Construction of pdCas9-w

pdCas9-w was constructed using PCR and Gibson assembly from the following plasmids:

• pdCas9 (pdCas9-bacteria, plasmid #44249, Addgene): dCas9 controlled by a Tetracyclin inducible promoter and a Chlorampheicol resistance gene
• pWJ66 (pWJ66, plasmid #46570, Addgene): 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-w
Open 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-w
Extract 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-w
Gibson 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.

Construction of pdCas9-w
Site-directed mutagenesis of dCas9-w

It was noticed after assembly of pdCas9-w that dCas9 contains an EcoRI restriction site, which makes it "un-BioBrick-able". Since it is an important part for our project, we decided to remove the restriction site by site-directed mutagenesis to be able to submit it as a functional BioBrick.

Materials and method

• Site-directed mutagenesis (cf. Protocols) of pdCas9-w with primers f_Mt_A2080C_pdCas9-w and r_Mt_A2080C_pdCas9-w
• Miniprep (cf. Protocols) of overnight liquid cultures in 7 mL LB with Chloramphenicol of colonies from site-directed mutagenesis plate to isolate mutated pdCas9-w plasmids
• Restriction digest (cf. Protocols) of mutated pdCas9-w plasmids with EcoRI to identify colonies for which the mutagenesis was successful
• Sequencing (Microsynth) of one pdCas9-w for which the mutageneis worked according to the restriction digest

Results

Coming soon

Construction of pdCas9-w
BioBrick mutated dCas9-w

To submit dCas9-w as a BioBrick, we extracted dCas9-w from the mutated pdCas9-w and inserted it into pSB1C3, a BioBrick assembly plasmid.

Materials and method

Coming soon

Results

Coming soon

Construction of pdCas9-w-sgRNAs

These experiments consist of inserting one or two sgRNA producing cassettes into pdCas9-w.
pdCas9-w was linearized by restriction digest. The sgRNA cassettes were synthesized (IDT) and necessary overlaps were added by PCR. The sgRNAs were inserted into pdCas9-w by Gibson assembly.

The sgRNA cassettes were synthesized with sequences "A" and "B" at its ends (cf. Fig.8a). These are the same sequences as those found at the ends of the linearized pdCas9-w.
When one cassette is inserted, no overlaps need to be added.
When two or three cassettes are added, sequences "C" and/or "D" need to be added at the ends of the cassettes (cf. Fig.8b), thus obtaining unique overlaps for the Gibson assembly.

Construction of pdCas9-w-sgRNAs
Open pdCas9-w by restriction digest

Materials and method

• Miniprep (cf. Protocols) of overnight liquid cultures in 5 mL LB with Chloramphenicol of pdCas9-w containing bacteria
• Restriction digest (Cf. Protocols) of pdCas9-w with BsrBI (blunt ends)
• Agarose gel electrophoresis of 2 µL of digested and undigested product (with 1kb Generuler)

Results

The digested and undigested plasmids should have the same size. Circular fragments migrates faster than linear fragments of the same size. This means that the undigested plasmid should migrate faster than the digested plasmid.
Even though the ladder is not clear, by looking at the relative heights we can see that the digested sample migrated more slowly than the undigested sample (cf. Fig.9). The restriction digest was successful.

Note that this was repeated several times since we needed a lot of linearized pdCas9-w.

Construction of pdCas9-w-sgRNAs
Amplify sgRNA cassettes by PCR

Materials and method

• 20 µL Phusion or Q5 PCR (cf. Protocols) of sgRNA cassette with primers as indicated in table below
• Agarose gel electrophoresis of 2 µL purified PCR products (with 1kb Generuler)
• PCR product purification (cf. Protocols)

Results

All amplified sgRNA cassettes are expected to be about 370 bp.

sgRNA cassette Z0 was amplified with primers f_Gbs_sgRNA-A + r_Gbs_sgRNA-BtoD and primers f_Gbs_sgRNA-A + r_Gbs_sgRNA-B with Q5 PCR. Both samples were successfully amplified (cf. Fig.10a).

sgRNA cassette Z4 was amplified with primers f_Gbs_sgRNA-A + r_Gbs_sgRNA-B with Phusion PCR. Samples 3 and 4 were successfuly amplified (cf. Fig.10b).

sgRNA cassette Z35 was successfuly amplified with primers f_Gbs_sgRNA-A + r_Gbs_sgRNA-B with Phusion PCR. However, it is not easily visible on the gel. (cf. Fig.10c)

sgRNA cassette X0 was successfuly amplified with primers f_Gbs_sgRNA-A + r_Gbs_sgRNA-B with Phusion PCR. (cf. Fig.10d)

sgRNA cassette X4 was successfuly amplified with primers f_Gbs_sgRNA-A + r_Gbs_sgRNA-B with Phusion PCR. (cf. Fig.10e)

sgRNA cassette X35 was successfuly amplified with primers f_Gbs_sgRNA-A + r_Gbs_sgRNA-B with Phusion PCR. (cf. Fig.10f)

