Rapid Characterization of Genetic Parts with Cell-free Systems

License jove.com August 2021 • 174 • e62816 • Page 1 of 18 Rapid Characterization of Genetic Parts with Cell-free Systems John B. McManus1,2, Casey B. Bernhards3,4, Caitlin E. Sharpes3,4, David C. Garcia2,4, Stephanie D. Cole4, Richard M. Murray2, Peter A. Emanuel4, Matthew W. Lux4 1 US Army Combat Capabilities Development Command Army Research Laboratory 2 Biology and Biological Engineering, California Institute of Technology 3 Excet, Inc. 4 US Army Combat Capabilities Development Command Chemical Biological Center

demonstrated the potential of CFPS for prototyping genetic components 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 whether for direct applications in cell-free systems or to predict the function of genetic constructs in cells, such as the relative activity of parts within a library 29 , metabolic pathway optimization 27 , and cellular burden 30 . Advantages to prototyping in CFPS versus cells highlighted by these studies include avoiding timeconsuming cloning, precise control over the concentration of DNA and other reaction components, and the ability to easily mix and match multiple DNA constructs. The advantage of avoiding cloning is especially apparent when using linear DNA templates, which enables new constructs to be assembled by in vitro methods that take hours instead of days 33 . The ability to manipulate the concentration of DNA constructs and other components simply by pipetting makes the approach even more attractive by enabling high-throughput experimentation powered by liquid handling robots 34 , 35 . While successes using CFPS for prototyping have been reported, it is important to note that it remains to be seen under what contexts CFPS results can reliably predict functionality in cells.
Here, we present a method for CFPS prototyping that emphasizes the advantages in speed, throughput, and cost compared to traditional cell-based approaches. The approach is derived from our previous work where we used CFPS to rapidly characterize a library of T7 promoter variants regulated by the transcription factor TetR 32 , significantly expanding on the small handful of regulated T7 promoter variants that were available in the literature at the time 36 , 37 .
Others have, since then, further expanded the range of such promoters 38 . In our method, genetic construct assembly is accelerated by using PCR to amplify template DNA via primers that add variant genetic parts to a reporter gene. Acoustic liquid handling in 384-well plates is used to increase throughput and decrease the volume of materials required. Previous work has demonstrated successful use of acoustic liquid handling at significantly lower volumes 39 , 40 with variability comparable to manual pipetting of larger volumes 41 . In addition to the method, we provide troubleshooting information and an assessment of potential cost and time savings. Note that while we include a protocol for producing cell-free lysates based on Sun et al. 42 here, numerous other commercial kits and protocols 43 , 44 should also work. Similarly, while we demonstrate the method for the characterization of promoter variants 32      2. Prepare the master mix according to Table 1 and store it on ice. Aliquot 30 or 40 µL (see Table 1) of the master mix into the determined number of PCR tubes and add 10 µL of each variable primer (i.e., primers encoding a part change, see Table 1) at 5 µM to appropriately labeled PCR tubes. 3. Purify the linear template using a commercial PCR purification kit or by the preferred PCR cleanup method.

Linear template preparation
If multiple bands were present by gel electrophoresis analysis, either optimize the PCR conditions or purify the correct molecular weight bands using a commercial gel extraction kit as per the manufacturer's recommendation. 4. Quantify each DNA template using a spectrophotometer.
Assess the DNA template quality by checking that the 260 nm/280 nm ratio is approximately 1.8.

(Optional)
Again separate a portion of the DNA template using a 1% agarose gel at 180 V for 20 min and ensure that any unwanted bands were removed during template purification.
6. Use purified DNA templates immediately or store at -20°C .

Representative Results
To demonstrate the utility of our methods, we present results

Discussion
The protocols described here provide a cost-effective and  Optimal programming of liquid transfers can also influence the accuracy and consistency of data generated; for transfers >1 µL from one source well to one destination well, we have found that sequential transfers of ≤1 µL should be programmed to reduce systematic well-to-well variability ( Figure 4) Beyond characterization of individual parts, the same method can be used to screen combinations of parts that form complex circuits, such as logic circuits 16 or oscillators 52 , 53 . This method can also be applied to screening and optimizing biosensors for applications in epidemiological diagnostics 54 , 55 , 56 , 57 or hazard detection and quantification 3 , 58 , 59 . The application of AI-driven techniques such as active learning 34 can also be paired with the high-throughput nature of this method to drive rapid exploration of complex biological design spaces.
Ultimately, we envision this approach supporting accelerated development times for new genetic designs in synthetic biology.

Disclosures
RMM has a financial stake in Tierra Biosciences, a private company that makes use of cell-free technologies such as those described in this article for protein expression and screening.
The other authors have nothing to disclose.