Precise Phage Mutagenesis with NgTET-Assisted CRISPR-Cas Systems

We study phage infection at the molecular level. This is really fundamental science. However, we are not only doing the fundamental science, we are also keen to translate this fundamental science into applications where we use this knowledge to study the phage infection, and to use this for phage engineering, and for therapeutic applications.

One of the most recent developments in the field of phage research is the discovery of a bacterial immune system. This immune system is present in all bacteria and they identified or generate tools to really fight against the phage. However, phage is quite clever.

A phage was able to identify tools as well and to develop tools that allow him to escape those immune systems. And one tool is actually the modification of its DNA by modification. And this is especially for us, really interesting because to study the chemical nature of these DNA modifications that phages have generated to escape immune systems are really interesting, and also allow us to become a tool to engineer the phage at the end.

In the last 10 years, wonderful technologies were developed by great colleagues around the whole globe. One of these technologies is for instance, next generation sequencing. Based on this technology, we can really sequence phage, but also the whole host genomes on the molecular level.

On the other hand, mass spectrometry, something like proteomics was developed. CRISPR is a wonderful tool that is now applied in various kingdoms of life. And also we are using this now to engineer the phage genome.

These are just a few wonderful technologies that have reshaped how we think about phages today. When we talk about current challenges, one of the challenge that we see is the engineering bio CRISPR of a phage genome. The phage genome is totally modified with DNA modifications, and the CRISPR nuclear ACEs are actually not able to cleave the DNA.

So this is really a challenge that we need to overcome if we want to engineer the genomes of bacterial phages. My group has discovered a totally novel building block of RNAs. We discovered that specific transcripts are carrying at their five prime terminus, the so-called redox specter, nicotinamide adenine dinucleotide short NAD, and we discovered this also recently now for the first time in E-coli T4 phage interactions.

Based on this discovery, we also identified that RNAs and proteins can interact with each other in a totally novel way. We discovered that this NAD that I described before is working like a molecule glue to link RNAs and proteins with each other. This RNA elation is a totally novel way of how RNAs and proteins interact with each other in nature.

And we made this discovery in eco anti phage interaction. We are also now keen to translate this fundamental science into applications, for instance, into the field of RNA-based therapeutics. To begin, treat 500 microliters of phage suspension with 20 units of DNA 1.

Add two microliters of RNAs AT1 mixed to the tube. Incubate the treated mixture at 37 degrees Celsius for 30 minutes. Next, load 500 microliters of the treated phage solution on top of a prepared sucrose gradient.

Centrifuge the gradient at 70, 000 G for 20 minutes at four degrees Celsius. Light the centrifugation tube from the bottom to visualize the turbid phage band in the gradient. Then with a blunt cannula, carefully remove the phage containing fraction into a new ultra ultracentrifugation tube.

Now, add 30 milliliters of ice cold TM buffer to the tube and centrifuge. Then resuspend the pellet in 500 microliters of TM buffer after discarding the supernatant. Store the resuspended phages in a glass vial at four degrees Celsius overnight.

Add one Microgram of proteinase K to the resuspended phages. Incubate the mixture at 37 degrees Celsius for 30 minutes. Then add an equal volume of phenol chloroform, isoamyl alcohol mixture to the sample.

Invert to mix well and centrifuge. Transfer the aqueous phase to a new reaction tube, and repeat the extraction three times. Perform three chloroform back extractions using an equal volume of chloroform to remove residual phenol.

Then precipitate the DNA by adding 0.1 volume of three molar sodium acetate at PH 5.5, and 2.5 volumes of ethanol. Incubate the mixture overnight at minus 20 degrees Celsius. The next day, centrifuge the sample at 15, 000 G for one hour at four degrees Celsius to pellet the DNA.

Wash the DNA pellet twice with 200 microliters of 70%ethanol. Gently shake the tube and centrifuge again. Carefully remove the supernatant.

Then resus, suspend the purified DNA in ultrapure water. Store the DNA at minus 20 degrees Celsius until further use. Inoculate Escherichia coli BL 21D 3 cells that are transformed with both the PET 28 NGTet plasmid, and a CAS 12 or CAS 9 expression plasmid.

Grow the culture in LB medium with supplemented with appropriate antibiotics at 37 degrees Celsius with shaking. Induce NG TED expression by adding 0.05 millimolar IPTG, and incubate for an additional two hours at 37 degrees Celsius. As a control, compare E.coli BL 21 D3 strains with and without NgTET overexpression.

Transfer 300 microliters of the E.coli cultures into a sterile tube. Infect the culture with either T4 wild type or NgTET treated phage at a multiplicity of infection of 0.01. Mix gently to ensure even phage distribution then incubate again.

Add the bacteria phage mixture to four milliliters of LB soft auger containing 0.75%auger and antibiotics. Mix thoroughly, but gently to avoid bubble formation. Pour the soft auger mixture onto a prewarm LB auger plate.

Let the plate solidify briefly at room temperature and incubate overnight at 37 degrees Celsius. The next day, count the plaques to determine the number of plaque forming units. For counter selection, infect E.coli cast 13 A spacer using the phages to be counter selected.

Perform the counter selection under the same conditions used for mutagenesis. After incubation, filter the SUP natant through a 0.45 micrometer filter to remove bacterial debris. Use the counter selected and filtered phages for a plaque assay on an E.coli B strain to isolate individual plaques for downstream validation.

The phage band was clearly visible when the T4 phage sample was sufficiently concentrated. Expression of NgTET protein in E.coli was confirmed by SDS phage showing its presence in both soluble, and insoluble fractions following cell lysis and centrifugation. Chromatograms showed that the relative abundances of dioxiadenosine, diiodohydroxyquinoline, and thymidine were unchanged between wild type and NgTET treated T4 phage DNA.

The abundance of modified cytosine derivatives was significantly reduced in the NgTET treated phage DNA compared to the untreated sample. A plasmid containing the NgTET gene, and homology regions for targeted mutagenesis was constructed using golden gate cloning. PCR screening confirmed successful amplification of the target gene mare in lanes two through nine of the agros gel, indicating positive hits among the picked plaques.

In wild type T4 phage DNA, the relative abundance of 5 glycosylhydroxyethyl 2 prime deoxidine was high. In NgTET treated T4 phage DNA, levels of 5 glycosylhydroxyethyl 2 prime deoxidine were strongly reduced compared to wild type. T4 phage DNA treated with the catalytic inactive mutant NgTET D234 A retained high levels of 5 glycolhydroxyethyl 2 prime deoxidine.

Recovered T 4 phage progeny showed nearly restored levels of 5 hydroxymethyl 2 prime deoxidine compared to wild type.