We added the new part BBa_K3352000 into the iGEM registry. The SplintR ligase coding sequence was obtained from the amino acid sequence in Paramecium bursaria Chlorella virus 1 (PBCV-1) (A544R - DNA ligase - Paramecium bursaria Chlorella virus 1 (PBCV-1) - A544R Gene & Protein, n.d.). We optimized this sequence for E. coli expression and removed the PstI cutting site. We also attached a 6x histidine tag (6x his-tag) upstream of the SplintR ligase sequence through a glycine-serine linker (GS linker).
Figure 1:Open reading frame for SplintR ligase expression and isolation
SplintR ligase catalyzes the ligation of adjacent single-stranded DNA splinted by complementary RNA strands (Biolabs, n.d.). Reported to proofread fully complementary in DNA/RNA hybrids down to the single nucleotide polymorph level, this enzyme is an essential component to viral diagnostic testing (Krzywkowski & Nilsson, 2017). In our project, it enables us to increase the specificity of our test by conducting direct RNA targeting.
In order to test this part, we flanked it with an upstream T7 promoter (BBa_J64997), strong RBS (BBa_B0034) and downstream double terminator (BBa_B0015). This formed our composite part BBa_K3352004).
Figure 2: Design of T7 Promoter SplintR ligase Expressing Construct (BBa_K3352004)
Cloning this part into the iGEM ampicillin backbone pSB1A3, the plasmid was transformed into DH5⍺ E. coli cells for plasmid replication and subsequently miniprepped for a high yield of plasmid. We then transformed these plasmids into BL21(DE3) E. coli cells. Growing an overnight culture and measuring the OD600 to dilute cells to standardized populations, we induced expression with 0.1M IPTG once the OD600 surpassed 0.5. Liquid cultures were grown for an additional 2 hours.
After protein expression, we harvested and lysed the cells with XTractor Lysis Buffer and centrifuged them for sample preparation (XTractorTM Buffer & XTractor Buffer Kit User Manual, n.d.). This was followed by protein purification through Ni sepharose affinity chromatography, which could isolate our his-tagged SplintR ligase enzymes.
Figure 3: SDS-PAGE on purified proteins with the T7 promoter SplintR ligase expressing construct (BBa_K3352004)
Our results in Figure #3 shows a protein band at around 35.2kDa, which is the molecular weight of our SplintR ligase enzyme (containing the 6x his-tag and GS linker attached to the enzyme). This provides evidence that SplintR ligase was expressed and purified.
Testing whether our purified SplintR ligase works as anticipated, we used it in our Viral Spiral Rolling Circle Amplification (RCA) viral diagnostic test. In our diagnostic test, SplintR ligase determines the complementary between the hybrid of DNA padlock probes bound to synthetic viral RNA targets before ligating and circularizing the padlock probe. Using the circularized padlock probe, signal amplification takes place through a pH and color change.
However, because SplintR ligase is stored in unfavorable elution buffer conditions with compounds such as imidazole, we conducted a dialysis buffer exchange. Using Vivaspin® 6 Centrifugal Concentrators, we further enriched our SplintR ligase sample (Vivaspin® 6, n.d.). The new buffer conditions contain 10mM Tris-Hcl, 300mM NaCl, 1mM DTT, 0.1mM EDTA, and 50% glycerol at a pH of 7.4, which is the optimal storage conditions and pH starting point for our RCA test.
Video 1: SARS-CoV-2 RNA RCA Test with Purified SplintR ligase
Conducting the RCA test on SARS-CoV-2, the PCR tube under letter b is a negative control with no SARS-CoV-2 RNA Target and the PCR tube under letter c is our test group with a SARS-CoV-2 RNA Target. Both tubes use our purified SplintR ligase. The test group yielded a color change from purple to orange in just one hour and ultimately ended at yellow while our negative control remained purple. This result indicates that our SplintR ligase enzymes enabled an accurate detection of the presence of SARS-CoV-2 through its synthetic RNA fragment in our RCA diagnostic test.
Figure 4: pH over Time Analysis of both tubes in the SARS-CoV-2 RNA RCA Test with Purified SplintR ligase. The RCA test with SARS-CoV-2 target and purified SplintR ligase. The orange line shows the functional RCA test with SARS-CoV-2 target and purified SplintR ligase. The blue line shows the negative control with no SARS-CoV-2 target and SplintR ligase.
Using our modeling team’s software to transform our qualitative viral detection readout to quantitative data, we constructed a pH over time graph through the change in hues of both tubes in the video. As shown in Figure #4, there is only minimal fluctuation around the same pH for our negative control and a rapid significant drop in pH of our test group. This is expected due to the production of hydrogen ions from our RCA test that ultimately results in the color change. This provides further validation that SplintR ligase enzyme was expressed, purified, and functioned its intended use.