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The Baccelerator: Back on track…

The Baccelerator: Back on track…. The Vector – pSB1C3. High copy BioBrick assembly plasmid. 3A Assembly Resistance gene Homology regions for integration. 3A assembly. Homology Regions. AmyE Alpha-amylase Starch metabolism PyrD Dihydroorotic acid dehydrogenase Pyrimidine metabolism

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The Baccelerator: Back on track…

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  1. The Baccelerator: Back on track…

  2. The Vector – pSB1C3 High copy BioBrick assembly plasmid • 3A Assembly • Resistance gene • Homology regions for integration

  3. 3A assembly

  4. Homology Regions • AmyE • Alpha-amylase • Starch metabolism • PyrD • Dihydroorotic acid dehydrogenase • Pyrimidine metabolism • EpsE • Inhibitor of motility

  5. RipX/CodV system • Remove resistance • Ethics

  6. Surface Display Possible Candidates

  7. LytB • Modifier protein • 2115bp • Expressed in stationary phase

  8. LytC • Autolysin • 1488bp

  9. LytD • Autolysin • 2640bp • Orientation unknown

  10. WapA/ WprA • Protein precursors • 7002bp/2682bp respectively • Heavily involved in cell wall turnover • C terminus known to be on the surface • Unsuitable

  11. Unknowns • YqgA – 426bp • YwsB – 534bp • YocH – 861bp – Amidase • PgdS – 1239bp – Hydrolase

  12. Signaling: ComD and ComE

  13. ComD • Total size: 1440bp • We’ll need to split this construct into two for synthesis • A PCR assembly method will then be used to join the two halves 25bp overlap with plasmid 25bp overlap with plasmid ComD Prefix RBS Scar Suffix

  14. ComE • Total size: 867bp 25bp overlap with plasmid 25bp overlap with plasmid ComE Prefix RBS Scar Suffix

  15. Notes on Synthesis • pSBC13 will be the vector • 25bp of overlapping sequence will be added to the end of the ComD and ComE constructs • This means that PCR can be used to BioBrick it into the vector, instead of using ligation

  16. Sequence Analysis ComD (S. pneumoniae) ComP (B. subtilis) BLAST search with ComD protein and NT sequence failed against the B.sub genome (cannot alter BLAST parameters in genome search) Alignment of aa-sequence ComP vs ComD showed very little homology No conserved aa-sequences between these molecules.

  17. General membrane targeting in B. sub: N: contains one R or K residues for interaction with negative charges in membrane H: hydrophobic helix core interrupted by glycine or proline which allow insertion into the membrane. C: can contain Spase I cleavage site. If not, equivalent to membrane retention signal.

  18. Signal recognition peptide - ribozyme

  19. Different ComD entries aligned. N terminal R and K residues ComP contains a KK at 5,6

  20. TMHMM analysis of topology: Inside to Inside: 8.

  21. ComD entries including R6 laboratory strain and 3 NCBI listed ones Outside to outside; 6.

  22. Problems: Little homology between ComD and ComP S. Pneumonia membrane targeting uncharacterized So far no other B.sub transmembrane protein detected which starts outside. Unknown how In-out orientation is determined. Unkown whether TMHMM is appropriate for gram + prediction. Positive: Nothing suggests that is should not work. Further, manual analysis of protein sequence of ComD might help to determine whether it would be targeted to the membrane…

  23. Testing of ComD membrane localization? SecA-GFP – inner membrane

  24. Gold Medal - Judging Criteria

  25. 9. Characterize or improve an existing BioBrick Part or Device and enter this information back on the Registry. • Parts that we could use from the Registry: Promoters for B.subtilis

  26. 10. Help another iGEM team by, for example, characterizing a part, debugging a construct, or modelling or simulating their system. Possible teams for collaboration: • Sheffield – use quorum sensing to detect V.cholerae

  27. 11. Develop and document a new technical standard that supports the: (i) design of BioBrick Parts or Devices, or (ii) construction of BioBrick Parts or Devices, or (iii) characterization of BioBrick Parts or Devices, or (iv) analysis, modeling, and simulation of BioBrick Parts or Devices, or (v) sharing BioBrick Parts or Devices, either via physical DNA or as information via the internet.

  28. 12. Outline and detail a new approach to an issue of Human Practice in synthetic biology as it relates to your project, such as safety, security, ethics, or ownership, sharing, and innovation. Consider Ethics Panel with Claire. Possible collaboration with other teams (We were thinking of a joint Ethics discussion).

  29. GFP

  30. GFP GFP TEV

  31. GFP TEVs TEV

  32. GFP TEVs HIV1

  33. TEVs TEV t=[0:0.1:20]; [t,p_ts]=ode45(@fp_tsprime,t,0); [t,p_t]=ode45(@fp_tprime,t,0); function [p_tprime,p_t] = fp_tprime(t,p_t) s_t = 1; d_t = 1; p_tprime = s_t - d_t*p_t; end function p_tsprime = fp_tsprime(t,p_ts) d_ts = 1; k_ts=1; p_tsprime = ((k_ts * p_t_max * p_st_max) / (km_ts + p_st_max)) - d_ts*p_ts ; end

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