Editing In vitro translation (section)

=== Discussion ===
<div>
Cell-free protein synthesis has emerged as a powerful and enabling technology for a variety of applications ranging from biomanufacturing to rapid prototyping of biochemical systems. The breadth of applications is supported by the capacity to monitor, manipulate, and augment cellular machinery in real-time. In spite of the expanding impact of this platform technology, broad adaptation has remained slow due to technical nuances in the implementation of the methods. Through this effort, we aim to provide simplicity and clarity for establishing this technology in new labs. Toward this end, our protocol for an ''E. coli''-based cell-free protein synthesis platform can be achieved within a startup time of four-days by laboratory trained non-experts, such as undergraduate students ('''Figure 1'''). Additionally, once a stock of reagents and extract are produced, subsequent CFPS batch reactions can be set up, incubated, and quantified in just 5 h. A single, 1 L cell growth can result in enough extract for four hundred 15 µL CFPS reactions, while single batch preparations of the other cell-free reagents can support thousands of reactions. Reagent preparations can also be scaled up if an even larger stock is needed. The CFPS reactions can be setup in a high-throughput manner, by using a 96-well plate or PCR tubes for testing of a variety of conditions in parallel. Volumetric yields will decrease when using smaller vessels as seen in '''Figure 2C'''. CFPS reactions can also be scaled up from microliters to tens of milliliters of total reaction volume in order to increase the total protein yield for a single condition. When scaling up volume, the primary consideration is that volumetric reaction yields decrease as the surface area-to-volume ratio of the reaction decreases'''<sup>37</sup>'''<sup>,</sup>'''<sup>52</sup>'''. In order to scale-up while maintaining similar volumetric yields of protein expression, users should split the reaction volume into numerous reaction vessels and/or increase vessel size. For reaction scales ranging from 15 µL - 100 µL in volume, numerous 15 µL reactions in parallel are recommended. For reactions exceeding 100 µL in volume, flat-bottom 24-well plates are recommended, and 12-well plates are recommended for reaction volumes exceeding 600 µL. Such pairings of reaction volumes and vessels provide consistency in volumetric reaction yields upon scale-up'''<sup>17</sup>'''<sup>,</sup>'''<sup>31</sup>'''<sup>,</sup>'''<sup>37</sup>'''<sup>,</sup>'''<sup>52</sup>'''. Scaling up beyond these volumes can be accomplished by utilizing multiple wells of the plate in parallel. Using this format, the reaction can be scaled to over 10 mL total volume. Optimizing the reaction volume-reaction vessel combination can support applications of biomanufacturing without sacrificing the productivity of the reaction.

When performing this protocol, there are a few key considerations that impact volumetric reaction yields as well as indicators associated with poorly performing extract. In order to ensure proper lysis and to prevent denaturation of functional transcription/translation machinery, it is important to mitigate the heat produced during lysis. Immerse the cell resuspension in an ice water bath during sonication to rapidly dissipate heat during sonication ('''Figure 3A'''). An indicator of effective cell lysis is the emergence of a darker appearance of the cell lysate compared to pre-sonicated samples ('''Figure 3B'''). For user flexibility, the sonicator and probe shown in '''Figure 3A''' is adaptable to a range of volumes from 100 µL to 15 mL of resuspended cells. To accomplish this, the user can adjust the number of joules delivered for lysis of the desired volume of cells. Additionally, larger volumes of extract can be prepared through two complementary approaches. Users can sonicate multiple tubes in parallel, and/or sonicate larger volumes of cell resuspension, scaling the amount of energy proportionally with the volume as previously described'''<sup>29</sup>'''<sup>,</sup>'''<sup>45</sup>'''. Another step that indicates extract quality is the centrifugation step following cell lysis. Post cell lysis, we recommend centrifugation at 18,000 x g to provide a clear division between the supernatant (transcription/translation machinery, fragmented genomic DNA which no longer functions to template transcription/translation) and the pellet (undesired cellular components such as the cell membrane and precipitated proteins) ('''Figure 3C'''). We have found that centrifugation at 18,000 x g improves separation, resulting in improved reproducibility compared to spins at lower speeds such as 12,000 x g. For convenience, we recommend using a table-top refrigerated centrifuge, capable of achieving a minimum of 12,000 x g. This step is also commonly performed at 30,000 x g, which should be considered if the appropriate equipment is available'''<sup>54</sup>'''<sup>,</sup><sup>55</sup><sup>,</sup><sup>56</sup><sup>,</sup><sup>57</sup><sup>,</sup><sup>58</sup><sup>,</sup><sup>59</sup><sup>,</sup>'''<sup>60</sup>'''. Extract performance is not affected by centrifugation speeds at this step given that proper separation is achieved. When removing the desired supernatant, it is best to avoid any cloudy materials that exist at the boundary between the supernatant and pellet since this contamination will reduce the productivity of the extract. Aiming for purity of the supernatant results in more productive extracts and is worth the reduced quantity of extract obtained for new users.

