ftp_upload/58882/supplemental_figure_2.jpg

'''Supplementary '''Figure 3''': Standard curve for sfGFP on Cytation 5.''' This curve was determined using the methods outlined above. All data collected for this manuscript was converted from endpoint fluorescence readings to [sfGFP] in µg/mL using this standard curve. https://www.jove.com/files/ftp_upload/58882/supplemental_figure_3.jpg
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