Category:Team T4

Figure 1 shows the SDS-PAGE of T5 protein components. DIT is a distal tail protein which is a small component of a tail tip protein complex. Figure shows that pb9 has a low molecular weight around 25 kDa.

Figure 1 shows the SDS-PAGE of T5 protein components including pb3 protein. Since pb3 is one of the larger proteins that make the viral tail complex, the SDS-PAGE shows that pb3 has a high molecular weight of more than 75 kDa than other tail protein components.

Figure 1 shows the SDS-PAGE of T5 protein components including a straight tail fiber (pb4). The SDS-PAGE shows that the molecular weight of the straight tail fiber protein (pb4) to be around the 75 kDa marker.

Fig 3

T7gp0.3 (deleted) mutants grew on non-restricting hosts but failed to overcome DNA restriction of other E. coli hosts.

. In these two bacteriophages, LTF function is essential to penetrate the shield of the host’s O-antigens. We also demonstrate that LTF-mediated adsorption becomes superfluous when the non-specific cell protection by O-antigen is missing, allowing the phages to bind directly to their common secondary receptor, the outer membrane protein BtuB.

figure 4. The similarity of the a.a. sequence regions in LTFs of T5-like phages with other viruses are indicated by similar colors.

In figure 6, y-branched linear dsDNA is incubated with a GST/uvsW fusion protein and single-stranded products are observed, indicating the unwinding (i.e., helicase) activity of the protein. In lane 9 of the gel, removal of ATP from the reaction mixture abolishes the observed unwinding activity.

In Figure 4, a reaction mixture including DNA primase was incubated to ensure initiation of DNA replication. After incubation, RNAseH was added and hydrolyzed, radiolabeled primers were observed by gel electrophoresis.

In figure 3, bands corresponding to radiolabeled RNA oligonucleotides are observed after gel electrophoresis. DNA with replication machinery including a polymerase modified to exclude proofreading activity was incubated with RNAseH and the reaction products were analyzed by gel electrophoresis.

Fig 5 -- Removal of RNA primers radiolabeled with phosphorus was quantified by gel electrophoresis.

FIG. 4. Oligonucleotide products of the 5’ to 3’ DNA exonuclease activity of T4 RNase H

Figure 7B shows that globomycin inhibits the processing of lipoproteins which is shown as no expression of llp (no bands) at 20 ug/ml and 120 ug/ml. At 5 ug/ml globomycin, processing of lipoprotein still occurs but is still inhibited since there is still expression of precursor lipoprotein (p-Llp). With no globomycin, Llp is processed as shown with a band near the 8kDa marker but no expression at around 9 kDa. This suggests that Llp is a lipoprotein and must be processed in order to bind to the FhuA protein on the periplasmic side of the cell outer membrane.

The data from Table 2 suggested that Llp blocks the adsorption of phage T5 to the FhuA receptor protein on the host cell membrane. T5 adsorption was carried out for 10 minutes on medium plates at 37 deg C. T5 adsorption was measured by using standard cell and phage concentrations from another experiment. For JM101/(pVK3 – vector with no mutations and carries functional oad and llp genes), 82% of phages were not adsorbed to FhuA. For JM 101/(pVK3/IS1-2 – vector with an insertion downstream of oad and llp genes), 85% of phages were not adsorbed to FhuA. JM101/(pLG339 – vector with no llp gene), only 12% of phages were not adsorbed. This data suggests that llp blocks adsorption of T5 to FhuA.

Figure 8 shows the expression of T5 proteins during infection of E. coli F cells. All proteins were separated on a SDS-polyacrylamide gel and samples were analyzed at six time points. Llp was synthesized 5 minutes after infection and Llp showed the most expression at 10 minutes. In later time point, Llp was expressed less. This suggests that Llp was produced during early transcription.