Construction of pdCas9-w-sgRNAs
Assemble pdCas9-w-sgRNA constructs by Gibson assembly

Materials and method

Part 1: pdCas9-w + sgRNA assembly

• Gibson assembly (cf. Protocols) with linearized pdCas9-w and purified sgRNA PCR products:
1. Z0 / Z4 / Z35 / X0 / X4 / X35 (overlaps A and B)
2. Z0+Z4 / Z0+Z35 / Z4+Z35 / X0+X4 / X0+X35 / X4+X35 (overlaps A and C/D for 1st cassette and C/D and B for 2nd cassette)

Part 2: pdCas9-w-sgRNA + sgRNA assembly

• Coming soon

Results

Part 1: pdCas9-w + sgRNA assembly

In colony PCR, primers were placed such as amplicons are 445 bp if the plasmid self-ligated, 723 bp if Gibson assembly of 1 sgRNA cassette worked and 1120 bp if Gibson assembly of 2 sgRNA cassettes worked.

pdCas9-w-Z0 was obtained by a faulty assembly of pdCas9-w-Z0-Z4 that only took up 1 of the 2 sgRNA cassettes. Sample 20 of the colony PCR (cf. Fig.11a) was confirmed by sequencing to be pdCas9-w-Z0 and to not contain any mutations that may have an effect on its activity. Sample 13 seems to be pdCas9-w-Z0-Z4 according to colony PCR (cf. Fig.11a), but sequencing showed that it is pdCas9-w-X0-Z0, probably due to contamination of a tube. (This sample will be used again in Part 2.)
Colony PCR showed that all colonies tested for pdCas9-w-Z4 has 1 inserted sgRNA cassette (cf. Fig.11b). Sequencing comfirmed that sample 5 is in fact pdCas9-w-Z4. Colony PCR showed that the Gibson assembly for sample 5 of pdCas9-w-Z35 seems to have worked (cf. Fig.11c) and sequencing confirmed that is the case. Sequencing also showed some mutations or deletions in the promoter and/or terminator for these 2 samples. We decided to keep working with these and test whether these mutations are significative with an activity assay.
Colony PCR and sequencing showed that Gibson assembly was successful for sample 5 of pdCas9-w-X0 (cf. Fig.11d). However, it contains a mutation inthe promoter. A 2nd pdCas9-w-X0 without mutations will be constructed in part 2, after which we will be able to compare the activity of this promoter with/without a mutation.
Colony PCR of pdCas9-w-X4 showed many potentially good samples (cf. Fig.11e). Sequencing of sample 10 confirmed that it is in fact pdCas9-w-X4 and that it does not contain any mutations.
Colony PCR and sequencing showed that Gibson assembly was successful for the colony of pdCas9-w-X35 (cf. Fig.11d) and no mutations were found in the sequence.

Construction of pWJ89alt

pWJ89alt was constructed using PCR and Gibson assembly from the following DNA fragments:

• pWJ89: low copy plasmid with GFP under a J23117 promoter with and GG-rich upstream regulating sequence (URS) and a Kanamycin resistance gene, received from David Bikard's lab
• J23117alt: alternative promoter with randomely generated sequences except for the -35 and -10 sequences (ordered from IDT)

pWJ89 was amplified without its J23117 promoter by PCR and the J23117alt promoter was amplified by PCR. These fragments were assembled by Gibson assembly, the final product is pWj89alt.
The J23117alt promoter was BioBricked.

PCR pWJ89

We received plasmid pWJ89 in bacteria and did a Miniprep (cf. Protocols) on overnight cultures to isolate it. This step consists of amplifying pWJ89 without the J23117 promoter by PCR.

Materials and method

• 20 µL Phusion PCR and 25 µL Q5 PCR (cf. Protocols) of pWJ89 using primers f_Rmv_J23117_of_pWJ89 and r_Rmv_J23117_of_pWJ89.
• Agarose gel electrophoresis of 2 µL PCR products (with 1kb Generuler)
• PCR product purification (cf. Protocols)

Results

Successful PCR reactions are expected to yield 4400 bp amplicons.
Both negative control lanes (cf. Fig.13) are empty, which means there is no contamination. Phusion PCR did not work, but Q5 PCR did (cf. Fig.13). We will work with this sample for next steps.

PCR J23117alt promoter

Coming soon

Gibson assembly of pWJ89alt

Coming soon

BioBrick J23117alt promoter

Coming soon

Transistor activity assay

Coming soon

Primer table