Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

The overall goal of the following experiment is to screen for and characterize phage genes with unknown functions. First, putative phage. Viral open reading frames called ORFs are expressed in e coli.

Clones of the phage. Orff expressing e coli are inoculated into multi phenotype assay plates or maps, which contain growth, medium and various substrates. Bacterial growth is then monitored over time by spectrometry as a second method of analysis, phage gene expressing e coli are grown in either continuous culture or by serial batch culturing.

The resulting growth metabolites are then collected and sent for metabolomic analysis by gas chromatography, time of flight mass spectrometry. The resulting data offers a phenotypic profile associated with expression of a single putative phage open reading frame. This method was initially developed to elucidate the function of unknown vial proteins.

It can also be applied to studies looking at the link between genotypes and phenotypes, and in examining the interactions between bacteria phage and host across biomes. Begin this procedure by labeling sterile 96 well microtiter plates with the e coli clone identification number and map schematic type, which indicates the substrates that are being tested against in the assay. Aseptically transfer sterile water into a liquid reservoir.

Then using a multi-channel pipette transfer 60 microliters of water into each well of the microtiter plate. Next aseptically transfer three X basal medium into a liquid reservoir. Then using a multi-channel pipetter transfer 50 microliters into each well of the microtiter plate.

Using the same technique pipette 50 microliters of each basal medium used in the map schematic. Next, transfer 30 microliters of each substrate into the appropriate. Well then using a multi-channel pipetter transfer 10 microliters of bacterial cells into each well.

Be sure to change tips before reintroducing the pipette back into the culture stock. Cover each plate with an adhesive plate film. Firmly press the film atop the wells of the plate and along the edges to create an even and tight seal.

Using a sterile razor blade, remove any excess film from the edges of the microtiter plate. Place the prepared map in the input sleeve of a multipl spectrophotometer. Open the plate reader software and create a protocol to measure the absorbance every 30 minutes for a total of 32 hours.

With 60 seconds of shaking between reads, set the temperature in the apparatus to 37 degrees Celsius. Once the map is equilibrated to the set temperature, start the protocol after 32 hours, remove the map from the outputs sleeve of the plate reader. Label each data file with the e coone identification number, date, and the map schematic type.

Analyze the data as described in the accompanying document to assess growth and biologically relevant growth. Parameters From metabolite analysis cells may be cultured by either continuous or serial passage batch Turing. To maintain a steady state.

Here we'll demonstrate a continuous culture. Sterilize the components of a 75 milliliter continuous culture reactor, and a two liter feeding bottle as described in the accompanying document Following sterilization, place all of the autoclave materials into a biological safety cabinet and turn the ultraviolet light on while the medium cools. Once the medium is cooled, add 0.1%L arabinose, and 100 micrograms per milliliter.

Ampicillin to 75 milliliters of 0.5 XLB broth and transfer it to the continuous reactor. Unwrap the continuous culture reactor cap and screw it onto the reactor. Avoid touching the sampling tube, unwrap the feeding bottle cap and screw it onto the feeding bottle.

Avoid touching the sampling tube, keeping the 18 inch tubing attached to port epsilon sterile. Unwrap the 18 inch tubing and peristaltic pump tubing. Adapter fit one end of the 18 inch tubing onto one end of the peristaltic pump tubing adapter fit the other end of the peristaltic pump tubing adapter to the 18 inch tubing.

Attached port epsilon of the feeding bottle cap attach zero point 22 micron filter units onto ports, beta, and delta of the continuous culture reactor. Attach a zero point 22 micron filter unit on the adapter of port alpha. And to that, attach the 18 inch tubing in the biosafety hood.

Inoculate the continuous culture reactor with 100 microliters of an overnight culture of e coli prepared as described in the accompanying document. Tighten the reactor and feeding bottle lids and then move the system into a 37 degree Celsius incubator. Equipped with a magnetic stir plate and mini peristaltic pump fit the peristaltic pump tubing adapter into the peristaltic pump.

The tubing from the feeding bottle goes into the pump and leads out to the reactor. Set the pump to fast then to start it. Switch to forward check to ensure that medium begins to flow through the tubing.

Place the reactor on the magnetic stir plate and begin mixing. Allow the continuous culture reactor to equilibrate for 24 hours before sampling the next day. To sample the continuous culture, screw a five milliliter lure lock syringe onto port gamma and withdraw 4.5 milliliters of culture.

Use 500 microliters of the culture to measure the optical density at 600 nanometers. Then use the remainder of the aliquot to make four one milliliter cultures at an OD 600 of 0.35 and 1.7 milliliter micro fuge tubes to prepare the samples for gas chromatography, time of flight mass spectrometry or GC to F ms. Centrifuge the cells at 16, 900 times G for two minutes to pellet them.

After the spin decant, the supernatant then wash with 500 microliters of PBS. Perform this wash. Step a second time, decant the supernatant to final time, and then submerge the micro fuge tubes containing pelleted cells in liquid nitrogen.

Until the bubbling stops, store the samples in a minus 80 degree Celsius freezer. Repeat the sampling every 12 hours for four days after all of the samples have been collected. Ship three samples per clone on dry ice to a metabolomics core facility for sample processing analysis and normalization of GC to f ms to obtain viral open reading frames.

Seawater was collected from Starbuck Island and Caroline AOL of the Southern Line Islands below the coral boundary layer. Bacterial growth under 72 carbon specific conditions was assessed for 47 clones using maps. The resulting data were then used to categorize the clones as expected growth, gain of function, loss of function, and no growth phenotypes.

To test the hypothesis that clones harboring functionally similar proteins have metabolomics profiles. Median metabolite abundances were determined for 84 clones grown in continuous culture by GC TOF ms. The resulting data were then used to hierarchically cluster the clones based on their relative metabolite abundances, which are indicated by color.

The most abundant metabolites are shown in red, while the least abundance are shown in dark blue. Preliminary metabolomics analysis revealed that while structural and metabolic genes do not clearly separate from one another, those genes exhibiting similar effects on the host do correlate. For example, the annotated capsid gene clusters closely with the putative metabolic genes highlighted in this study EDT 24 40 and EDT 24 41 investigations using a publicly available transmembrane topology and signal peptide predictor program showed evidence that both putative metabolic genes harbor a single transmembrane domain.

Interestingly, five out of the nine clones in the first cluster group have predicted transmembrane domains using the same topology. Taken together these data that this method is able to provide information on clone metabolite profiles, potential clones with related functions and clone metabolite outliers. While attempting this procedure is important to remember that the methods presented are heavily influenced by bacterial physiology.

Considerations need to be made to ensure clonal independent groups are experimented with contamination is prevented and a single variable is being tested using the appropriate controls.