Team:EPF Lausanne/Notebook/Ecoli

EPFL 2015 iGEM bioLogic Logic Orthogonal gRNA Implemented Circuits EPFL 2015 iGEM bioLogic Logic Orthogonal gRNA Implemented Circuits

E. Coli Laboratory Notebook

Construction of pdCas9-w
May 7th to June 1st 2015

pdCas9-w contains a gene that produces dCas9 fused to the w subunit of RNA polymerase (RNAP), which recruits RNAP.

Materials and method

  • Open pdCas9-bacteria by Phusion PCR (cf. Protocols) 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)
    pdCas9-bacteria was a gift from Stanley Qi (Addgene plasmid # 44249).
  • Extract w subunit from pWJ66 by Phusion PCR (cf. Protocols) with 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)
    pWJ66 was a gift from Luciano Marraffini (Addgene plasmid # 46570).
  • Gibson assembly (cf. Protocols) of pdCas9-w using purified PCR products of pdCas9-bacteria and w subunit + transformation (cf. Protocols) of ultra-competent DH5a cells (NEB)
    Control with colony PCR (cf. Protocols) with primers f_Gbs_w and r_Scq_pdCas9_w_sgRNA primers + agarose gel electrophoresis of 2 µL PCR products (with 1kb Generuler) + restriction digest (Cf. Protocols) of pdCas9-w (isolated by Miniprep (cf.Protocols)) with BamHI and KpnI seperately + agarose gel electrophoresis of 2 µL digested products (with 1kb Generuler) + sequencing (Microsynth)
  • Site-directed mutagenesis (cf. Protocols) of dCas9-w with primers f_Mt_A2080C_pdCas9-w and r_Mt_A2080C_pdCas9-w (necesary for pdCas9-w to be BioBrick compatible)
    Control with restriction digest (cf. Protocols) of mutated pdCas9-w (isolated by Miniprep (cf. Protocols)) with EcoRI + sequencing (Microsynth)
  • BioBrick mutated dCas9-w

Results

Open pdCas9 by PCR
May 10th 2015

Linearized pdCas9-w is expected to be 6705 bp.
We tested many parameters: HF vs. GC buffer, different annealing temperatures and different extension times. Many, but not all, of our samples were successfully amplified (cf. Fig.1).
For next steps, sample from lane 1 (cf. Fig.1) was used.

Figure 1
Fig.1 - Gel of linearized pdCas9

Extract w subunit from pWJ66 by PCR
May 7th 2015

Successful PCR reactions are expected to yield 340 bp fragments.
PCR was succesful for sample visible on gel. (cf. Fig.2)

Figure 2
Fig.2 - Gel of w subunit extracted from pWJ66

Gibson assembly of pdCas9-w
May 29th and June 1st 2015

Colony PCR - 05/29/2015

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. It is empty which means there is no contamination.
Gibson assembly seems to have worked for some 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 next steps. We did overnight liquid cultures of these colonies.

Figure 3a Figure 3b Figure 3c Figure 3d
Fig.3 - Gels of colony PCR of dCas9-w Gibson assembly products

Restriction digest - 06/01/2015

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).

Figure 4a Figure 4b Figure 4c
Fig.4 - Gels of restriction digest of pdCas9-w Gibson assembly products

Sequencing - (date?)

As dCas9-w is very long, only part of it was sequenced (the w subunit and its surrounding base pairs). No mutations were detected.

Site-directed mutagenesis of dCas9-w - (date?)

BioBrick mutated dCas9-w - (date?)

Construction of pdCas9-w-sgRNAs - July 9th to August 7th 2015

These experiments consist of inserting one or two sgRNA producing cassettes (cf. Fig.8) into pdCas9-w. In part 1, one sgRNA (X0, X4, X35, Z0, Z4 or Z35) was inserted into pdCas9-w by Gibson assembly. For technical reasons, we were unable to make most constructs with two inserted cassettes using this method. In part 2, we extracted sgRNA cassettes from existing constructs and inserted them in other constructs that already contained one other sgRNA cassette.

