Fluorescence microscope image of RV308 cells, electroporated with fluorescein labeled oligonucleotides

Introduction
In vivo use of programmable restriction enzymes can provide the opportunity of genome engineering. Here we develop and test strategies for the application of our programmable restriction endonuclease.

In our strategy we cotransformed Fok_i and Fok_a constructs fused to binding domains into a certain bacteria strain leading to the basal expression of the Fok heterodimers. Transformation of single-stranded bacteriophage M13 DNA hybridized with modified oligonucleotides enables the Fok heterodimers to bind the DNA. Inside the cells the phage DNA should be cut by the dimerized and thereby activated Fok_a should result in a decreased progeny of phages within the bacterial culture in comparison to control assays. Two ways of detection were envisioned. A qPCR should reveal successful cutting of the phage DNA by the lack of amplified phage DNA. In addition an adjacent phage titer determination via blue white screening  by plating the cells to IPTG/Xgal plates for an adjacent phage titer determination via blue white screening should lead to a decreased number of plaques in comparison to the control assays.

in vivo scheme

Methods

In detail two heterodimeric Fok constructs His-Dig-Split-Fok_a (BBa_K243036) and His-FluA-Split-Fok_i (BBa_K243010) are encoded by two different plasmids, plasmid pJS419 with a chloramphenicol and pEx with an ampicillin resistance. This enables a positive selection of the bacteria strain XL1 blue cotransformed with both plasmids. Subsequent to the preparation of electrocompetent cotransformed XL1 blue, the M13 ssDNA hybridized with the modified oligonucleotides were inserted into these bacteria by electroporation at 1.7 kV. The infected cells were incubated at 37°C for 1.5 h. At different dilutions the phages were mixed with 180μl ER2738 cells with an OD600 of about 0.5. After an incubation time of 10 minutes they were plated in 3 ml top agar on IPTG/X-Gal plates.

XL1 Blue as well as ER2738 harbour a F-plasmid coding for the omega-fragment of beta-galactosidase. The M13 phages in turn encodes the lacking alpha-fragment. Consequently, alpha-complementation can be detected by a blue/white screening. After incubation overnight at 37°C the infected cells were inspected for blue plaques fomration because of the enzymatic cleavage of X-Gal and production of the blue dye 5,5'-dibromine-4,4'-dichloride-indigo and plaques can be seen where the phages lysed the ER2738 cells. In comparison with plated bacteria only transformed with M13 ssDNA, M13 ssDNA with other oligos not modified or infected with entire phages directly the number of pfu (plaque forming units) should be decreased.

Another possible approach is a qPCR after the insertion and incubation of the phage DNA hybridized with a modified oligo or pure ssDNA into the XL1 blue. This quantitative real time polymerase chain reaction allows to amplify and simultaneously quantify a certain sequence of the phage DNA. The additional feature of the qPCR is the quantification of the accumulating DNA happens in real time after each amplification step. The phage DNA would be extracted by boiling preparation, a method faster than conventional alkaline lysis minipreps leaving the DNA somewhat dirtier, but adequate for qPCR. After the qPCR with appropriate primers, the DNA can be quantified with a fluorescent dye, intercalating in the double-stranded DNA. The expected result would be a lower amount of double-stranded phage DNA for our XL1 blue transformed with DNA and oligo in comparison to the control assay.

Results and Discussion

After the first test attempt to plate cotransformated XL1 Blue on IPTG/X-Gal plates no blue plaques could be seen, indicating that we plated the wrong dilutions, the M13 ssDNA wasn’t good, our cells aren’t electrocompetent or the basal activity of Fok_a and Fok_i is already sufficient to destroy the whole phage DNA without an adapter-oligo. A transformation of a test plasmid in the electrocompetent XL1 blue and another electrocompetent strain, RV 308, showed in comparison a weaker but existing transformation rate in XL1 blue. Another test assay was to test our infection method by infecton of ER2738 and XL1 blue with phages from the ph.d 7 phage display peptide library, i.e. incubation of cells with OD600 of about 0.5 at different phage dilutions and plating them in prewarmed top agar on IPTG/X-Gal plates. Only the ER2738 but not XL1 blue showed entire blue coloration of the plates. (Bedeutung für Xblue?) At another test assay we transformed the XL1 blue with new M13 ssDNA and incubated them for 1.5h on a 37°C shaker at 750rpm. Then we mixed them at different concentrations with ER2738 of an OD600 of 0.42 and plated them after 10 min incubation time in top agar on IPTG/X-Gal plates. As result all the plates were totally blue. Also with a shorter incubation time of XL1 blue of 0.5h on the 37°C shaker we obtained the same result indicating that our ER2738 are already contaminated with phages. Now the next is to try the boiling prep and the qPCR or redo the same assay with new ER2738.