1. Designing the gene fragment

NOTE: The gene fragment should have all the necessary genetic elements for transcription/translation, including promoter, ribosome binding site (RBS), start codon, the gene of interest, and terminator. While the terminator is not necessary for a linear expression template (LET), it will be important if the user decides to insert the sequence into a plasmid. These sequences were lifted from the pJL1-sfGFP plasmid⁵⁵ (gift from Michael Jewett's lab), which uses a T7 promoter. In addition to these necessary genetic elements, a restriction enzyme cut site is added six base pairs before the promoter (5' cut site) and another six base pairs after the terminator (3' cut site), in this case using HindIII (other restriction enzymes can be used, but it is helpful to standardize the sequences with one high fidelity restriction enzyme to reduce the number needed to keep in the library). Primer sites are added ten base pairs upstream of the 5' cut site and ten base pairs downstream of the 3' cut site, in this case using standardized M13 primer sequences (primers are inexpensive stock items). The restriction enzyme site and primers used are at the discretion of the user. However, the user must ensure the sequences are not present anywhere else in the template (do not want to create unwanted cuts or sites of amplification initiation). The sequences for the templates used in this work are detailed in the supplemental material. These steps are used to modify from this base template.

1. Determine the desired gene to be expressed and obtain the amino acid sequence or the genetic sequence if it has been expressed in E. coli.
2. If it is an amino acid sequence, perform codon optimization for E. coli using one of many standard vendor tools⁵⁶. If using the template provided in the supplement, ensure the optimized sequence has no HindIII restriction sites (AAGCTT). In the case that it does, continue to optimize the sequence until there is no longer a HindIII site.
3. Copy the sequence and paste it into the provided template for Supplementary Sequence #1 where the gene of interest is indicated. If expressing sfGFP, use Supplementary Sequence #1 as is. If expressing subtilisin, use Supplementary Sequence #2 as is.
4. Order the minimal template and the necessary primers from the preferred DNA synthesis service.

2. Resuspending the gene fragment and the primers

NOTE: Upon receipt of the gene fragment, follow the manufacturer's protocols for resuspension or use this simple guide to create a DNA stock.

1. Centrifuge the tube (300 x g for 5 s) to collect the DNA pellet at the bottom.
2. Add double distilled water (ddH₂O) to make a final concentration of 10 ng/µL of DNA template.
3. Vortex the solution on a medium setting for 5-10 s.
4. Dissolve the entire pellet by incubating at 50 °C for 20 min.
5. Briefly vortex again
6. Centrifuge at 300 x g for 5 s to collect the solution at the bottom of the tube.
7. Store at -20 °C or use in PCR.
8. Prepare a 100 µM primer stock by resuspending the primers in nuclease-free water. To determine the amount of water to add, multiply the number of nanomoles of lyophilized primer by 10. For example, if the tube contains 45 nM of lyophilized primer, add 450 µL of ddH₂O and vortex the solution.
9. Store the primer stock solutions at -20 °C or continue to perform the amplification.

3. Amplifying the gene fragment via PCR

NOTE: Decide which PCR kit is right for the gene of interest. Smaller genes (<1,000 kb) may be more amenable to a cheaper Taq polymerase, while larger genes (≥1,000 kb) may benefit from high fidelity polymerase to reduce errors. It is important to note that this initial PCR amplification is not necessary if the user is not concerned with preserving the initial gene fragment (It provides multiple attempts at circularization and allows for comparative studies of LET vs. RCA product). It is also important to note that this PCR amplified LET can be used directly in reactions; however, as mentioned in the introduction, it would only allow for a limited number of reactions if the further amplification steps were disregarded. Digestion and ligation can be performed on the resuspended gene fragment directly⁵⁷ (if one is certain, they will not need more LET to perform additional circularization stocks). If this is the case, skip section 3 and continue to section 4. For performing PCR, follow these steps.

