Results
This page presents the key findings and outcomes of our project work.
Infusion Cloning
We used Infusion cloning to insert the linker sequence and Dam into the same reading frame as dCas9 in the pdCas9 vector. The PCR results showed bands in 5 of the wells at a size equal to the weight of dCas9-Dam.
We obtained positive sanger sequencing results for cloning showing the linker sequence as well as Dam downstream of dCas9 without any scars.
Co-transformation
XL-1 Blue cells were co-transformed with dnaAP2-GFP and dCas9-Dam along with negative controls (cells with dnaAP2-GFP on Ampicillin + Chloramphenicol agar plates and cells with dCas9-Dam on Chloramphenicol + Tetracycline agar plates).
The next day, we observed small translucent colonies of the XL1 Blue strain. All the plates were contaminated, the contamination could have happened during transformation or the competent cell aliquots that we prepared could be contaminated.
XL1 Blue colonies (Translucent colonies):
- GFP (6kb) cells on Ampicillin + Tetracycline: ~ 10 × 10³
- S2 (10kb) cells on Chloramphenicol + Tetracycline: ~ 3-4 × 10³
- GFP + S2 cells on Ampicillin + Chloramphenicol: ~ 0.5-0.6 × 10³
- GFP cells on Ampicillin + Chloramphenicol: nil
- S2 cells on Ampicillin + Tetracycline: nil
Top row (Single Transformation): Had lots of colonies. GFP transformation was more efficient as compared to dCas9-Dam.
Middle row (Co-transformation): Had 6-fold less and 20-fold less colonies as compared to single transformation with GFP and dCas9-Dam plasmids respectively.
Bottom row (Negative controls): Had no colonies (except satellite colonies).
The results were in line with expected trends for heat-shock transformations:
- Large plasmid = lower transformation efficiency
- Co-transformation = probability drop due to competition for the same delivery machinery for entry of plasmids into the cell
BsaI Digestion and Gel Electrophoresis for Plasmid Confirmation
- The dCas9-Dam plasmid is expected to give a 10.2kb band after digestion with BsaI
- The dnaAP2-gfp plasmid is expected to give a 6.2kb band after single digestion and 1.5kb and 4.6kb bands after double digestion
- Digested the plasmid samples from co-transformation with BsaI
- We ran the two digestion mix on 1% agarose at 100V for 1 hour
- Observed four bands (~10kb, ~6kb, ~4.5kb, 1.5kb) in both the dual plasmid samples thus confirming the presence of both dCas9-Dam and dnaAP2-gfp plasmids
- Co-transformation was successful
gRNA Cloning
Our initial gRNA cloning of dCas9-Dam with gRNA after sequencing showed absence of gRNA (only the dCas9-linker-Dam was present). However, we successfully cloned gRNA into the dCas9 plasmid (confirmed by sequencing results).
Our second gRNA cloning with the modified protocol showed a band with weight approximately 220 bp which potentially indicates successful cloning and was sent for sequencing.
GFP Readings
GFP expression in Dam(-) cells was reduced nearly five-fold as compared to Dam(+) cells. This matches with our visual observations with the cell pellet.
