Baccelerator• Overview• Assembly strategy• Testing strategy• Issues• Modelling

Coloured products

Viral Protease

Current MechanismActivationsignal

Pre-made FRET pair

C

Ex IntA1 CMD stop LacI stop Suffix Ex IntA2 sp

1 2

3

p SB1C

K 74

IntA2 sp

1 23Ex IntA CMD stop

p SB1C

IntA1

stop

IntA2

SPEC1

2

IntA1

SPEC stop

IntA2

SPECIntA1 IntA2stop

IntP1 IntP2SPEC stop

p SB1C3

3

Com E Com DSurface Protein

Coil (Reporter)

Pyrd

LacI stopCoil

(Reporter)

TEV Lac Ibinding

AmyE

RC

RC

Surface Display

Cell Wall Proteins CwlC

765bp 2 cell wall binding repeats

LytC 1488bp 3 cell wall binding repeats

Previous Research Everything except the CWBs removed Both successfully attached to a lipase Retained function on the cell surface When both combined, 36% of cell wall protein

B. sub native so can perform PCR Linker/peptide can be attached to primer

Options

Testing

Increased salt concentration disrupts CWB Can test for production and rough localisation

Attach His-tag and linker Can test for precise localisation and cleavage

Modelling

10 ng/ml is the required conc. of the peptide to activate ComD

2.24 kDa is the mass of the peptide

If there is total cleavage in a small area, resultant conc. is 1000x required

750nm200nm

Additional

Two BioBricks Schistosoma protease specific linker/signal, which

could be formed into a series of parts responding to different parasites

Cell wall binding segment, which when attached would allow you to display anything on the surface of B. sub

Modelling

GFP

GFP

TE

V

GFP

TE

V

TE

Vs

GFP

TE

Vs

GFP

HI

V1

GFP

0 10 20 30 40 50 600

5

10

15

time

conc

entra

tion

of G

FP

GFP

TE

V

0 10 20 30 40 50 600

10

20Production of TEV

0 10 20 30 40 50 600

5

10Production of split GFP

0 10 20 30 40 50 600

500

1000Production of GFP

TEV

TEVs

GFP

0 20 40 600

10

20Production of TEV

0 20 40 600

5

10Production of split TEV

0 20 40 600

1000

2000Production of fused split TEVs

0 20 40 606

7

8Production of split GFP

0 20 40 600

5

10x 10

4 Production of GFP

TEVs

GFP

HIV1

0 20 40 600

10

20Production of HIV1

0 20 40 600

5

10Production of split TEV

0 20 40 600

1000

2000Production of fused split TEVs

0 20 40 606

7

8Production of split GFP

0 20 40 600

5

10x 10

4 Production of GFP

The System1. Transcription and translation of Protease detector Peptide

2. Transcription and translation of ComD

3. Transcription and translation of ComE

4. Cleavage of AIP, activated by Protease

5. Activation of ComD receptor, reaction activated by AIP

6. Phosphorylation of ComE, activated by ComD receptor

* Release of Protease (artificially triggered)

Equations

• 1. Transcription and translation of Protease detector Peptide

• 2. Transcription and translation of ComD

• 3. Transcription and translation of ComE

Equations

• 4. Cleavage of AIP, activated by Protease

• 5. Activation of ComD receptor, reaction activated by AIP

• 6. Phosphorylation of ComE, activated by ComD receptor