Reporter Plasmid Construction
Figure 1: Recipient bacteria carrying our reporter plasmid (AmpR and RFP)
Killer Plasmid : design and construction
DesignWe received a mobilizable plasmid (pK18mob) carrying Kanamycin resistance gene and OriT. Our purpose in to combine its conjugative capabilities with the Crispr/Cas9 system, so the final killer plasmid will be able to conjugate and to cut a target gene (the RFP gene and Ampicillin resistance gene). So, from the pK18mob, we have inserted the Crispr/Cas9 system : the Cas9 gene, and a single guide RNA.
We have design two single guide RNA, to target the RFP gene and the Ampicillin resistance gene by Crispr/Cas9 system. The sequence was design in Benchling, and carry : cut/ligation sites at extremities, promoter pJ23119, the guide RNA and the terminator. Three DAM site (GATC) was cleaned, to avoid methylation. This sequence was synthetized by IDT.
Figure 2: Schematic representation of our system
Construction Step 1: integration of Cas9 geneFrom the plasmid pCAS, we have cut the Cas9 gene (4469bp with its promoter and terminator) with NEB digestion enzyme, and we have inserted it by ligation (with T4 ligase) in the plasmid pK18mob (3793 bp, received from C.E.N). We verify the presence of Cas9 sequence insertion in our cloned bacteria by performing a Miniprep and an enzyme digestion to linearize the plasmid, and check its size.
Figure 3: Linearizing digestion of the pK18mob vector with Cas9.
Construction Step 2: Integration of the sgRNAAfter digestion of our sgRNA, the ligation was done with the plasmid pK18mob with the CAS9 sequence. The result was transformed in TOP10 strain. We verify the presence of our sgRNA insertion by performing PCR, and then, after Miniprep, by enzyme digestion for verification.
Figure 4: PCR of the pK18mob vector with Cas9 with sgRNA.
Step 3: transformation of our plasmid in a conjugative strainWe transformed our final killer plasmid in E. coli S17-1 strain. We used S17 electrocompetent cells.
- We have now a conjugative strain (S17-1) carrying a killer plasmid, capable of conjugation and targeting the RFP gene, and a killer plasmid targeting the AmpR gene.
- The inserted sequence of the Cas9 and of the sgRNA were sequenced by Macrogen, and are all correct.
- To have a better biosafe system, we also have design the same killer plasmid, but in an other conjugative plasmid (pSW23T) which have de R6K Origin of Replication. (See Perspective / Improvement / Biosafety section)
Efficiency of conjugation.
Test #1 conjugation efficiency: transfer of the mobilizable plasmid pK18mobTo verify the efficiency of transfer of the mobilizable plasmid pK18mob, we conjugated the recipient strain DH5ɑ containing the plasmid pSB4A5_J04450 with the donor strain S17-1 containing the plasmid pK18mob (WT). The result of the conjugation was spread on LB/Xgal/IPTG box without selection pressure. The counting of strains is based on the expressed phenotype. Donors include lactose operon and are blue in appearance, recipient cells that have not conjugated express RFP. While the transfer of the plasmid pK18mob (WT) restores the lactose operon in DH5ɑ by transferring the intact LacZɑ gene. The simultaneous expression of the lactose operon and the rfp gene produces a purple phenotype with DH5ɑ bacteria that have received the plasmid that can be mobilized by conjugation.
Figure 5: Differentiation of strains based on the expressed phenotype.
After 2 hours of conjugation on solid LB medium, the cells are collected and diluted in PBS 1X before being spread on LB/Xgal/IPTG plate per 100µl. Colony counts are carried out three times for each dilution.
Table 1: Count after conjugation (transfer of mobilizable plasmid pK18mob).
Efficiency of our Crispr/Cas9 system.
Test #1 of our Crispr/Cas9 efficiencyTo verify the efficiency of Crispr/Cas9 system of our plasmid, we have transformed the killer plasmid in a DH5ɑ strain carrying the RFP reporter plasmid (pSB4A5_J04450). If our CRISPR/Cas9 plasmid system work well, it will cut the RFP gene and the recipient bacteria (red and fluorescent) will appear white.
Verification of our Crispr/Cas9 efficiency.
Table 2: Count after transformation by pK18mob_CAS9 with or without sgRNA anti RFP.
- As we can see, all of our transformed bacteria appears in red. Our Crispr/Cas9 system seems to have an efficiency of 100%
Biomolecular confirmation of our Crispr/Cas9 systemAn enzyme digestion (PstI) was performed on the same strains to check that the recipient plasmid was correctly cut after the transformation of our killer plasmid.
