Difference between revisions of "Team:Munich/Results"

In the last step we made an effort to scale up the sonication step. Preliminary experiments (results not shown) indicated 4 mL as the maximal sample volume that could efficiently be lysed with the device we had at our disposal. As indicated in the publication by Yong-Chan Kwon & Michael C. Jewett2 the number of sonication cycles was increased in proportion to the increase of sample volume. The presented results shown in the figure below shows that this approach was successful: Using higher sample volumes for sonication increased protein content and expression quality of the cell extract. This proves that upscaling of cell extract preparation with cell lysis by sonication is indeed feasible.

Even though previous attempts by Shresta et al.[ref 3] to use Lysozyme in cell extract preparation have been unsuccessful, we tested the effect of adding the cell-wall degrading enzyme at 1 mM concentration to the cell lysis reaction. In our experiments lysozyme increases the efficiency of cell lysis for all tested settings. Moreover, the expression quality was likewise increased after Lysozyme addition, indicating that the remaining enzyme does not interfere with protein biosynthesis.

Cultivation and cell lysis are the apparent most crucial steps in preparation of cell extract but there are other steps that can have a great impact onto cell extract quality. Some of our findings regarding those steps are:

• Freezing cell pellets overnight for next-day processing does not interfere with extract quality but confers a major simplification of cell extract preparation
• Preliminary results indicate that omitting the run-off reaction after lysis does not significantly impair the extract quality
• Dialysis of cell extract can be substituted by diafiltration in centrifugal filters without reducing expression yield of the resulting extract

With regard to global application of Phactory it is crucial to enable long-term storage and shipping of our cell extract. In order to fulfill these requirements we chose to freeze-dry our cell extract. Lyophilized extract can be stored at ambient temperature and can be reactivated by addition of only nuclease-free water. Initial tests for the optimal lyophilization conditions revealed several important points:

• cell extract quality is only preserved when cell-extract is mixed with the TXTL reaction buffer prior to lyophilization
• the preservation of quality does not depend on the size of lyophilized extract aliquots

Phages were manufactured based on three components: the cell extract, an energy solution (ATP, GTP, NAD+ etc.) and a supplement solution (aminoacids, tRNAs, pholic acid etc.). The only additional component for phage assembly is pure phage DNA. We expanded the assembly platform by

• producing the first clinically relevant phages at therapeutic concentrations
• manufacturing of therapeutic phages independent of a living pathogen
• achieving similar phage titers as the commercial cell extract
• determining the amount of DNA necessary for sufficient phage assembly
• calculating the amount of DNA produced in the cell extract by replication

Phactory has is the ability to assemble various bacteriophages, in a bacteria-independent manner. To underline this feature and demonstrate universal applicability, we assembled a variety of different E. coli phages, both DNA and RNA-based.

To prove the influence of the DNA concentration on the bacteriophage titer, cell extract reactions were prepared with varying T4 DNA concentrations. The bacteriophage production was performed. The titer of the bacteriophages was measured with the top agar method and the formed plaques were counted. The increase in DNA concentration results also in an increase in the bacteriophage concentration. This increase is nonlinear and our model predicted. This finding is probably due to a critical concentration of phage proteins, which have to be reached for capsid head assembly (similar to a critical micelle concentration).

In the TX-TL system should be possible to modify phage proteins without altering their genome. This was attempted by modifying HOC (highly immunogenic capsid protein), which is part of the capsid protein structure of the T4-phage. Therefore, His-TEV-YFP-HOC was separately expressed and the purified protein was applied to our phage assembly.

For protein modification of the T4 capsid protein HOC, a plasmid for protein expression was cloned and His-YFP-HOC (82kDa) was expressed. The plasmid was transformed into BL21, expressed and puryfied by nickel affinity chromatography and gelfiltration.

FILM

MODEL

After the two chromatography steps, the purified protein had a final concentration of 70µM and no by-products were visible by silver staining. The CD-spectra (minima of 218nm) of the protein corresponds to the model and the Ramachandran plot indicates that the protein was not denatured during purification. Then, the purified protein was intentionally denatured by thermal transition. Thereby, it was confirmed that the protein is stable below 40°C. This is of importance, as phage assembly is performed at 29°C.

The purified protein was added to the assembly mix. Bacteria were transfected with the modified phages. Unbound phages and protein were removed by centrifugation. Fluorescence was measured in dependence of the proximity to the bacteria. Theoretically, YFP intensity should correlate with the binding of YFP-HOC modified phages to the bacteria.

Plaque Assay verifies functionality of our manufactured phages and cell extract

• we are able to produce functional phages in our cell extract
• the cell extract of our optimized strain has an endotoxin content below the detection limit and our regular self-produced cell extract has fewer endotoxins than the commercial cell extract
• next-generation sequencing allowed us to accurately quantify contamination
• phenol-chloroform extraction leads to a large amount of contaminating DNA which complicates phage assembly
• next-generation sequencing helped us to improve our purification protocols, leading to improved phage assembly

We performed a Plaque Assay to determine the activity of the viable phages (titer) in our assembly batch. By creating serial dilutions, we were able to calculate a plaque forming units/milliliter (PFU/ml) value. The plaque assay protocol was used.