It is important to note that while the methods we have presented are reproducible and can be executed by scientists with minimal expertise, there can be batch-to-batch and reaction-to-reaction variation. This may be attributed to variation in the proteomic composition of the lysate post-sonication'''<sup>61</sup>'''. The batch-to-batch variability that we have observed is generally diminished upon supplementation with T7RNAP and optimization of magnesium concentrations. Exogenous addition of T7RNAP is common among CFPS reactions to support optimal protein expression, and we find that having two sources of T7RNAP - endogenous expression in BL21*(DE3) and the supplemental T7RNAP to a final concentration of 16 µg/mL - improves batch-to-batch reproducibility for new users'''<sup>45</sup>'''<sup>,</sup>'''<sup>46</sup>'''. With experience, users can modify their experiments to utilize only a single source of T7RNAP if desired. Quantification of total protein content of a new batch of extract and appropriate adjustment of Mg<sup>2+</sup> concentration may also help to diminish batch-to-batch variation in volumetric protein expression yields. Variations in protein expression can also be due to differences in the size and structure of the protein of interest, the codon usage of the gene and its corresponding ribosome binding site of the gene of interest, as well as the type of expression vector used'''<sup>62</sup>'''<sup>,</sup>'''<sup>63</sup>'''. For these reasons, some proteins may not express as well as the model protein sfGFP, resulting in reduced volumetric yields from CFPS reactions.

Limitations of the presented CFPS technique include that it may not be directly suitable to all applications of cell-free, such as metabolic engineering and tuning of expression conditions, without additional modifications to the protocols. However, we believe that this protocol will provide a basis for establishing the CFPS platform in new laboratories and provide non-experts with the ability to implement introductory cell-free reactions in their labs. After initial implementation, researchers can experiment with the platform to make their own modifications for more specific applications based on other literature in the field.

The CFPS platform costs $0.021/µg protein (excluding the cost of labor and equipment), making our system competitively priced with commercial kits without compromising ease of reaction setup. Assessments of comparative costs per µL of reaction show similar trends ('''Supplemental&nbsp;''''''Figure 1'''). We estimate startup costs to be ~$4,500 for all reagents, and an additional $3,200 for specialized equipment, such as a sonicator. Person hours to complete this procedure are estimated to be ~26 h for all reagent prep from the ground up. However, once large stocks of reagents have been prepared, demands on labor diminish substantially. Additionally, as experience with the platform is gained, we recommend scaling up the size of the cell growth, extract preparation, and reagent preparation to maximize time efficiency. Given the startup costs, we recommend the CFPS platform for applications in synthetic biology, high-throughput efforts, and protein expression conditions that are incompatible with traditional protein expression platforms due to conflict with the cell's biochemistry and viability constraints. In these specialized cases where the desired technique is enabled by the CFPS platform, the greater cost of CFPS over ''in vivo ''expression is justified.

Continued development of the CFPS platform is likely to provide broader utility to biotechnology efforts such as the metabolic engineering of enzymatic pathways, production and characterization of traditionally intractable proteins, nonstandard amino acid incorporation and unnatural protein expression, stratified medicine manufacturing, and expanding beyond the laboratory into the classroom for STEM education'''<sup>64</sup>'''<sup>,</sup><sup>65</sup><sup>,</sup>'''<sup>66</sup>'''.&nbsp;These efforts will be further supported by the ongoing efforts for detailed characterization of the CFPS platform. A better understanding of the composition of the cell extract will lead to continued refinement toward improved reaction yields and flexibility in reaction conditions'''<sup>61</sup>'''<sup>,</sup>'''<sup>67</sup>'''<sup>,</sup>'''<sup>68</sup>'''.
</div>
&nbsp;Materials