Biomolecular confirmation of the effect of CRISPR/Cas9 on the reporter plasmid: miniprep on colony and linear digestion with PstI.
- This plasmid digestion confirms that the reporter plasmid carrying the rfp and TEM-1 genes has been correctly cut by our Crispr/Cas9 system, both by the RNA guide targeting RFP and the one targeting the resistance gene.
Tests of ConjugationDifferent test were performed to verify the conjugation and/or Crispr/Cas9 system efficiency.
Conjugation “Cross” testthe purpose of this test is the verify the conjugation and the efficiency of our system by crossing, in a agar plate, a recipient strain (carrying our RFP reporter plasmid) with both our control donor strain (S17-1 with our plasmid without sgRNA) and our donor strain (S17-1 with our killer plasmid with the sgRNA).
Cross test 1 with RFP Killer plasmidRecipient strain: DH5ɑ strain (E. coli) with the RFP rapporter plasmid.
Donor strain : S17-1 strain with our RFP killer plasmid.
Control strain : S17-1 strain without our RFP sgRNA.
Cross test 1.
Cross test 2 with AmpR killer plasmidRecipient cell: DS984 strain (E. coli CmR) with the RFP rapporter plasmid (AmpR).
Donor strain : S17-1 strain with our Amp killer plasmid.
Control strain : S17-1 strain without our AmpR sgRNA.
Cross test 2. Vertically : the recipient cell (with RFP reporter plasmid). Horizontally : Up: the donor strain (cutting AMP gene) Down: the control donor strain (without cutting Amp gene)
Conjugation “in plate” testBefore conjugation test, our strain (donor, recipient and control) was precultured O/N from glycerol stock.
The different strain used are:
- Donor : S17-1 with our killer plasmid (targeting RFP or AmpR)
- Control : S17-1 with our conjugative plasmid pK18mob, with Cas9 BUT without the sgRNA (no targeting)
- Recipient : Dh5alpha, or DS984 (with CmR gene in chromosome) or TOP10 (with a TEM1 gene in chromosome). Each of the recipient strain have our reporter plasmid (with RFP or GFP)
Classic conjugation assay used for our test
Conjugation in plate test 1 (non quantitative) targeting AmpR (TEM1)
- Donor (D) : S17-1 with our killer plasmid (sgRNA targeting AmpR, resistance: KanR)
- Control (CTRL) : S17-1 with our conjugative plasmid pK18mob, with Cas9 BUT without the sgRNA (no targeting). KanR
- Recipient (R) : TOP10 (with a TEM1 gene in chromosome)
- Conjugation time : 3 hours 30 min
- D.O. (600 nm) = about 1 (for recipient strain), about 0,5 (for donor/control strain)
Conjugation in plate test 1: On left : Donor and recipient strain after conjugation. On right : control and recipient strain after conjugation.
- First result seems to show that conjugation append (at least in control strain) and that donor strain (if have conjugated) seems to kill the AmpR gene as expected. This first test is non quantitative, so we don’t know exactly how much bacteria we have, and more test are needed.
Efficiency of Crispr/Cas9 system by conjugation.To verify the effectiveness of our system using Cas9 carried by a mobilizable plasmid, we contacted the number of events or a recipient cell (TOP10 PyrF::TEM-1) lost its red phenotype when losing its plasmid pSB4K5_bbaJ04450 after conjugation with a donor cell (S17-1) containing the mobilizable plasmid containing Cas9 and an anti-RFP RNA guide. A negative control was performed using a donor strain that did not include guide RNA. Donor cells were counted after dilution on LB kanamycin and recipients on LB ampicillin. Colony counts are carried out three times for each dilution. The effectiveness of our system using Cas9 carried by a mobilizable plasmid give 27% for a D:R ratio 1:169.
Table 3: Averages of the counts before and after conjugation and transfer of the mobilizable plasmid pK18mob_CAS9. Averages achieved over three spreads per sample. The interval is given by twice the standard deviation.
Conjugation in fluorescence microscopyTo visualize at the scale of a cell, the effect of the transfer of a mobilizable plasmid, containing Cas9 and its RNA anti RFP guide, by conjugation: we performed a 60-minute time lapse on a mixture containing the donor strain (S17-1 with pK18mob_CAS9_sgRNA anti RFP and pSB4C5_R0010_E0840 (GFP)) and the recipient strain (DH5a with pSB4A5_J04450 (RFP)).