Part:BBa_K5087012
SapF
Introduction
This primer was designed to incorporate SapI recognition sites into the reporter module (BBa_K5087025) that we wanted to incorporate into our SynLOCK Cassette (BBa_K5087017).
Our goal was to insert the reporter expression module into the Cassette Spine to create the complete SynLOCK Cassette, while preserving the SapI recognition sites. These sites would later enable the reporter to be excised and replaced with the crRNA spacers. Additionally, the amplified product needed to have sticky ends after SapI digestion that would allow it to be ligated into the Cassette.
Experimental Validation
PCR Setup
A dilution of the matrix DNA (tsPurple Reporter Unit in the pJUMP29-1A backbone) for the PCR was prepared by mixing 2 µl of isolated plasmid (254 ng/µl) with 38 µl H₂O.
After pipetting the master mix into PCR tubes, 1 µl of the plasmid dilution was added to all samples except for the negative control (substituted with H₂O).
Table 1: PCR Reaction Master Mix Components (for a single reaction)
| Component | Volume (µl) |
|---|---|
| Q5 mix | 12.5 |
| SapF (BBa_K5087012) primer (10 µM) | 1.25 |
| SapR primer (BBa_K5087013) (10 µM) | 1.25 |
| H₂O | 9.0 |
| Total | 24 |
Thermocycler Program
- Initial denaturation: 98°C for 30s
- Denaturation: 98°C for 10s
- Annealing: 70°C (for negative control and one sample), 65°C (for another sample) for 15s
- Extension: 72°C for 25s
- Final extension: 72°C for 2min
- Hold: 4°C indefinitely
Steps 2, 3, and 4 were looped 30 times.
Note: The annealing temperature was calculated using the NEB Tm Calculator tool and was estimated at 72°C.
Figure 1. Electrophoresis of the BBa_K5087025 reporter module (indicated as TU5 for translational unit 5) amplified using SapF and SapR primers.
Conclusion: An anticipated 939 bp band was obtained. As expected, the 72°C annealing temperature yielded higher reaction efficiency, as indicated by the brighter band on the gel. However, the product was also successfully created at 65°C.
Sequence
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 30
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 6
Biosafety
We used the Asimov's tool — Kernel — to check the sequence's safety with the Biosecurity Sequence Scanner. The results showed no flagged sequences, confirming that this part is safe to use.
| None |