1. Use the 100 µM stocks from step 2.8 to create 10 µM working solutions. Many PCR kit protocols call for 10 µM solutions of primers.
2. Program the thermal cycler to conduct the reaction according to the kit manufacturer's protocols. Different kits call for slightly varied cycling parameters. For the kit listed in the Table of Materials, the conditions are 94 °C for 30 s of initial denaturation; 30 cycles of 94 °C for 30 s of denaturing, 45 °C for 30 s of primer annealing, and 68 °C for 60 s of extension; with a final extension at 68 °C for 5 min; and finally, a 10 °C indefinite hold.
   1. Ensure to select the correct elongation time (variable depending on the length of the gene to be amplified). Have an elongation time of 1 min for every 1,000 bp.
   2. Ensure to enter the correct annealing temperature for the primers. Use an online Tm calculator that uses both primers as inputs to determine the best annealing temperature⁵⁸. An annealing temperature of 45 °C is sufficient when using M13 primers.
   3. When determining the number of cycles, refer to the manufacturer's protocol, but 30 cycles will most often result in sufficient amplification.
3. If performing PCR, thaw and vortex the dNTPs. Use the PCR buffer provided in the kit.
4. In a single PCR tube, combine all the kit components as directed in the manufacturer's protocol. To ensure successful amplification, add 1 µL of resuspended DNA stock (step 2.6).
5. Gently homogenize the mixture by vortexing on medium setting for 5-10 s. Alternatively, pipette half the volume up and down 10-20 times to vortex.
6. Perform the PCR reaction.
7. If the PCR protocol did not include a final cooling step, allow the reaction to cool for 5 min at 10 °C before removing to drive condensation to the bottom of the tube.
8. Purify the reaction using a PCR clean-up kit following the vendor's instructions.
   1. In a 1.5 mL tube, add DNA binding buffer and PCR sample at a ratio of 5:1, respectively.
   2. Transfer this mixture to the spin column and centrifuge at 16,000 x g for 1 min. Discard the flow-through.
   3. Add 200 µL of DNA wash buffer to the column and incubate at room temperature for 1 min.
   4. Centrifuge for 1 min at 16,000 x g and discard the flow-through.
   5. Repeat steps 2.8.3 and 2.8.4 without the 1 min incubation step.
   6. Centrifuge for an additional 1-2 min at 16,000 x g to remove any remaining buffer.
   7. Elute the DNA in 46 µL of ddH₂O.
9. Quantify the purified DNA using a spectrophotometer.
10. Store the purified DNA at -20 °C or proceed to the next step.

4. Digestion and circularization

NOTE: Further amplification can be achieved by circularizing the DNA followed by RCA. Digest the DNA to prepare the template for circularization. This will remove the primer sequences and create sticky ends at both the 5' and 3' ends of the template. Reattach these ends via ligation reaction.

1. In a PCR tube, combine 5 µL of the necessary buffer, 20 U of HindIII, and 45 µL of the purified DNA from step 3.8.
2. Gently homogenize this mixture with a pipette.
3. Incubate the mixture in a thermal cycler for 15 min at 37 °C.
4. Heat inactivate HindIII by incubating for 20 min at 80 °C.
5. Allow the reaction to cool to 10 °C before removing to drive condensation to the bottom of the tube.
6. Add 5 µL of T4 ligase buffer and 800 U of T4 ligase to the newly digested DNA.
   1. Use T7 ligase, if desired.
7. Gently homogenize this mixture with a pipette.
8. Incubate the mixture for 1 h at 25 °C to perform the circularization reaction.
9. Purify the reaction using a PCR clean-up kit following the vendor's instructions. Use the same protocol detailed in step 3.8.
10. Quantify the DNA using a spectrophotometer. The expected values are ~20 ng/µL.
11. Store at -20 °C or proceed to the next step.

5. Isothermal rolling circle amplification

NOTE: The Rolling circle amplification (RCA) can be performed using a commercial kit or with individually purchased components. Following the manufacturer's protocol will ensure a successful amplification. Kits typically contain a sample buffer, reaction buffer, and strand displacing polymerase, such as φ29 polymerase. Multiple reaction tubes can be combined to produce a large amount of DNA for cell-free expression (4 µg from 20 pg of starting material). The following protocol works efficiently.

