Editing In vitro translation (section)

=== Introduction ===
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Cell-free Protein Synthesis (CFPS) has emerged as a technology that has unlocked a number of new opportunities for protein production, functional genomics, metabolic engineering, and more within the last 50 years'''<sup>1</sup>'''<sup>,</sup>'''<sup>2</sup>'''. Compared to standard ''in vivo'' protein expression platforms, CFPS provides three key advantages: 1) the cell-free nature of the platform enables the production of proteins that would be potentially toxic or foreign to the cell'''<sup>3</sup>'''<sup>,</sup><sup>4</sup><sup>,</sup><sup>5</sup><sup>,</sup>'''<sup>6</sup>'''; 2) inactivation of genomic DNA and the introduction of a template DNA encoding the gene(s) of interest channel all of the systemic energy within the reaction to the production of the protein(s) of interest; and 3) the open nature of the platform enables the user to modify and monitor the reaction conditions and composition in real time'''<sup>7</sup>'''<sup>,</sup>'''<sup>8</sup>'''. This direct access to the reaction supports the augmentation of biological systems with expanded chemistries and redox conditions for the production of novel proteins and the tuning of metabolic processes'''<sup>2</sup>'''<sup>,</sup>'''<sup>9</sup>'''<sup>,</sup>'''<sup>10</sup>'''. Direct access also allows the user to combine the CFPS reaction with activity assays in a single-pot system for more rapid design-build-test cycles'''<sup>11</sup>'''. The capacity to perform the CFPS reaction in small volume droplets or on paper-based devices further supports high-throughput discovery efforts and rapid prototyping'''<sup>12</sup>'''<sup>,</sup><sup>13</sup><sup>,</sup><sup>14</sup><sup>,</sup><sup>15</sup><sup>,</sup>'''<sup>16</sup>'''. As a result of these advantages and the plug and play nature of the system, CFPS has uniquely enabled a variety of biotechnology applications such as the production of proteins that are difficult to solubly express ''in vivo'''''<sup>17</sup><sup>,</sup>'''<sup>18</sup><sup>,</sup><sup>19</sup><sup>,</sup>'''<sup>20</sup>''', detection of disease'''<sup>21</sup>'''<sup>,</sup><sup>22</sup><sup>,</sup>'''<sup>23</sup>''', on demand biomanufacturing'''<sup>18</sup>'''<sup>,</sup>'''<sup>24</sup>'''<sup>,</sup><sup>25</sup><sup>,</sup><sup>26</sup><sup>,</sup>'''<sup>27</sup>''', and education'''<sup>28</sup>'''<sup>,</sup>'''<sup>29</sup>''', all of which show the flexibility and utility of the cell-free platform.

CFPS systems can be generated from a variety of crude lysates from both prokaryotic and eukaryotic cell lines. This allows for diverse options in the system of choice, each of which have advantages and disadvantages depending on the application of interest. CFPS systems also vary greatly in preparation time, cost, and productivity. The most commonly utilized cell extracts are produced from wheat germ, rabbit reticulocyte, insect cells, and ''Escherichia coli ''cells, with the latter being the most cost-effective to date while producing the highest volumetric yields of protein'''<sup>30</sup>'''. While other CFPS systems can be advantageous for their innate post-translational modification machinery, emerging applications using the ''E. coli''-based machinery are able to bridge the gap by generating site-specifically phosphorylated and glycosylated proteins on demand'''<sup>31</sup>'''<sup>,</sup><sup>32</sup><sup>,</sup><sup>33</sup><sup>,</sup><sup>34</sup><sup>,</sup>'''<sup>35</sup>'''.

CFPS reactions can be run in either batch, continuous-exchange cell-free (CECF) or continuous-flow cell-free (CFCF) formats. The batch format is a closed system whose reaction lifetime is limited due to diminishing quantities of reactants and the accumulation of inhibitory byproducts of the reaction. CECF and CFCF methods increase the lifetime of the reaction, and thereby result in increased volumetric protein yields compared to the batch reaction. This is accomplished by allowing the byproducts of protein synthesis to be removed from the reaction vessel while new reactants are supplied throughout the course of the reaction'''<sup>2</sup>'''. In the case of CFCF, the protein of interest can also be removed from the reaction chamber, while in CECF, the protein of interest remains in the reaction chamber comprised of a semi-permeable membrane'''<sup>36</sup>'''<sup>,</sup>'''<sup>37</sup>'''. These methods are especially valuable in overcoming poor volumetric yields of difficult-to-express proteins of interest'''<sup>38</sup>'''<sup>,</sup><sup>39</sup><sup>,</sup><sup>40</sup><sup>,</sup><sup>41</sup><sup>,</sup><sup>42</sup><sup>,</sup>'''<sup>43</sup>'''. The challenges in implementing the CECF and CFCF approaches are that 1) while they result in more efficient use of the bio machinery responsible for transcription and translation, they require notably larger quantities of reagents that increases overall cost and 2) they require more complex reaction setups and specialized equipment compared to the batch format'''<sup>44</sup>'''. In order to ensure accessibility for new users, the protocols described herein focus on the batch format at reaction volumes of 15 µL with specific recommendations for increasing the reaction volume to the milliliter scale.

The methods presented herein enable non-experts with basic laboratory skills (such as undergraduate students) to implement cell growth, extract preparation, and batch format reaction setup for an ''E. coli''-based CFPS system. This approach is cost-effective compared to commercially available kits without sacrificing the ease of kit-based reaction setup. Furthermore, this approach enables applications in the laboratory and in the field. When deciding to implement CFPS, new users should thoroughly evaluate the efficacy of conventional protein expression systems for startup investment, as CFPS may not be superior in every case. The CFPS methods described here enable the user to directly implement a variety of applications, including functional genomics, high-throughput testing, the production of proteins that are intractable for ''in vivo ''expression, as well as field applications including biosensors and educational kits for synthetic biology. Additional applications such as metabolic engineering, tuning of protein expression conditions, disease detection, and scale-up using CECF or CFCF methods are still possible but may require experience with the CFPS platform for further modification of reaction conditions. Our methods combine growth in enriched media and baffled flasks, with relatively rapid and reproducible methods of cell lysis through sonication, followed by a simplified CFPS reaction setup that utilizes optimized premixes'''<sup>45</sup>'''. While the cellular growth methods have become somewhat standardized within this field, methods for cell lysis vary widely. In addition to sonication, common lysis methods include utilization of a French press, a homogenizer, bead beaters, or lysozyme and other biochemical and physical disruption methods'''<sup>46</sup>'''<sup>,</sup><sup>47</sup><sup>,</sup><sup>48</sup><sup>,</sup>'''<sup>49</sup>'''. Using our methods, approximately 2 mL of crude cell extract are obtained per 1 L of cells. This quantity of cell extract can support four hundred 15 µL CFPS reactions, each producing ~900 µg/mL of reporter sfGFP protein from the template plasmid pJL1-sfGFP. This method costs $0.021/µg of sfGFP produced ($.019/µL of reaction), excluding the cost of labor and equipment ('''Supplemental Figure 1'''). Starting from the scratch, this method can be implemented in 4 days by a single person and repeat CFPS reactions can be completed within hours ('''Figure 1'''). Additionally, the protocol can be scaled up in volume for larger batches of reagent preparation to suit the user's needs.Importantly, the protocol presented here can be implemented by laboratory trained non-experts such as undergraduate students, as it only requires basic laboratory skills. The procedures described below, and the accompanying video have been specifically developed to improve accessibility of the ''E. coli'' CFPS platform for broad usage.
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