Category:Team Blue B 2018

The T4 SegB protein is a site-specific endonuclease that recognizes a 27-bp sequence. 666 bp segB ORF was placed under the control of T7 promoter in pET-15b vector. SegB protein was overproduced in E. coli T7 lac promoter system and purified. The purified protein was incubated with a linear plasmid DNA and products were analyzed of the reaction through gel electrophoresis (Figure 3). SegB cleaved DNA by introducing double-strand breaks in reactions requiring Mg2+, Mn2+, or Ca2+ cations. With Mg2+ and Ca2+ cation, SegB cleaved the DNA at 1 point, creating 2.2 kb and 1.6 kb fragments. In the presence of Mn2+, multiple cleavage sites were present.

An in-vitro complementation assay was used to study incorporation of gp17 during assembly of viral particles. Each extract was separately mixed with 4 different components; water, complete tails purified from SPP1sus31 with gp17, tails without gp17 purified from SPP1sus82, and purified gp17. After incubation, the titer of the progeny was calculated in Figure 7. The expected result was sus31 tails in both extracts allowed it to join to the capsids for virion assembly. When mixing SPP1sus82 tails without gp17 with SPP1sus45, virion particles assembled, suggesting endogenous gp17 was able to join head and tail. SPP1sus82 had a low titer, thus tails weren’t able to attach. Finally, purified gp17 mixed SPP1sus82 extracts was sufficient for tail to head stickage. This assay shows that gp17 is involved in binding to the tail and later attaching to the DNA-filled capsid.

Figure 3. S105, holin, and S107, antiholin, are two transmembrane proteins produced by alternate starts on gene S in phage lambda. S105 accumulates into rafts, which are associated with the formation of large holes in the membrane. These holes allow endolysin to reach the peptidoglycan and result in cytolysis. S105 has three transmembrane domains. The difference between S105 and S107 is an extra N-terminal positive charge on S107, which prevents the first transmembrane domain from entering the membrane. The researchers created a mutant allele of S105 with the first transmembrane domain deleted, such that it would act like S107, and fused it to mCherryFP. Through deconvolution fluorescence imaging, they showed that the fusion protein blocked S105 raft formation, suggesting that antiholins prevent lysis by blocking the formation of holin rafts.

The R lysis cassette, including R endolysin and S holin genes was synthesized and inserted into a plasmid under regulation of arabinose and temperature sensitive promoters. The plasmid was introduced into Δasd E. coli χ6212 and the construct was named JOL1675. Under conditions inducing expression of the plasmid, presence of the R endolysin was confirmed by Western Blot and shown in Figure 2. A band at 17.7 kDa, the estimated size of the endolysin, was seen in cells with the plasmid expressed, but not in cells grown under conditions repressing expression. Lysis efficiency was confirmed by monitoring the number of viable cells, which declined to 0 after 48 hours of lysis, as shown in Figure 4. No viable cells remained, indicating that the endolysin effectively ruptured and killed the hosts.