1. In a single tube, combine 20 µL of the sample buffer, 20 µL of the reaction buffer, 0.8 µL of the enzyme, and 1 µL of the circular expression template (CET) from step 4.9.
   NOTE: This will have a total DNA mass of ~20 ng, but RCA can work with picogram amounts, thus allowing the dilution of the CET and extreme enzymatic amplification if there is a significant need for material in the high throughput screening.
2. Homogenize the mixture with a pipette and aliquot 10 µL of the mixture into four separate tubes.
3. Incubate at 30 °C for 4-18 h.
4. Heat inactivate the enzyme by incubating at 65 °C for 10 min. Reduce the temperature to 12 °C for 5 min to encourage condensation at the bottom of the tube.
   NOTE: It's easiest to combine all temperature steps in an automated protocol on a thermal cycler.
5. Dilute the resulting solution by adding 15 µL of ddH₂O to each tube.
6. Combine all tubes and add directly to a cell-free reaction.
7. If desired, use a PCR clean-up kit to purify the product and elute it in 36 µL of ddH₂O to quantify. Ensure that the template concentration is ~100 ng/µL.

6. Cell-free reaction

NOTE: Perform cell-free expression by combining energy buffer, extract, and RCA template. A typical cell-free reaction using the PANOx-SP energy buffer consists of 1.2 mM ATP, 0.85 mM each of GMP, UMP, and CMP, 30 mM phosphoenolpyruvate, 130 mM potassium glutamate, 10 mM ammonium glutamate, 12 mM magnesium glutamate, 1.5 mM spermidine, 1 mM putrescine, 34 µg/mL of folinic acid, 171 µg/mL of E. coli tRNA mixture, 2 mM each of 20 unlabeled amino acids, 0.33 mM NAD, 0.27 mM Coenzyme A (CoA), 4 mM potassium oxalate, 57 mM HEPES-KOH buffer (pH 7.5), 0.24% volume of the E. coli extract, and variable amounts of DNA^23,49. The volume of reaction can vary but 15 µL reactions can save on reagent usage and are small enough for use in a 384 black-walled microplate^49,50.

1. If expressing a fluorescent protein such as sfGFP, prepare a plate reader to read at the desired excitation/emission, temperature, and agitation.
2. If using a 384-well plate, aliquot 60 µL of H₂O into the wells bordering an empty sample well to maintain humidity and reduce the edge effect.
3. Add the various required components into a tube for each sample. Add enough to perform triplicates. Replicates within the plate can help identify causes of variability.
   1. Add the extract, the energy buffer and then the DNA.
   2. Dilute to the final desired volume with ddH₂O.
4. Mix this solution thoroughly by pipetting half of the solution volume up and down 10-20 times.
5. Transfer the reaction mixture in 15 µL aliquots to the desired wells in the microtiter plate.
6. Seal the plate with a colorless sealing film to maintain humidity and prevent evaporation.
7. Place the sealed plate in the plate reader and allow the reaction to complete.
   1. If expressing a protein that does not have the capability of being monitored live, use another temperature-controlled apparatus such as a thermoblock to incubate the plate.

7. Subtilisin assay

NOTE: If expressing the subtilisin BPN' (SBT(n)) gene in Supplementary Sequence #2, follow this protocol to assay the activity.

1. Prepare a 10 µM stock solution of N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide in dimethylformamide (DMF).
2. Set a plate reader to measure absorbance at 410 nm every 20 s for 10 min while maintaining a temperature of 25 °C.
3. In a flat bottom, colorless 96-well plate, aliquot 94 µL of ddH₂O and 1 µL of N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide from step 7.1.
4. Add 5 µL of the finished cell-free reaction from step 6.7 and read using a plate reader set to the protocol described in step 7.2.