1. The Baccelerator: Back on track…

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

4. 3A assembly

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

6. RipX/CodV system
Remove resistance
Ethics

7. Surface Display
Possible Candidates

8. LytB
Modifier protein
2115bp
Expressed in stationary phase

9. LytC
Autolysin
1488bp

10. LytD
Autolysin
2640bp
Orientation unknown

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

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

13. Signaling: ComD and ComE

14. 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

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

16. 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

17. 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.

18. 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.

19. Signal recognition peptide - ribozyme

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

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

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

23. 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…

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

25. Gold Medal - Judging Criteria

26. 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

27. 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

28. 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.

29. 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).

30. GFP

31. GFP
GFP
TEV

32. TEV
TEVs
GFP

33. TEVs
GFP
HIV1

34. [t,p_ts]=ode45(@fp_tsprime,t,0);
TEV
TEVs
t=[0:0.1:20];
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