Team:UCSC/Proof of Concept
Proof Of Concept
Gene Elimination Strategy
To model gene elimination within a bacterial genome, we engineered a strain of E. coli that has the mcherry gene chromosomally integrated. This mcherry-E. coli strain will be the target of Progenie, proving our system’s mechanics without working directly with dangerous Shiga toxin-producing bacteria. Because the INTEGRATE system is highly sequence specific, we predict that a working mcherry -elimination system can be translated to stx2 by changing guide RNA sequences. This section outlines our laboratory workflow using an mcherry model to demonstrate the three core aspects of Progenie. We first verified the gene elimination mechanism, then developed a procedure for infecting a target population with phage, and finally demonstrated that the mechanism can spread through conjugation.
Integration of mCherry
We integrated mcherry into the E. coli chromosome to generate our model. To do this, we used Clonetegration [2], a two-plasmid system designed to integrate a gene into a specific site in the genome. The first plasmid—pOSIP-KL-mcherry—gets integrated into the bacterial chromosome. The second plasmid—pE-FLP— “flips out” all the integrated plasmid except for the mcherry gene. The pOSIP-KL-mcherry plasmid contains both the cloning and integration modules. The cloning module holds the mcherry gene to be ultimately integrated. The integration module encodes for the lambda phage integrase, a temperature sensitive lambda repressor, a kanamycin resistance gene, and the R6Kγ origin of replication. The integration module is flanked by FRT sites which allow for its excision. The pE-FLP plasmid expresses FLP, which makes the recombinase protein Flippase, along with an ampicillin resistance gene, a replication protein, and the pSC101 origin of replication [2].
When pOSIP-KL-mcherry is transformed into competent E. coli, the entire plasmid is integrated into the bacterial chromosome. pE-FLP is then transformed into the cell and expression of Flippase begins, which excises the entire integration module of pOSIP-KL-mcherry through FLP-FRT recombination. Only the mcherry gene is left inside the bacterial chromosome. We performed the Clonetegration protocol on two strains of E. coli: DH5α, and DH5αF’. The result is two chromosomal mcherry strains of E. coli which can be targeted with the Progenie system [2].
The Destination Vector
We generated a destination vector, dubbed pMADRID, that can be easily adjusted to target a gene of interest. We designed pMADRID to have BsaI restriction enzyme sites in a placeholder spacer region. Cutting out the placeholder spacer with BsaI produces overhangs on the edges on the spacer region; a new guide sequence with complementary overhangs can then be ligated in. The plasmid pMADRID was derived from an aliquot of pSPAIN donated to UCSC by Leo Vo at Columbia University.
Targeting pMINT
We will alter the destination vector to create the mcherry -targeting vector pMINT. Our proof of concept relies on our engineered plasmid successfully knocking out the mcherry gene. To do this, we designed variations of pMADRID that contain unique, targeting, spacers. The mcherry targeting plasmids are henceforth referred to as pMINT1, 2, or 3. The three spacers—designated by the suffix 1-3— are restriction cloned into pMADRID and will guide integration of the entire INTEGRATE system into the chromosomal mcherry promoter.
We tested our gene elimination mechanism by transforming each pMINT construct into DH5α- mcherry cells and measuring the change in mCherry fluorescence over time. The Progenie mechanism was successful because we observed a significant decrease in relative fluorescence in the DH5α- mcherry cells that were treated with pMINT compared to the non-targeting control. For specific results, see: here
Phage Delivery System
We will use a modified form of the bacteriophage M13 to deliver the mcherry -targeting plasmid to DH5α-F’- mcherry cells. To create a plasmid delivery system that would work in both our current model target DH5α F’- mcherry and our future plans target E. coli O157:H7, we chose M13 phage as our mode of initial delivery. Both cell types have F or F-like pili, which is the binding site for M13 [1]. To avoid using fully a infectious virus, we will utilize the modified phage M13KO7 (Thermo Fisher) which recognizes and carries circular single-stranded DNA containing an F1 origin of replication. To get the phage to package our plasmid, we will have to modify pMINT to contain an F1 ori. We will term the mcherry -targeting phagemid system φMINT. We will transform φMINT into a strain of E. coli , then infect the culture with M13KO7 phage. Infected cells will secrete packaged phage holding φMINT into the media, where we will isolate it. We will then titrate the phage-containing media to determine what dosage of packaged φMINT to use to target DH5α-F’- mcherry cells. We will transduct them then measure the mCherry fluorescence of the DH5α-F’- mcherry culture over time. The phage delivery system will be successful if there is a significant decrease in relative fluorescence in the DH5α-F’- mcherry that were transduced with φMINT when compared to a non-targeting control.
Conjugation
We will make the mcherry -targeting plasmid mobilizable through bacterial conjugation. Since the M13 phage targets cells containing an F or F-like plasmid, the cells that will first be infected by our plasmid already have the ability to be donors in bacterial conjugation. To alter φMINT so it can be conjugated by these cells, we will add an Origin of Transfer (ori T). This new mobilizable form of our plasmid will be called φMINTO. We will transform φMINTO into DH5α F’ cells and perform conjugation experiments by mixing transformed non-fluorescent DH5α F’ donors and DH5α- mcherry recipients and measure the mCherry fluorescence of the recipients over time. We will determine that φMINTO was successfully conjugated if there is a significant decrease in relative fluorescence in the DH5α F’- mcherry recipients compared to a non-targeting control.
Creating the Progenie Application
We combine the three aspects of Progenie into a multi-step experiment to show how Progenie can work in application. In a mixed culture containing DH5α- mcherry and DH5α F’- mcherry , we will add phage carrying φMINTO and measure the mCherry fluorescence over time. We will determine that Progenie successfully knocked out a gene from a mixed population if there is a significant decrease in relative fluorescence in the mixed flask compared flasks that had only DH5α F’- mcherry and phage, DH5α- mcherry and phage, and mixed population but no phage.
