Fig 1.
A general strategy to measure restriction digest efficiency.
(A) DNA cleavage-triggered loss of function of marker proteins. A modular template DNA used for CFPS consists of T7P, RBS, and a marker gene encoding for fluorescent proteins. The restriction site of interest can be either within the marker gene (scissor 1) or upstream of the marker gene (scissor 2). The restriction digest terminates the peptide synthesis prematurely, hence no or only truncated peptides can be produced. (B) Basic experimental procedure for quantifying the undigested DNA molecules. A defined volume of the digest solution is directly mixed with CFPS aliquot. The mixture is introduced into FemDA. Only intact (i.e., undigested) DNA in the droplets can produce functional proteins that can be detected using fluorescence microscopy. The fraction of positive droplets over the array is used to derive the concentration of the undigested DNA. The residual template DNA that cannot be detected by gel electrophoresis (upper-right scheme) can be detected at the single-molecule level using FemDA. The dashed line on FemDA (lower-left) indicates a position of the cross-section view.
Fig 2.
Assessment for restriction digest efficiencies.
(A) Agarose gel electrophoresis of restriction digests. 80 ng DNA was applied to each lane. Lane M: DNA marker; lane 1&5: mNeonGreen template DNA (prior to digestion); lane 2: DNA digested by NcoI for 1 h; lane 3: DNA digested by NcoI-HF for 1 h; lane 4: DNA digested by NcoI-HF for 15 min; lane 6: DNA digested by NcoI-HF for 24 h; lane 7: DNA digested by BtgI for 24 h. (B) Digital CFPS using DNA digest solution. The NcoI-HF (left) or NdeI (right)-digested (for 15 min) mNeonGreen DNA solution was mixed with CFPS components and introduced into FemDA. Fluorescent proteins were synthesized in the droplet containing undigested DNA. Scale bar: 10 μm. (C) Simultaneous measurement of restriction digest efficiency of multiple enzymes with multiplexed digital CFPS. BamHI-HF-digested mTurquoise2 (cyan) DNA, NdeI-digested mNeonGreen (green) DNA, and EcoRI-HF-digested mScarlet (red) DNA were mixed and used for a single CFPS reaction on FemDA. The digestion time for each was 15 min. Scale bar: 10 μm. (D) Sum of Gaussian distributions of equal peak-to-peak intervals of fluorescence intensities of droplets. The NdeI-digested (for 1 h) DNA solution was used for this digital CFPS. The blue square represents the observed probability of droplets containing different numbers of template DNA molecules. The red cross represents the corresponding probability fitted by the Poisson distribution. The observed probability and the fitted probability were consistent with each other, proving the Poisson distribution of single DNA molecules. (E) Restriction digest efficiencies (digested versus the initial quantity of template DNA, expressed in %) of NcoI-HF at different time points. (E) Measurement of enzyme kinetics and Michaelis-Menten curve fitting for NdeI. (G) Restriction digest efficiencies (left y-axis marked in black) of NdeI at different time points and the number of false-positive colonies (right y-axis marked in red) caused by the undigested DNA in a model transformation experiment. Each reaction or transformation was performed in triplicate. Error bars: 1 standard deviation.
Table 1.
Restriction digest efficiency of different REs.
Fig 3.
DNA digest analysis using capillary electrophoresis (Agilent Bioanalyzer) and slab gel electrophoresis.
The measured digest efficiency (in % unit, from Table 1) was labeled beside the corresponding RE site on the template DNA map. On capillary electrophoresis (middle), lane 1~15: mNeonGreen DNA restriction digest with (in the order from left to right) XbaI, NdeI, NheI-HF, BmtI-HF, BamHI-HF, XcmI, PflMI, BstEII-HF, HpaI, BbsI-HF, BsgI, AfeI, BstXI, StuI, and BsrGI-HF, respectively. On agarose slab gel electrophoresis (bottom), lane M1: Hi-Lo DNA marker; lane 16: template DNA (prior to digestion, 1008 bp); lane 2~17: digested by (in the order from left to right) XbaI, NdeI, NheI-HF, BmtI-HF, BamHI-HF, XcmI, PflMI, BstEII-HF, HpaI, BbsI-HF, BsgI, AfeI (newly purchased), AfeI (old stock), BstXI, StuI, BsrGI-HF; lane M2: 100 bp DNA ladder; label “Y”: undigested DNA was detectable on the slab gel; label “N”: undigested DNA was not detectable on the slab gel. 100 ng (calculated based on the stock concentration of template DNA solution measured using NanoDrop) DNA was applied to each lane of the slab gel.
Fig 4.
The modular template DNA to interrogate the digest efficiency of various DNA-cleaving/degrading enzymes.
The protein-coding region is colored in green. (A) Site-specific cleavage of DNA by CRISPR-Cas nucleases with the aid of sgRNA. (B) ssDNA preparation using exonucleases. Both of λ exonuclease and exonuclease III recognize dsDNA’s specific ends and digest one of the strands along the chain. This consecutive digestion disables the transcription and translation, as the duplex DNA sequence of the T7P region (pink colored) is essential for the binding of T7 RNA polymerase. (C) Non-specific cleavage of DNA by DNase I. Either the truncated open reading frame or the defective promoter or 5’ untranslated region can lead to the premature termination of transcription and translation.