Publication protocol
"Cloning of NdsDNA Gates into Plasmids Note: This section describes the Gibson cloning method for inserting 4 copies of the gate into a plasmid backbone.
Order ndsDNA gate templates as double-stranded genomic blocks from a DNA manufacturer (gate template sequences are shown in Table 1; strands occur in ndsDNA gates are shown in Table 2; domain level sequences are shown in Table 3). After receiving the ordered DNA, spin the tubes containing genomic blocks at 10,000-14,000 x g for 1 min to ensure that all dried DNA is at the bottom of the tube. Resuspend the dried genomic blocks in DNase-free water to achieve a final concentration of 10 ng/µl. Note: Alternatively, DNA can be resuspended using 1x Tris ethylenediaminetetraacetic acid (EDTA) buffer (TE buffer: 10 mM Tris and 1 mM EDTA, pH 8.0). However, EDTA is a chelating agent for divalent cations and could inhibit PCR. Generate 4 gate fragments with different overlap regions through a standard PCR with a High-Fidelity DNA polymerase (see Figure 3A). The primer sequences are detailed in Table 4 (melting temperature of these primers is 62 °C). Run a 2% agarose gel at 140 V for 30 min at RT (for a detailed agarose gel electrophoresis protocol see 42) and cut the bands corresponding to each PCR amplified fragment out of the gel. Then purify the gel slices using a gel extraction kit (please refer to the Materials) following the manufacturer’s instructions. Digest a high copy number plasmid backbone (see materials) with PvuII-HF and PstI-HF at 37 °C for 1 hr (see Table 5) according to the manufacturer’s protocol. PvuII-HF and PstI-HF are high fidelity restriction enzymes, which dramatically reduce unspecific cuts. Run a 1.5% agarose gel and cut the linearized backbone (typically run the gel at 140 V for 30-40 min at RT). Then extract the DNA from the gel slice using gel extraction kit following the manufacturer’s instructions. Perform Gibson assembly43 with the linearized vector and purified PCR fragments (see Table 6 and Figure 3B) at 50 °C for 1 hr. Transform the Gibson assembly product from step 2.8 into Escherichia coli (E. coli) and plate on a Lysogeny Broth (LB) agar plate containing Ampicillin antibiotics (at a concentration of 100 µg/ml). Perform transformation through electroporation or a heat shock method 44,45, and use the appropriate E. coli strain. For example, use E. coli strain JM109 for heat shock transformation, and use DH5α electrocompetent E. coli cells for electroporation. Note: The plasmid backbone used contains an Ampicillin resistance cassette. If using a different selection marker, use the appropriate antibiotics instead of Ampicillin. 3. Bacterial Culture Amplification and Quality Control Note: This section describes the mass production and isolation of plasmids containing the DNA gates after quality control. Pick a single colony from the Ampicillin selective plate from step 2.9 and incubate a culture of 3 ml enriched medium containing Ampicillin antibiotics (at a concentration of 100 µg/ml). Mark the colony such that it can be utilized again in subsequent experimental steps. Grow the culture at 37 °C O/N with vigorous shaking (200-300 rpm). Typically, incubate for 16-24 hr. Extract the plasmid DNA from the bacterial culture using a Mini-prep kit following the manufacturer’s instructions.
Measure the purified plasmid DNA using a spectrophotometer following the manufacturer’s instructions. Typical yield ranges from 50-1,000 ng/µl. Get the extracted plasmid DNA sequenced by sending the sample to a DNA sequencing company. Sequencing primers should be located about 100 nucleotides upstream and downstream of the region to be sequenced; the sequencing primer for the plasmid (see materials for plasmid) has the following sequence: ATTACCGCCTTTGAGTGAGC. Note: If there is sequence error or recombination in the inserted ndsDNA gates, select a different colony from the plate from step 2.9. Follow steps 3.1-3.4 to verify that the sequences of the inserted gates are correct. After verifying that the sequences are correct, pick the corresponding colony from the Ampicillin selective plate (from step 2.9), and incubate a culture of 800 ml Terrific Broth (TB) containing Ampicillin antibiotics (at a concentration of 100 µg/ml). Grow the culture at 37 °C for 16-24 hr with vigorous shaking (200-300 rpm). TB particularly is well suited for high yield plasmid production. Note: Alternatively, LB could also be used to grow bacteria although the plasmid yield can be an issue. Purify the DNA using a Maxi-prep kit following the manufacturer’s instructions. Follow step 3.3-3.4 to check whether the sequences are correct. If any recombination occurred, see the following note. Otherwise, move on to step 4. Note: One possible issue here is that multiple copies of inserted gates in the plasmid may recombine due to DNA repair. To address this problem, use an E. coli strain lacking the recA protein (a protein related to DNA repair) such as JM109 or DH5α to transform a previously sequence-verified plasmid (i.e., without any sequence errors and recombination). Then pick one colony from this plate and verify the plasmid sequence by sending the sample to a DNA sequencing company.Digest the purified plasmid DNA from step 3.7 with restriction enzyme PvuII-HF for 1 hr at 37 °C (see Table 7). Typically digest the plasmid with 4 units of PvuII-HF per 1 mg of plasmid. High fidelity restriction enzymes are recommended for use because they dramatically reduce unspecific cuts. Perform ethanol precipitation on the sample. Add 2 equivalent volumes of ice-cold absolute ethanol to the sample. Incubate the mixture at -80 °C for at least 1 hr (this mixture can also sit at -80 °C for O/N). Centrifuge at 10,000-14,000 x g at 0 °C for 30 min. Remove the supernatant. Add 1,000 µl of RT 95% ethanol to the sample, and invert 10-15 times. Centrifuge at 10,000-14,000 x g at 4 °C for 10 min. Remove the supernatant and air dry on bench for 10-20 min. Resuspend the DNA pellets in an appropriate volume of Nuclease free H2O (typically 100-200 µl). Adding more than 200 µl will generally make the sample too dilute for use in kinetics experiments. Measure the resuspended DNA using a spectrophotometer following the manufacturer’s instructions. Digest join gates with nicking enzyme Nb.BsrDI at 65 °C for 1 hr using 4 units of enzyme per 1 µg of plasmid (see Table 8); digest fork gates with nicking enzyme Nt.BstNBI at 55 °C for 1 hr using 8 units of enzyme per 1 µg of plasmid (see Table 9). Note: Step 4.2 removes enzyme digestion buffer and helps concentrate the gates for kinetics experiments. Step 4.2 can be skipped for join gates because both restriction enzyme PvuII-HF and nicking enzyme Nb.BsrDI share the same digestion buffer. In step 4.2.8, Nuclease free H2O is used instead of TE because EDTA is a chelating agent for divalent cations and can inhibit restriction enzymes that need these ions to function. Note: The addition of excess amounts of enzymes may lead to high amounts of initial circuit leakage (Figure 4), which is most likely caused by over-digestion46. This issue can be addressed by optimizing the enzyme amounts (see Figure 4). Typical range of enzymes is from 1-10 units/1 µg plasmid."
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