{|
|-
| '''Name'''
| '''Company'''
| '''Catalog Number'''
| '''Comments'''
|-
| Luria Broth
| ThermoFisher
| 12795027
|-
| Tryptone
| Fisher Bioreagents
| 73049-73-7
|-
| Yeast Extract
| Fisher Bioreagents
| 1/2/8013
|-
| NaCl
| Sigma-Aldrich
| S3014-1KG
|-
| Potassium Phosphate Dibasic
| Sigma-Aldrich
| 60353-250G
|-
| Potassium Phosphate Monobasic
| Sigma-Aldrich
| P9791-500G
|-
| D-Glucose
| Sigma-Aldrich
| G8270-1KG
|-
| KOH
| Sigma-Aldrich
| P5958-500G
|-
| IPTG
| Sigma-Aldrich
| I6758-1G
|-
| Mg(OAc)2
| Sigma-Aldrich
| M5661-250G
|-
| K(OAc)
| Sigma-Aldrich
| P1190-1KG
|-
| Tris(OAc)
| Sigma-Aldrich
| T6066-500G
|-
| DTT
| ThermoFisher
| 15508013
|-
| tRNA
| Sigma-Aldrich
| 10109541001
|-
| Folinic Acid
| Sigma-Aldrich
| F7878-100MG
|-
| NTPs
| ThermoFisher
| R0481
|-
| Oxalic Acid
| Sigma-Aldrich
| P0963-100G
|-
| NAD
| Sigma-Aldrich
| N8535-15VL
|-
| CoA
| Sigma-Aldrich
| C3144-25MG
|-
| PEP
| Sigma-Aldrich
| 860077-250MG
|-
| K(Glu)
| Sigma-Aldrich
| G1501-500G
|-
| NH4(Glu)
| MP Biomedicals
| 02180595.1
|-
| Mg(Glu)2
| Sigma-Aldrich
| 49605-250G
|-
| Spermidine
| Sigma-Aldrich
| S0266-5G
|-
| Putrescine
| Sigma-Aldrich
| D13208-25G
|-
| HEPES
| ThermoFisher
| 11344041
|-
| Molecular Grade Water
| Sigma-Aldrich
| 7732-18-5
|-
| L-Aspartic Acid
| Sigma-Aldrich
| A7219-100G
|-
| L-Valine
| Sigma-Aldrich
| V0500-25G
|-
| L-Tryptophan
| Sigma-Aldrich
| T0254-25G
|-
| L-Phenylalanine
| Sigma-Aldrich
| P2126-100G
|-
| L-Isoleucine
| Sigma-Aldrich
| I2752-25G
|-
| L-Leucine
| Sigma-Aldrich
| L8000-25G
|-
| L-Cysteine
| Sigma-Aldrich
| C7352-25G
|-
| L-Methionine
| Sigma-Aldrich
| M9625-25G
|-
| L-Alanine
| Sigma-Aldrich
| A7627-100G
|-
| L-Arginine
| Sigma-Aldrich
| A8094-25G
|-
| L-Asparagine
| Sigma-Aldrich
| A0884-25G
|-
| Glycine
| Sigma-Aldrich
| G7126-100G
|-
| L-Glutamine
| Sigma-Aldrich
| G3126-250G
|-
| L-Histadine
| Sigma-Aldrich
| H8000-25G
|-
| L-Lysine
| Sigma-Aldrich
| L5501-25G
|-
| L-Proline
| Sigma-Aldrich
| P0380-100G
|-
| L-Serine
| Sigma-Aldrich
| S4500-100G
|-
| L-Threonine
| Sigma-Aldrich
| T8625-25G
|-
| L-Tyrosine
| Sigma-Aldrich
| T3754-100G
|-
| Fisherbrand Premium Microcentrifuge Tubes: 2.0 mL
| Fisher Scientific
| 05-408-138
|-
| Fisherbrand Premium Microcentrifuge Tubes: 1.5 mL
| Fisher Scientific
| 05-408-129
|-
| Fisherbrand Premium Microcentrifuge Tubes: 0.6 mL
| Fisher Scientific
| 05-408-120
|-
| PureLink HiPure Plasmid Prep Kit
| ThermoFisher
| K210007
|-
| Ultrasonic Processor
| QSonica
| 
Q125-230V/50Hz

3.175 mm diameter probe

| <br/>
|-
| Avanti J-E Centrifuge
| Beckman Coulter
| 369001
|-
| JLA-8.1000 Rotor
| Beckman Coulter
| 366754
|-
| 1L Centrifuge Tube
| Beckman Coulter
| A99028
|-
| Tunair 2.5L Baffeled Shake Flask
| Sigma-Aldrich
| Z710822
|-
| Microfuge 20
| Beckman Coulter
| B30134
|-
| New Brunswick Innova 42/42R Incubator
| Eppendorf
| M1335-0000
|-
| Cytation 5
| BioTek
|-
| Strep-Tactin XT Starter Kit
| IBA
| 2-4998-000
|}



pJL1-sfGFP Addgene 69496&nbsp;

BL21(DE3) New England BioLabs