Difference between revisions of "Team:Munich/preResults"

To assess functionality of cell extract, testing of protein expression is necessary, which can be achieved by expressing fluorescent proteins and measuring the fluorescence time trace in a plate reader. To find the optimal quality control we compared the performance of different fluorescent proteins in cell extract.

We chose expression of mTurquoise – a variant of Cyan Fluorescent Protein (CFP) – as our favorite quality control for cell extract, as it results in high fluorescence intensity in a reproducible manner.

In addition, to examining protein expression we decided to analyze transcription uncoupled from translation by transcribing an RNA aptamer that binds a fluorescent dye.

We compared the fluorescence time trace of a Malachite Green binding aptamer in different cell extract samples. The fluorescence time traces decline after 30-50min indicating, that RNA degradation starts to prevail over transcription. Differences in the observed kinetics can be explained by variations in cell extract composition.

The main question was, if the different performance of the cell extracts (P15, E10, myTXTL), especially concerning phage titers, is due to a divergent composition or caused by a discrepancy in activity of the relevant proteins e.g. transcription and translation related proteins Therefore, one student was sent to Bavarian Biomolecular Mass Spectrometry Centre (BayBioMS), where he analyzed the samples under the supervision of Dr. Christina Ludwig.

The results of the Mass Spectrometry gave us insight into the composition of the different TX-TLs. We were able to identify 1771 proteins in all the extracts. The results indicate a high homogeneity between all three TX-TLs, as illustrated in the heatmap. Based on the LFQ values (Label Free Quantification) a volcano plot of two samples (Arbor/E10, Arbor/P15 and E10/P15) was generated. In general, there is no protein more frequent in Arbor TX-TL in comparison to E10 and P15. In contrast, some translation related proteins were slightly more abundant in E10 and P15 like certain tRNA ligases (PheS and PheT). The RecBCD subunits (the dsDNA degrading complex) is of similar abundance in all TX-TLs, showing the importance to consider its negative impact on the assembly. The results were further validated with DAVID, a tool for finding metabolic pathways based on proteomic data. The identified pathways indicate no correlation with transcription/translation related pathways (data not shown).

We concluded that the performance difference is most likely due to decreased activity during TX-TL preparation rather than a change in composition.

We tested the expression quality of several of our home-made cell extracts from fresh and lyophilized aliquots. This comparison also includes the commercially available myTXTL® cell-free expression system (arbor biosciences) and highlights two important results of our efforts to optimize production of cell extract:

• our improved cell extract can be lyophilized and retains 40-70 % of its performance
• our final settings for cell extract preparation yield extract that clearly outperforms the commercial system, with up to 9-fold higher protein expression

An essential part of the bacteriophage manufacturing is the DNA. For the extraction of the bacteriophage DNA we started with a Phenol-Chloroform extraction protocol (link). To purity was controlled with an agarose gel, where two distinct bands where present in for all four samples the proper high mass band and a low mass band.

To reduce the amount of the low mass bands the protocol was changed to a column based technique (link). With this technique the incorrect band where significantly reduced in all samples, which was proven with an agarose gel.

We determined the concentration of phages assembled in our cell extracts P15 and E10, which had the most promising results in our cell extract quality control. We compared our extracts to the commercial extract (myTXTL) and saw a similar performance.

All in all our extract has the same phage assembly potential as the commercial cell extract. Moreover, lyophylization, which makes the cell extract more long lived, does not reduce the effiency of bacteriophage assembly. We concluded that phages could be assembled on site in a durable lyophilized cell extract.

The successful assembly of all phages was confirmed by plaque assay and transmission electron microscopy (TEM). In addition, DNA encoding for NES and FFP phages was used to perform assembly of these phages in our cell extract. However, we were not in possession of the respective host bacterial strains and therefore could not demonstrate successful assembly.

Pathogenic bacteria such as salmonella, pseudomonas and staphylococcus are prone to develop multi-drug resistance and pose an urgent or serious threat (Centers for Disease Control and Prevention, 2013. Antibiotic/Antimicrobial Resistance.). Therefore, to fulfill this medical need, phages specific for these bacteria should be assembled next in our cell-free system. Potentially, this would require co-expression of the respective sigma factors that are needed for initiation of transcription.

The DNA sequence added to the cell-free system serves as the template for the required phage. We saw, that DNA-Polymerases can amplify the DNA segment, multiplying the amount of DNA in the cell-free reaction.

To assess this effect and its dependence on deoxynucleotide triphosphates (dNTPs), we performed an absolute quantification of T7 DNA in the cell-free reaction by quantitative PCR (qPCR). A standard curve with a serial dilution of T7 DNA. We used the TXTL qPCR protocol (add link). We used two selected cell extracts from us (P15 and E10), which reached similar phage titers compared to the commercial cell extract (myTXTL) in previous experiments. We compared the replication potential in comparison to myTXTL.

The addition of dNTP to the reference reaction leads to an increase in DNA concentration by a factor of 15 in the reaction after 4 hours (290 ng compared to 19 ng). This is higher than in the myTXTL reaction without additional dNTPs, in which there is a 1.8-fold increase in DNA (91 ng compared to 51 ng) after the 4-hour reaction. The home-made cell extracts P10 and E15 however do not resemble this behavior.

It would be desirable to increase DNA amplification in our cell extracts. We therefore conducted a cause analysis, focusing on the T7 replication system. A more than 250-fold increase in processivity of the T7 DNA polymerase is achieved by its binding behavior to E. coli thioredoxin1. We suspected reduced presence of this factor in our cell extract. Thioredoxin could be added to a phage assembly reaction to further test these assumptions. However, our proteome analysis did not confirm that there were low levels of thioredoxin present in our cell extract.

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.

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 × 10⁷ 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.

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.

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.

In order to achieve defined phage concentrations and therefore defined doses, we optimized the monodispersity of our alginate droplets.

In our initial attempts to create alginate droplets the size within a batch often varied significantly. Additionally, due to aggregation a lot of droplets were lost. Optimization of parameters such as flow rate, alginate concentration and N2 pressure led to an increase of monodispersity for all tested sizes (50-300 μm). Specifically, an alginate concentration of 1.8 % alginate and 0.2 % low-viscosity alginate proved to be ideal. Pressure and flow rate determine the droplet sizes.

The main problem of oral application is the acidic environment in the gastric fluid, necessitating protective measures against degradation. The other requirement of phage protection is the release of functional phages in the intestines. For this reason we compared the behavior of the encapsulated phages and non-encapsulated phages in simulated gastric fluid (SGF) and simulated intenstinal fluid (SIF).

In SGF, the number of active non-encapsulated phages decreases by more than 99.99 % within an hour. This shows the urgent need of a form of protection against degradation to make oral application of bacteriophages possible. As a reference, we used phages that were chemically released by citrate from alginate droplets.

In comparison, the encapsulated phages were tested in SGF for the same time as the non-encapsulated phages. Afterwards, the same droplets were exposed for two hours to simulated intestinal fluid to test the release of functional bacteriophages in this environment.

The encapsulated phages were barely released in an hour of exposure to SGF. After transfering the capsules to SIF the number of active phages reached that of the undegraded reference. This indicates that the encapsulation of bacteriophages in alginate capsules enables the possibility of an oral application. Further experiments could test the alginate capsules in an animal model system.