4.15 w13 final presentation


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4.15 w13 final presentation

  1. 1. Selecting for Small Small Φ group W13 final presentation
  2. 2. Outline  Background  Application  T7 structure  Genome  In vivo assembly  In vitro assembly  Project  The big picture  Recent results  Plans for spring
  3. 3. Background – Application Mateu M. G., Protein Engineering, Design & Selection, 2011, 24(1–2), 53–63  Natural viruses and their capsids are not optimal.  Size is an important issue  Two methods for virus capsid studies  Site directed mutagenesis  Direct evolution
  4. 4. Background – T7 structure  Icosahedral shape  ~60nm in diameter  Mature capsid is 2nm thick  415 capsid proteins  90% gp10a and 10% gp10b  Volume:120 x 103 nm3
  5. 5.  Has double stranded DNA  Has 39,937 base pairs  Encodes for all the proteins necessary for DNA replication  Has many non-essential genes that can be removed Background – T7 Genome Enterobacteria phage T7 Minor capsid protein Major capsid protein DNA polymerase Connector protein Assembly/scaffolding protein
  6. 6. Background – in vivo Assembly  Capsid proteins bind the connector protein ring (gp8)  The capsid radially extends outward with the help of scaffolding proteins (gp9)  Gp9 is somehow ejected from the capsid  Terminase stuffs the DNA into the capsid  The capsid irreversibly expands as DNA enters  The tail proteins attach
  7. 7. Background – in vitro Assembly a: proheads Isolated from WT T7 b: 9-10 heads Scaffolding and head proteins c: converted heads Isolated from WT T7 d: 10 heads Scaffolding, head, and connector proteins Figure 1. Gel electrophoresis of purified proteins, intact heads, and dissociated heads. a, Head protein and scaffolding protein purified after expression individually from the cloned genes, and c, 10-heads isolated after co-expression of connector, scaffolding and head protein in the same cell, 9-10 heads isolated after co-expression of scaffolding and head proteins in the same cell, and proheads and converted heads isolated from wild-type T7 lysates were analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. b, The same set of heads, without disruption, were analyzed by electrophoresis through a 1% agarose gel (pH 8.0). of the purified proteins into prohead shells. Therefore, we expressed and purified these proteins individually from the cloned genes, as described in Materials and Methods. The purified scaffolding protein eluted at a position corresponding to a molecular mass of 49 kDa upon gel filtration chromatography in buffered 50 mM NH4Cl, whereas the size calculated Figure 2. Electron micrographs of proheads, converted heads, 9-10 heads, and 10-heads. Samples of a, proheads, b, 9-10 heads, c, converted heads, and d, 10-heads, described in more detail in the legend to Figure 1, were stained with 2% uranyl acetate and examined by conven- tional transmission electron mi- croscopy (the bar represents 100 nm). The small round particles apparent in the 10-heads, and to a lesser extent in the converted heads, are believed to represent a cellular component that bands at approxi- mately the same position as con- verted heads in the CsCl step gradients used. These particles do not have the characteristic appear- ance of T7 connectors (see Cerritelli & Studier, 1996) and the 10-heads contained little if any gp8 (Figure 1c). Cerritelli M. E., J. Mol. Biol., 1996, 258, 286-298  T7 capsids can be assembled in vitro utilizing plasmids  Basis for site-directed mutagenesis  Stability of such capsid require more research.
  8. 8. Project – Big Picture Capsid in vitro assembly Determine mutant capsid size Compare genome and capsid size Compare structure subunits Identify sites for mutation Design primer/plasmid for SDM Apply mutagen Select for plaque size Determine mutant capsid size Determine titer Site directed mutagenesis Direct evolution
  9. 9. Project – Big Picture Site directed mutagenesis Direct evolution
  10. 10. Project – Recent results 3/1 3/15 3/30 4/15 Start of iGem Start of small Φ group Select Φ for study • T7 • Qβ Design plan of attack SDM design for T7 and Qβ T7 arrive! T7 spot test and tittering experiment  Mutation design  Sequence comparison  Viability comparison  Spot test  Tittering result
  11. 11.  Mutation Design  Major/Minor Capsid Proteins  Minor capsid protein produced from ribosomal slippage in a series of T’s  Alter Genome Size  Knock out DNA polymerase Project – Recent results
  12. 12.  Sequence comparison (partial) Project – Recent results 57-59nm ~60nm
  13. 13.  Basic experimental procedure Project – Recent results Spot test Titering test
  14. 14.  Viability comparison  Top: BL21; bottom: W3110 Project – Recent results
  15. 15.  Spot test on BL21 Project – Recent results T7+ T7 new
  16. 16.  Titer result on BL21 Project – Recent results -5 titer -15 titer control -10 titer
  17. 17.  Determine concentration of the phage with titering experiments  Direct evolution  N-methyl-N’-nitro-N-nitrosoguanidine?  Site-directed mutagenesis  Isolate phage genome and clone genes into plasmid Plans for spring