Figure ¨8
Fig.8:Schematics of structure of sgRNA cassettes

Materials and method

Part 1: Assemble pdCas9-w + sgRNA

  • Open pdCas9-w by restriction digest (cf. Protocols) with BsrBI (blunt ends) + agarose gel electrophoresis of 2 µL of digested and undigested product (with 1kb Generuler)
  • Amplify sgRNA cassettes by Phusion or Q5 PCR (cf. Protocols) with primers f_Gbs_sgRNA-A and r_Gbs_sgRNA-B + agarose gel electrophoresis of 2 µL PCR products (with 1kb Generuler) + PCR product purification (cf. Protocols).
  • Gibson assembly pdCas9-w-sgRNA (cf. Protocols) with linearized pdCas9-w and purified sgRNA PCR products + transformation (cf. Protocols) of ultra-competent DH5a cells (NEB)
    Control with colony PCR (cf. Protocols) with primers f_ColPCR_sgRNAs and r_ColPCR_sgRNAs + agarose gel electrophoresis of 2 µL PCR prodcuts (with 1kb Generuler) (one colony PCR (cf. Fig11a) was done with primers f_Gbs_pdCas9 and r_Scq_pdCas9_w_sgRNA) + sequencing (Microsynth)

Part 2: Assemble pdCas9-w-sgRNA + sgRNA

  • Extract sgRNA from pdCas9-w-sgRNA by Q5 PCR (cf. Protocols) with primers f_Gbs_sgRNAinPd and r_Gbs_sgRNAinPd + agarose gel electrophoresis of 2 µL PCR products (with 1kb Generuler) + PCR product purification (cf. Protocols)
  • Open pdCas9-w-sgRNA by Q5 PCR (cf. Protocols) with primers f_opn_d9w and r_opn_d9w + agarose gel electrophoresis of 2 µL PCR products (with 1kb Generuler) + PCR product purification (cf. Protocols)
  • Gibson assembly of pdCas9-w-sgRNA, as describes in table below, + transformation (cf. Protocols) of ultra-competent DH5a cells (NEB)
    Control with colony PCR (cf. Protocols) with primers f_Sq_CoP_pdCas92ndIns and r_Sq_CoP_pAraC2ndInsert + agarose gel electrophoresis of 2 µL PCR prodcuts (with 1kb Generuler or 2-log DNA ladder) + sequencing (Microsynth)
sgRNA insert sgRNA in pdCas9-w-sgRNA (backbone)
Z4 pdCas9-w-Z0
Z35 pdCas9-w-Z0
Z35 pdCas9-w-Z4
X4 pdCas9-w-X0
X35 pdCas9-w-X0
X35 pdCas9-w-X4

Results

Part 1: Assemble pdCas9-w + sgRNA

Open pdCas9-w by restriction digest
July 16th, 19th and 26th 2015 (repeats)

The digested and undigested plasmids have the same size. Circular fragments migrates faster than linear fragments of the same size. This means that the undigested plasmid will 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.

Figure 9
Fig.9 - Gel of digested pdCas9-w (lane 1) and undigested pdCas9-w (lane 2)

Amplify sgRNA cassettes by PCR
July 9th, 10th, 13th, 14th, 16th and August 4th 2015

Amplified sgRNA cassettes are expected to be about 370 bp.

sgRNA cassette Z0 was amplified with Q5 PCR. Both samples were successfully amplified (cf. Fig.10a).
sgRNA cassette Z4 was amplified with Phusion PCR. Samples 3 and 4 were successfuly amplified (cf. Fig.10b).
sgRNA cassette Z35 was successfuly amplified with Phusion PCR. However, it is not easily visible on the gel. (cf. Fig.10c)
sgRNA cassette X0 was successfuly amplified with Phusion PCR. (cf. Fig.10d)
sgRNA cassette X4 was successfuly amplified with Phusion PCR. (cf. Fig.10e)
sgRNA cassette X35 was successfuly amplified with Phusion PCR. (cf. Fig.10f)

Figure 10a Figure 10b Figure 10c Figure 10d Figure 10e Figure 10f
Fig.10: Gels of sgRNA cassettes amplified by PCR with primers f_Gbs_sgRNA-A + r_Gbs_sgRNA-B
Fig.10a - Gel of sgRNA cassette Z0 amplicon (lanes 1-2), successful for both samples
Fig.10b - Gel of sgRNA cassette Z4 amplicon, successful for samples in lanes 3 and 4
Fig.10c - Gel of sgRNA cassette Z35 amplicon, successful for sample in lane 1
Fig.10d - Gel of sgRNA cassette X0 amplicon, successful for sample
Fig.10e - Gel of sgRNA cassette X4 amplicon
Fig.10f - Gel of sgRNA cassette X35 amplicon, successful for sample

Assemble pdCas9-w-sgRNA constructs - July 26th, 29th and August 7th 2015

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 have the 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 in the 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.

