Team:EPF Lausanne/Notebook/Ecoli
Construction of pdCas9-w
pdCas9-w was constructed using PCR and Gibson assembly from the following plasmids:
- pdCas9 (pdCas9-bacteria was a gift from Stanley Qi, Addgene plasmid # 44249): dCas9 controlled by a Tetracyclin inducible promoter and a Chlorampheicol resistance gene
- pWJ66 (pWJ66 was a gift from Luciano Marraffini, Addgene plasmid # 46570): tracrRNA, w-dCas9 (dCas9 fused at its C-terminal to rpoZ, which encodes for the w subunit of RNA polymerase) and CRISPR array. (The w subunit recruits RNAP)
pdCas9 was opened by PCR and the w subunit was extracted from pWJ66 by PCR. The w subunit was then fused to the C-terminal of dCas9 by Gibson assembly, the final product is pdCas9-w.A site-directed mutagenesis was done on this plasmid to eliminate an EcoRI restriction site. This will allow us to submit pdCas9-w as a BioBrick.
Construction of pdCas9-wOpen pdCas9 by PCR
We received plasmid pdCas9 in bacteria and did a Miniprep (cf. Protocols) on overnight cultures to isolate it. This step consists of linearizing pdCas9 by PCR.
Materials and method
- 20 µL Phusion PCR (cf. Protocols) of pdCas9 with primers f_Gbs_pdCas9 and r_Gbs_pdCas9
- PCR product purification (cf. Protocols)
- Agarose gel electrophoresis of 2 µL purified PCR products (with 1kb Generuler ladder)
Results
Linearized pdCas9-w is expected to be 6705 bp.The first try of this PCR was unsuccessful (gel is not shown here). For our second try, we tested many parameters: HF vs. GC buffer, different annealing temperatures and different extension times. This time, many, but not all, of our samples were successfully amplified (cf. Fig.1). The difficulty of this PCR is probably due to the fact that the size of the ampicon is very long, almost 7 kb.For next steps, sample from lane 1 (cf. Fig.1) was used.
Construction of pdCas9-wExtract w subunit from pWJ66 by PCR
We received plasmid pWJ66 in bacteria and did a Miniprep (cf. Protocols) on overnight cultures to isolate it. This step consists of extracting rpoZ, the w subunit, from pW66 by PCR.
Materials and method
- 20 µL Phusion PCR (cf. Protocols) of pWJ66 using primers f_Gbs_w and r_Gbs_w.
- PCR product purification (cf. Protocols)
- Agarose gel electrophoresis of 2 µL purified PCR products (with 1kb Generuler)
Results
Successful PCR reactions are expected to yield 340 bp fragments.PCR was succesful for sample visible gel. (cf. Fig.2)
Construction of pdCas9-wGibson assembly of pdCas9-w
This step consists of fusing the w subunit to dCas9 by Gibson assembly, using the PCR products from previous steps. We transformed cells with the Gibson assembly product and tested for colonies that contain the construct by colony PCR, restriction digest and sequencing.
Materials and method
- Gibson assembly (cf. Protocols) with purified PCR products:
- Open pdCas9: 0.02 pmol = 95 ng
- w subunit extracted from pWJ66: 0.06 pmol = 12.5 ng
- Transformation (cf. Protocols) of ultra-competent DH5a cells (NEB) with Gibson assembly product, spreading on Chloramphenicol plates
- Colony PCR (cf. Protocols) of colonies from plate used for culture of transformed cells with primers f_Gbs_w and r_Scq_pdCas9_w_sgRNA primers + agarose gel electrophoresis of 2 µL PCR prodcuts (with 1kb Generuler)
- Miniprep (cf. Protocols) of overnight liquid cultures in 5 mL LB with Chloramphenicol of colonies from plate used for culture of transformed cells to isolate pdCas9-w plasmids
- Restriction digest (Cf. Protocols) of pdCas9-w plasmids with BamHI and KpnI seperately + agarose gel electrophoresis of 2 µL digested products (with 1kb Generuler)
- Sequencing (Microsynth) of one colony for which Gibson assembly worked according to colony PCR and restriction analysis
Results
Colony PCR
Primers were placed such as amplicons are 666 bp if Gibson assembly worked and 396 bp if the plasmid self-ligated.Lane "C" is a negative control: PCR was run with all components except template DNA, ie. cells. It is empty which means there is no contamination.Gibson assembly seems to have worked for some of our samples. (cf. Fig.3)To avoid working with too many samples, we kept the ones from lanes 16, 22, 25, 31, 37 and 43 for the Minipreps and restriction digest. We did overnight liquid cultures of these colonies.
Restriction digest
pdCas9-w samples from different colonies are present on gels in triplicates in the following order:
- Undigested - expected to yield 7 kb circular plasmid (migrates faster than linear fragments of the same size)
- Digested by BamHI - expected to yield two fragments of 6147 bp and 834 bp if insert is present or one 6705 bp fragment if it is not
- Digested by KpnI - expected to yield two fragments of 45334 bp and 2447 bp if insert is present or one 6705 bp fragment if it is not
BamHI and KpnI are both unique cutters in pdCas9 (without the insert) and double cutters in pdCas9-w (with the insert).Too much ladder was loaded so it is difficult to estimate the size of the fragments. The smaller fragments are also very difficult to see.By looking at the relative heights, we can say that all colonies seem to have the w subunit insert. There is only the undigested sample for colony 16 that is not visible, probably due to an error while loading the gel. Sequencing confirmed that sample 22 is in fact pdCas9-w. We used this sample for the next steps and stored it as a glycerol stock (c.f. Protocols).
Sequencing
As dCas9-w is very long, only part of it was sequenced (the w subunit and its surrounding base pairs). No mutations were detected.