A calibration curve using known endotoxin concentrations is required for the LAL-Test. A dilution series ranging from 0.625 EU/ml to 5 EU/ml. The fitting curve is used to interpolate the concentrations in the unknown sample. The linear fit of the calibration curve had a R2 of 0.98, an intersection with the y-axis at 0.38 and a slope of 0.39 ml/EU. These values are in accordance with the requirements of the LAL-Test manufacturer.

Removal of endotoxins is impeded by their tendency to form stable interactions with other biomolecules2. Our method of preventing the lipid A biosynthesis is therefore superior to extensive isolation steps required for removing endotoxins in conventional phage production.

Besides the cell extract, the DNA quality is the key to phage assembly. Unpure DNA (Host DNA, proteins contamination) inhibits the assembly of the phage within the cell lysate.

Our first attempts with classical chloroform/phenol extraction failed because of low molecular DNA (agarose gel). Therefore, we decided to search for other purification protocols, DNAseI and the Norgene kit (46800) tremendously increased DNA purity. With this DNA, we were finally able to achieve high titers by assembly.

As the results indicate, the main contaminant in chloroform phenole extratction is E.coli, as the origin for the native phage DNA. DNAseI and the Norgene kit led to reduction of E.coli contamination.

We sequenced several phage genomes after preparation using …. After receiving the phage genomes, one of the first steps to do is analyse the received sequences. This has been done using an inhouse software poreSTAT [1]. For each sequencing sample, it is analysed how many bases are sequenced, what bp yield has been achieved and how many pores were used. Considering the sequence in which the reads have been acquired, it can nicely be seen how the used chip gets worn out with each sequencing experiment. While for the T7 sequencing almost all pores are usable, the used pores decreases with every sequencing experiment … _*_* 4 missing figures _*_* While the T7 experiment produced most reads and most base-pairs sequenced, the remaining three experiments produced less data. The read and base-pair yield can be seen in Table 1.

TABLE

The actual task here was to assemble the phage genomes from the Nanopore reads. Particularly Nanopore sequencing is well suitable for genome assembly, since its long reads allow to reduce ambiguity of highly similar and repetitive sequences. Figure 2 shows the reads distributions of the sequencing experiments. _*_* 4 missing figures _*_* However, during the initial screening it could be seen, that the read lengths do not approach the thought length of the phage genomes in the 50-70kbp range for T7 and 100 kbp range for the remaining phages. Smaller reads generally lead to more ambiguity and thus are to be avoided from a bioinformatics point of view. However, since no bioinformatician can change the sequenced data afterwards, we went on with the given data. There are several tools available for de-novo genome assembly in general, however most approaches are used for short-read genome assembly (2nd generation sequencing, Illumina) and employ a de bruijn graph approach. Here we have 3rd generation sequencing data which must be handled totally different from old short-read sequencing data: the reads are less perfect in terms of sequencing errors. While short-reads nowadays have error-rates of about 1% (e.g. 1 base out of 100 is reported incorrectly), this error is up to 15% for nanopore sequencing data using newest sequencing chemistry (R9.4 at the time of wiki-freeze). Thus the number of available assemblers drops dramatically, where canu [2] and miniasm [3] are the most prevalent ones. Both rely on a overlap-layout-consensus approach, which, historically, can be seen as the father of all assemblers (see celera assembler [5]). We first used canu to assemble our genomes. Unfortunately we have been confronted with a major problem: contamination. We thus developed sequ-into to first detect the contamination and also get rid of contamination-originated reads. More on the performance and finding while using sequ-into can be found at …[link to /Software]. Particularly for assembly, contamination is bad because it can lead the assembler into wrong directions – depending on the phylogenetic distance of the original sample and the contamination. After getting rid of the contamination we noticed some strange patterns in the phage genome assemblies after re-aligning the reads to the assembly, which can be seen in Figure 3. *_*_ missing figure *_*_

In theory we can expect a uniform coverage over the full genome, since there is no bias for read template generation during sample preparation (thanks to random primers). However, what we saw here is that the first part has lower coverage than the remaining part and there is a high-coverage region in the middle of the assembled genome. Since we know phage genomes may have repitions, we thought to splite the genome in the middle and reorganise the structure to no avail. We thus tried to use the other assembler, miniasm, which is known for very fast assemblies, with little error correction. However, this error correction can be achieved by combining miniasm with minimap for read mapping and racon for polishing the sequences. Thus, the assembly pipeline changed to the following calls:

MAY BE COLLAPSIBLE #!/usr/bin/env sh INREADS=$1 ASMFOLDER=$2 ASMPREFIX=$3 THREADS=$4 if [ -z "$4" ] then THREADS=4 fi # path to used executables MINIMAP2=minimap2 MINIASM=miniasm GRAPHMAP=graphmap RACON=racon # first we must overlap all reads with each other $MINIMAP2 -x ava-ont -t$THREADS $INREADS $INREADS > $ASMFOLDER/$ASMPREFIX.paf # then miniasm can create alignment $MINIASM -f $INREADS $ASMFOLDER/$ASMPREFIX.paf > $ASMFOLDER/$ASMPREFIX.gfa # extract unitigs from miniasm awk '$1 ~/S/ {print ">"$2"\n"$3}' $ASMFOLDER/$ASMPREFIX.gfa > $ASMFOLDER/$ASMPREFIX.unitigs.fasta # align reads with unitigs $MINIMAP2 $ASMFOLDER/$ASMPREFIX.unitigs.fasta $INREADS > $ASMFOLDER/$ASMPREFIX.unitigs.paf # find contigs from unitigs $RACON $INREADS $ASMFOLDER/$ASMPREFIX.unitigs.paf $ASMFOLDER/$ASMPREFIX.unitigs.fasta > $ASMFOLDER/$ASMPREFIX.contigs.fasta ~/progs/minimap2/minimap2 -x map-ont -a -t$THREADS $ASMFOLDER/$ASMPREFIX.contigs.fasta $INREADS > $ASMFOLDER/$ASMPREFIX.reads.mm2.sam $GRAPHMAP align -r $ASMFOLDER/$ASMPREFIX.contigs.fasta -d $INREADS -o $ASMFOLDER/$ASMPREFIX.reads.gm.sam

And can be started simply from the command-line using: ./assemble.sh "FQ file" "PATH TO ASM FOLDER" "PREFIX of output" This finally led to a good assembly after rearranging the middle part which initially was “over-expressed”.

After having a core genome we want to check how many protein-coding genes we can find on the genome. For this task, again several programs exist. Two of the more common programs are glimmer [7] and genemark [8]. Because the reputation among the target audience is higher for genemark [9] we used this tool for the genome annotation. We ran the tool on the assembled genome in FASTA format generating a gene annotation file (gff3) for the genome highlighting all coding sequences. For easier and more compact usage, we transformed the genome in fasta format with the annotation in gff3 into the embl flat file format. Finally, we can describe the assembled genomes as follows: TABLE

Using the embl flat file format we visualized the phage genomes in a circular genome diagram plot (Figure 5). *-+-+* figures *_*_* Here we can see see several things. For the 3S genome, we can see that at certain position we see a high decrease in the coverage (at 58kbp, 72kbp and 110kbp). At these positions no reads align to the reference genome. For the NES genome we can see a similar behaviour at 330kbp. Additionally we can see two spikes at the ends of the genome. However, we could also see … Finally the FFP genome again has the same problems as the NES genome ends. Some final bliblubb …

Phactory yields phages with toxicity levels that allow oral administration to the patient. However, oral delivery requires protection of the phages from rapid degradation in the acidic gastric juice, while direct intravenous application requires additional purification steps. To overcome these hurdles, we prototyped two 3D-printed fluidic devices that can be assembled for less than $5. For oral application, we built a nozzle to encapsulate the phages in monodisperse calcium-alginate microspheres protecting them in the stomach. The alginate solution, ejected from a dispenser needle with a syringe pump, was sheared off by a parallel stream of air. Our results show that after 1 hour incubation in simulated gastric fluid, active phages are successfully released in simulated intestinal fluid. For intravenous administration, we can purify the bacteriophages from the remaining cell-extract via fractionation in a pressure-driven size-exclusion filter system. Additionally, we built microfluidic hardware for our human practice project OraColi.

- Alginat für lokale pH wert erhöhung im magen und auflösung in chelatoren(darm), da Ca2+ Ionen quervernetzen

- Enkapsulierung durch Co-Flow System. Druckluft schert Tropfen von Spritzennadel ab, Düse == Hardware  Quervernetzung 1h in CaCl2 Lsg  Waschen  Verifikation durch Mikroskopie  Fertig für Einsatz (insg. Ca 1.5-2h)

Experimente 1. 3D Modell von SYBR Gold gelabelten Phagen im Droplet durch z-Stack (BF/GFP) 2. Darkfield Bilder von gelabelten Droplets (bild) 3. Droplet Zusammensetzung (Viskosität zweier Alginate) 4. Droplet Größen bei Flussrate und Druck 5. Verhalten im Magen bzw. saurem Millieu (pH: 1) und Pepsin (1h @37°C, 10h @ RT) -> Phagen Degradation (barplot) 6. Verhalten im Darm bzw. pH 7 und Pankreatin (2h @ 37°C) ->Phagen relase über Zeit (plot)

1. Zetsche, B, Gootenberg, J.S., Abudayyeh, O.O., Slaymaker, I.M., Makarova, K.S., Essletzbichler, P., Volz, S.E., van der Oost, J., Regev, Aviv, Koonin, E.V., Zhang, F., 2015. Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell 163, 759-771.