Figure 11a Figure 11b Figure 11c Figure 11d Figure 11e
Fig.11: Gels of colony PCR of pdCas9-w-sgRNA Gibson assembly products
Fig.11a - Gel of colony PCR of pdCas9-w-Z0-Z4 (lanes 1-10) and self-ligation control pdCas9-w (lane "C"). Lane 2 seems to be pdCas9-w-Z0-Z4 and lanes 5, 8 and 9 seem to only have 1 inserted sgRNA cassette, being either pdCas9-w-Z0 or pdCas9-w-Z4.
Fig.11b - Gel of colony PCR of pdCas9-w-Z4 (lanes 1-11). Gibson assembly seems to have worked for all colonies
Fig.11c - Gel of colony PCR of pdCas9-w-Z35 (lanes 1-8), seems to have worked for lane 5, self-ligation for all others
Fig.11d - Gel of colony PCR of pdCas9-w-X0 (lanes 1-5) and pdCas9-w-X35 (lane 6). Gibson assembly of pdCas9-w-X0 seems to have worked for lanes 2-5, but sample in lane 5 looks slightly better than the others. The faint band in lane 1 is self-ligation. Gibson assembly of pdCas9-w-X35 seems to have worked.
Fig.11e - Gel of colony PCR of pdCas9-w-X4 (lanes 1-11). All samples seems to have the sgRNA insert, even though the bands are not very precise.

Part 2: Assemble pdCas9-w-sgRNA + sgRNA

Extract sgRNA from pdCas9-w-sgRNA and open pdCas9-w-sgRNA by Q5 PCR

Amplified sgRNA cassettes (Z4, Z35, X4 and X35) are expected to be 400 bp. All were successfully amplified (cf. Fig.12a and Fig.12b).
Amplified pdCas9-w-sgRNA (Z0, Z4, X0 and X4) are expected to be about 7000 bp. All were successfully amplified (cf. Fig.12a and Fig.12b).

Figure 12a Figure 12b
Fig.12 - Gels of PCR products of pdCas9-w-sgRNA and sgRNA cassettes
Fig.12a - Gel of PCR products for the construction of pdCas9-w-sgRNA: pdCas9-w-Z0 (lane 1), pdCas9-w-Z4 (lane 2), sgRNA Z4 (lane 3), pdCas9-w-X0 (lane 4), pdCas9-w-X4 (lane 5) and sgRNA X4 (lane 6), all were successfully amplified.
Fig.12b - Gel of PCR products for the construction of pArac-sgRNA: pArac (lanes 1-2), unsuccessfully amplified, and sgRNA Z0 (lanes 3+5, different primers), sgRNA Z4 (lanes 4+6, different primers), successfully amplified. Gel of PCR products for the construction of pdCas9-w-sgRNA: sgRNA Z35 (lane 7) and sgRNA X35 (lane 8), all successfully amplified.

Assemble pdCas9-w-sgRNA-sgRNA constructs

Colony PCR primers are placed such as amplicons are 724 bp if Gibson assembly worked and are 390 bp if it did not (self-ligation). All necessary constructs were successfuly obtained. Following colonies were kept for our constructs: colony #1 for pdCas9-w-Z0-Z4 (cf. Fig.13a), colony #4 (2nd attempt) for pdCas9-w-Z0-Z35 (cf. Fig.13c), colony #6 for pdCas9-w-Z4-Z35 (cf. Fig.13b), colony #4 for pdCas9-w-X0-X4 (cf. Fig.13c), colony #2 for pdCas9-w-X0-X35 (cf. Fig.13d) and colony #1 for pdCas9-w-X4-X35 (cf. Fig.13e).

Figure 13a Figure 13b Figure 13c Figure 13d Figure 13e
Fig.13 - Gels of colony PCR of pdCas9-w-sgRNAs
Fig.13a - Gel of colony PCR of pdCas9-w-Z0-Z4 (lanes 1-2), successful for colony #1, and colony PCR of pdCas9-w-Z0-Z35, self-ligation for all colonies (08/31/2015)
Fig.13b - Gel of colony PCR of pdCas9-w-Z4-Z35, successful for all colonies (08/31/2015)
Fig.13c - Gel of colony PCR of pdCas9-w-Z0-Z35 (2nd attempt), successful for colonies #1 and #4, and colony PCR of pdCas9-w-X0-X4 (part 1), successful for colonies #2 and #4 (09/02/2015)
Fig.13d - Gel of colony PCR of pdCas9-w-X0-X4 (part 2), maybe successful for colonies #8, #9 and #10 (gel unclear), and colony PCR of pdCas9-w-X0-X35, successful for colony #2
Fig.13e - Gel of colony PCR of pdCas9-w-X4-X35, succesful for colony #1 (maybe others, gel unclear) (09/02/2015)

Construction of pWJ89alt

pWJ89alt contains GFP controlled by the promoter J23117alt, which can be activated by dCas9-w bound to sgRNA X4 and inhibited by dCas9-w bound to sgRNA X0 or X35.

