Rapid, Enzymatic Methods for Amplification of Minimal, Linear Templates for Protein Prototyping using Cell-Free Systems

Summary

The study describes a protocol for creating large (µg-mg) quantities of DNA for protein screening campaigns from synthetic gene fragments without cloning or using living cells. The minimal template is enzymatically digested and circularized and then amplified using isothermal rolling circle amplification. Cell-free expression reactions could be performed with the unpurified product.

Abstract

This protocol describes the design of a minimal DNA template and the steps for enzymatic amplification, enabling rapid prototyping of assayable proteins in less than 24 h using cell-free expression. After receiving DNA from a vendor, the gene fragment is PCR-amplified, cut, circularized, and cryo-banked. A small amount of the banked DNA is then diluted and amplified significantly (up to 10⁶x) using isothermal rolling circle amplification (RCA). RCA can yield microgram quantities of the minimal expression template from picogram levels of starting material (mg levels if all starting synthetic fragment is used). In this work, a starting amount of 20 pg resulted in 4 µg of the final product. The resulting RCA product (concatemer of the minimal template) can be added directly to a cell-free reaction with no purification steps. Due to this method being entirely PCR-based, it may enable future high-throughput screening efforts when coupled with automated liquid handling systems.

Introduction

Cell-free gene expression (CFE) has emerged as a powerful tool with many applications. Such applications include disease detection^1,2,3,4,5,6, micronutrient and small molecule detection^{7,8,9,10,11,12}, biomanufacturing^{13,14,15,16,17,18}, education^19,20,21, manufacturing difficult proteins^{17,22,23,24,25,26,27}, and variant screening^{23,28,29,30,31,32,33}. This is due to the open nature of cell-free systems and the flexibility they confer. Many great review articles offer historical education and future perspectives on the technology^{34,35,36,37,38,39,40,41,42,43,44}.

A typical cell-free reaction consists of three major components: cell extract, energy mix, and genetic template. Active cell extract contains all the necessary machinery for transcription and translation (TXTL) and can be processed in a variety of ways³⁶. Glycolytic intermediates, electrolytes, amino acids, and cofactors in the energy mix support the TXTL process. It is a major source of variability in cell-free experiments⁴⁵ and can be prepared in many ways^34,46. Preparation of the genetic template has seen fewer improvements since traditional cloning methods result in plasmids with excellent expression characteristics. The downside to these traditional methods is the turnaround time and amount of biological expertise needed to construct and propagate them. Recent optimization efforts have resulted in simple 24-hour workflows for cell extract preparation^47,48 that can be performed in parallel with energy mix preparation^49,50. However, traditional cloning adds multiple days to the CFE prototyping timeline (Table 1)²³. Quickly amplified PCR products from the commercial gene fragment can be used directly⁵¹, but this limits the number of prototyping experiments as only 1 µg of DNA is produced, which corresponds to approximately five reactions (traditional 15 µL volumes). With these additional steps of circularization and isothermal amplification, greater than milligram quantities of the DNA is possible (~5,000 reactions for 1 mg). This dramatically increases the number of tests that can be made in high-throughput screening of proteins or combinatorial enzyme networks (cell-free metabolic engineering); it also allows for effective preservation of the linear template library as high concentration DNA. Furthermore, an increased amount of template would be necessary to prototype larger quantities of protein needed for material science applications (protein-based fibers and hydrogels). Some limitations of linear templates can be overcome by using an extract from BL21 DE3 Star or using recently discovered methods to protect linear templates from degradation^52,53,54. However, this does not address having limited stocks of vendor-produced DNA for PCR amplification or the issue of biological expertise and equipment needed for cloning.

This work presents a protocol explicitly designed to increase the amount of expression template that can be obtained from small quantities of vendor-produced gene fragments (typically 500-1000 ng of lyophilized powder). The described method does not require the skills necessary to perform traditional cloning in plasmids or transforming and propagating in living cells. Upon receiving a gene fragment in the mail, a user can produce enough templates for many cell-free reactions by employing isothermal rolling circle amplification (RCA) (Figure 1)²³. While the amount of DNA received from the vendor may be enough for limited screening efforts, it is quickly depleted, and re-purchasing gene fragments is time consuming and costly. The method is also especially well-suited for genes that are toxic and difficult to clone in E. coli.

Protocol

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.

Results

Expression of sfGFP from RCA templates was comparable to that of the pJL1 plasmid when using only 0.30 µL of unpurified RCA DNA in a 15 µL reaction (Figure 2A). In fact, doubling and tripling the amount of template appears to offer no benefit in BL21 DE3 Star extract, suggesting already saturated levels of the template at 0.30 µL per reaction. Conversely, there appears to be a benefit to increasing the amount of RCA template when added to cell extract sourced from the SHuffle strain (

Discussion

The gene of interest can be any desired protein, but it is best to start with a fluorescent protein as a convenient reporter for real-time or end-point readout on a well plate reader for new adopters of this method. For new protein sequences, copy the amino acid sequence of the desired protein and paste it into the desired codon optimization tool^61,62. There are usually many available organisms and strains of E. coli in the codon optimization tool, but c...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Alaline	Formedium	DOC0102
Ammonium glutamate	MP Biomedicals	MP21805951
Arginine	Formedium	DOC0106
Asparagine	Formedium	DOC0114
Aspartic Acid	Formedium	DOC0118
ATP	Sigma	A2383
Axygen Sealing Film	Corning	PCR-SP
CMP	Sigma	C1006
Coenzyme A	Sigma	C3144
CutSmart Buffer	NEB	B7204S	Provided with HindIII
Cysteine	Formedium	DOC0122
DNA Clean and Concentrator Kit	Zymo Research	D4004	Used for purifying DNA
dNTPs	NEB	N0447
E. coli tRNA	Sigma (Roche)	10109541001
Folinic Acid	Sigma	47612
Gene Fragment	IDT
Glutamic Acid	Formedium	DOC0134
Glutamine	Formedium	DOC0130
Glycine	Formedium	DOC0138
GMP	Sigma	G8377
HEPES	Sigma	H3375
HindIII-HF	NEB	R3104L
Histidine	Formedium	DOC0142
Isoleucine	Formedium	DOC0150
Leucine	Formedium	DOC0154
Lysine	Formedium	DOC0158
Magnesium glutamate	Sigma	49605
Methionine	Formedium	DOC0166
Microtiter Plate (384 well)	Greiner	781906
Microtiter Plate (96 well)	Greiner	655809
Multimode Plate Reader	BioTek	Synergy Neo2
NAD	Sigma	N8535
NanoPhotometer	Implen	NP80
OneTaq DNA Polymerase	NEB	M0480
PCR Tube	VWR	20170-012
Phenylalanine	Formedium	DOC0170
Phosphoenolpyruvate	Sigma (Roche)	10108294
Potassium glutamate	Sigma	G1501
Potassium oxalate	Fisher Scientific	P273
Proline	Formedium	DOC0174
Putrescine	Sigma	P5780
Serine	Formedium	DOC0178
Spermidine	Sigma	S0266
T4 DNA Ligase	NEB	M0202S
T4 DNA Ligase Reaction Buffer	NEB	B0202S	Provided with T4 DNA Ligase
TempliPhi Amplification Kit	Cytiva	25640010	Used for RCA
Thermal Cycler	Biorad	C1000 Touch
Thermoblock	Eppendorf	ThermoMixer FP
Threonine	Formedium	DOC0182
Tryptophan	Formedium	DOC0186
Tyrosine	Formedium	DOC0190
UMP	Sigma	U6375
Valine	Formedium	DOC0194