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
Assessing Phage Functionality by Plaque Assays
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.
Assessing Endotoxin Levels
Msb-B Knockouts Reduce Endotoxin Levels By 49-Fold
Endotoxins are pyrogens deriving from gram-negative bacteria. Their mini from any pharmaceutical product is mandatory. Therefore, or Phactory, we engineered an E. coli strain lacking lipid A, a major endotoxin component and used this bacterium to produce our cell extract To evaluate endotoxin content of different cell extracts, a Limulus Amebocyte Lysate (LAL)-test was performed according to the supplier manual. As a reference, we compared the cell extract from our msbB-deficient strain (K2) to a cell extract from a wild-type strain (K4) as well as a commercial cell-free system (myTXTL, Arbor Biosciences). A solution with live E. coli served as a positive control.
Compared to the K4 strain our msbB-deficient K2 cell extract had 49-fold reduced endotoxin levels (0.06 EU/ml compared to 2.94 EU/ml). Other cell extracts such as the P15 cell extract (3.83 EU/ml) and the commercial myTXTL (4.65 EU/ml) had even higher endotoxin contents.
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.
Nanopore Sequencing enhances DNA purification
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.
Phage Genome Engineering
An additional advantage of Phactory is the possibility of rebooting bacteriophages from their genomic template, which is especially important for the genetic engineering of bacteriophages. With the self-made cell extract it was possible to manufacture an engineered MS2 RNA phage, where a polyhistidine-tag was added on the phage RNA polymerase. The genome template was generated via simple cloning methods such as gibson assembly and PCR amplification. After purification of the engineered genome, phages were assembled in our self-made P15 cell extract. A plaque assay confirmed the successful assembly of functional phages with a titer of 3*10^7 PFU/ml.
To test the his-tag modification the Phages were amplified in a bacterial culture flask. After lysis of the bacteria the tagged polymerase was purified from the supernatant by nickel affinity chromatography. An SDS-PAGE proved, that the 62 kDa Protein remained in the Nickel column due to the successfully engineering of an inserted His-tag.
