Team:UiOslo Norway/Experiments
Experiments
Here you may find detailed information on our experiments and protocols.
dCas9 and β-lactamase
The sequences for the dCas9-lactamase were too large to be synthesized in one piece.
We therefore received them as separate g-blocks that had to be fused together before we could start expressing the proteins.Our goal was to fuse the dCas9 with either the C-terminal or N-terminal part of the split β-lactamase.
Fusing dCas9-β-lactamase parts
Perform a three-part Gibson assembly with pSB1C3 vector (submission vector), the two dCas9 g-blocks, one already linked to a split-β-lactamase.
Transform the assembly into competent DH5α cells, and let grow overnight to observe colonies.
After colonies are observed, make a gridplate with several of the colonies, followed by making overnight cultures from the gridplate colonies.
Miniprep the overnight cultures to extract the DNA, and send the DNA for sequencing.
Expressing and purifying dCas9-β-lactamase
After obtaining colonies with the right DNA sequence, clone the DNA into the pBAD vector (expression vector) using two-part Gibson assembly.
Transform the assembly into competent DH5α cells, and let grow overnight to observe colonies.
After colonies are observed, make a gridplate with several of the colonies, perform colony-PCR, followed by making overnight cultures from the gridplate colonies.
Miniprep the overnight cultures to extract the DNA, and send the DNA for sequencing.
After confirming which colonies have the right DNA sequence, insert the expression vector into a bacterial strain suitable for expression, like BL21, using the transformation protocol.
Make overnight cultures for subsequent small-scale protein expression.
Separate the cell fractions using an SDS gel.
Check protein expression with a Western blot using relevant detection antibodies or conjugates.
Run affinity chromatography of the different fractions using the His-tag, collect the fractions into Eppendorf tubes.
Run the fractions containing your protein of interest on an SDS gel.
CRISPR guide RNA
Transform the sgRNA generator from team Peking 2015 (BBa_K1689000) into DH5α cells.
After observing colonies from the transformation, make an overnight culture.
Miniprep the overnight cultures to extract the DNA, and send the DNA for sequencing.
The guide RNA oligos were ordered in form of forward and reverse DNA primers from Thermo Fisher.
Anneal the complementary primers and insert the guide RNA-oligo-DNA into the sgRNA generator using Golden Gate assembly.
Transform the Golden Gate assembly product and plate them on LB plates with chloramphenicol, X-gal and IPTG for blue white color selection.
After observing white colonies from the transformation, make an overnight culture.
Miniprep the overnight cultures to extract the DNA, and send the DNA for sequencing.
After confirming which colonies have the right DNA sequence, reverse transcribe the guide RNA-oligo-DNA into guide RNA using HiScribe T7 High Yield RNA Synthesis Kit.
Purify the guide RNA using RNeasy MinElute Cleanup Kit.
Lytic enzymes
Flowthrough of expressing glucanase
Perform a two-part Gibson assembly with glucanase and pSB1C3 vector (submission vector).
Transform the assembly into competent DH5α cells, and let grow overnight to observe colonies.
After colonies are observed, make a gridplate with several of the colonies.
Perform colony-PCR and make overnight cultures for the positive colonies.
Miniprep to purify the DNA.
Send the DNA for sequencing.
After obtaining colonies with the right DNA sequence, insert the DNA into the pBAD vector (expression vector) using two-part Gibson assembly.
Transform the assembly into competent DH5α cells, and let grow overnight to observe colonies.
Miniprep the overnight cultures to extract the DNA, and send the DNA for sequencing.
After confirming which colonies have the right DNA sequence, insert the expression vector into a bacterial strain suitable for expression, like BL21, using the transformation protocol.
Make overnight cultures for subsequent small-scale protein expression/middle-scale protein expression.
Separate the cell fractions using an SDS gel.
Check protein expression with a Western blot using relevant detection antibodies or conjugates.
Run affinity chromatography of the different fractions using their His-tag, collect the fractions into Eppendorf tubes.
Run the fractions containing your protein of interest on an SDS gel.
Use the GlycoSpot-Enzyme Standard assay to test the efficiency of the protein.
Protocols
Click to view our protocols in PDF-format.
- Agar plate format with enzyme preparations
- GlycoSpot-Enzyme screening standard
- Small-scale protein expression
- Medium-scale protein expression and purification (Affinity chromatography)
- Medium-scale protein expression and purification (Sonification and plate assay)
- SDS-PAGE 12%
- Western Blot
- Colony PCR
- DpnI digestion
- Gibson Assembly
- Golden gate assembly
- HiScribe T7 High Yield RNA Synthesis Kit
- Making chemically competent DH5α cells
- Miniprep
- PCR cleanup from Gel
- PCR cleanup
- Phusion PCR
- SOE splicing by overlap-overhang extension PCR
- Transformation protocol for DH5α