The Baccelerator: Back on track…

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

3A assembly

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

RipX/CodV system Remove resistance Ethics

Surface Display Possible Candidates

LytB Modifier protein 2115bp Expressed in stationary phase

LytC Autolysin 1488bp

LytD Autolysin 2640bp Orientation unknown

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

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

Signaling: ComD and ComE

ComD 25bp overlap with plasmid 25bp overlap with plasmid Prefix RBS Scar ComD Suffix 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

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

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

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.

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.

Signal recognition peptide - ribozyme

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

TMHMM analysis of topology: Inside to Inside: 8.

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

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…

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

Gold Medal - Judging Criteria

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

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

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.

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).

GFP

GFP GFP TEV

TEV TEVs GFP

TEVs GFP HIV1

[t,p_ts]=ode45(@fp_tsprime,t,0); TEV TEVs 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