Materials and method

  • Open pdCas9-w by restriction digest (cf. Protocols) with BsrBI (blunt ends) + agarose gel electrophoresis of 2 µL of digested and undigested product (with 1kb Generuler)
  • Amplify sgRNA cassettes by Phusion or Q5 PCR (cf. Protocols) with primers f_Gbs_sgRNA-A and r_Gbs_sgRNA-B + agarose gel electrophoresis of 2 µL PCR products (with 1kb Generuler) + PCR product purification (cf. Protocols).
  • Gibson assembly pdCas9-w-sgRNA (cf. Protocols) with linearized pdCas9-w and purified sgRNA PCR products + transformation (cf. Protocols) of ultra-competent DH5a cells (NEB)
    Control with colony PCR (cf. Protocols) with primers f_ColPCR_sgRNAs and r_ColPCR_sgRNAs + agarose gel electrophoresis of 2 µL PCR prodcuts (with 1kb Generuler) (one colony PCR (cf. Fig11a) was done with primers f_Gbs_pdCas9 and r_Scq_pdCas9_w_sgRNA) + sequencing (Microsynth)

Results

PCR pWJ89

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.

Figure 13
Fig.13 - Gel of pWJ89 amplified without J23117 promoter by PCR. Lane 1: Phusion PCR, lane 2: Phusion PCR negative control, lane 3: Q5 PCR, lane 4: Q5 PCR negative control

PCR J23117alt promoter

Successful PCR reactions are expected to yield 340 bp amplicons.
Both negative control lanes (cf. Fig.14) are empty, which means there is no contamination. Both Phusion and Q5 PCR worked (cf. Fig.13). We will work with the sample amplified by Q5 PCR.

Figure 13
Fig.14 - Gel of pWJ89 amplified without J23117 promoter by PCR. Lane 1: Phusion PCR, lane 2: Phusion PCR negative control, lane 3: Q5 PCR, lane 4: Q5 PCR negative control

Gibson assembly of pWJ89alt

Colony PCR showed two colonies for which Gibson assembly worked. However, images of gels of colony PCR were lost for technical reasons.
Sequencing confirmed that both colonies were in fact pWJ89alt and that one had a deletion but the other one did not have any mutations. We kept the mutation-free colony and stored it in a Glycerol stock (cf. Protocols).

BioBrick J23117alt promoter

Construction of pWJ89_mCherry

Open pWJ89 by restriction digest

PCR pWJ89alt

Extract mCherry by restriction digest

PCR mCherry

Gibson assembly of pWJ89_mCherry

Transistor activity assay

Primer table

Name Sequence Associated part
f_Gbs_pdCas9 CTCGAGTAAGGATCTCCAG pdCas9
f_Gbs_sgRNA-CtoA GTCGGCGATGGTGGTAGCTAATTATGTTCCctcgctcactgactcgctac sgRNA cassettes
f_Gbs_sgRNA-DtoA CTAGACCTAACTGAGATACTGTCATAGACGctcgctcactgactcgctac sgRNA cassettes
f_Gbs_sgRNA-A ctcgctcactgactcgctac sgRNA cassettes
f_Gbs_w ACACGCATTGATTTGAGTCA pWJ66
f_Mt_A2080C_pdCas9-w TGACTTTTCGcATTCCTTATTATGTTG pdCas9-w
r_Gbs_pdCas9 GTCACCTCCTAGCTGACTC pdCas9
r_Gbs_sgRNA-B tggcatcttccaggaaatc sgRNA cassettes/td>
r_Gbs_sgRNA-BtoC GGAACATAATTAGCTACCACCATCGCCGACtggcatcttccaggaaatc sgRNA cassettes
r_Gbs_sgRNA-BtoD CGTCTATGACAGTATCTCAGTTAGGTCTAGtggcatcttccaggaaatc sgRNA cassettes/td>
r_Gbs_w atttgatgcctggagatccttactcgagTTAACGACGACCTTCAGCA pWJ66
r_Mt_A2080C_pdCas9-w AGATTTTTTCAATCTTCTCACG pdCas9-w
r_Scq_pdCas9_w_sgRNA ctgatttgagcgtcagat pdCas9-w-sgRNA
EPFL 2015 iGEM bioLogic Logic Orthogonal gRNA Implemented Circuits

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