1. Encapsulation for Drug Delivery
7th August
iGEM Team 2009
Charles
Dave
Dineka
James
Kun
Nuri
Royah
Tianyi

2. The Project
“Auto-encapsulation of protein drugs for release in the small intestine.”

3. Gantt Chart ACTIVITIES Week 4 Week 5 Week 6 Week 7 Week 8 Week 9
MIT Project description
Assay Protocols
Cloning strategy
Genetic Circuits
Biobricks
Genes to PCR
Wet lab plan
Gantt Chart
Dry lab plan
Order genes
Order Biobricks
Start Wet Lab
PCR & Test Promoters
Timer + M1 integration M2 M3
Start Dry Lab
Genes delivered
Biobricks delivered
Complete
In progress
To do
Unknown

4. Modules 1 & 2: Update

5. M1 - Hunt for Cellulase
No clone readily available.
Professor input?

6. M2 - Sodium Acetate Food grade RAPID inducible crystallisation
Exothermic
Dissolves under acidic
conditions

7. The Timer Module Integration

8. Timer Design Rules
1. Try rely on activation, not repression. (Problem with degradation rates.) 2. Inputs must accumulate over time to see delays. 3. Last promoter must be as “non-leaky” as possible. 4. Tunability - linked to inducible promotor.

9. Timer - Logic A
PoPS
(Encapsulation) AB B
Inducible
Constitutive

10. Tunability LuxI M2 Lux R + I PoPS LuxR Start Timer – v1 PBAD PLux X
Kirsten’s
favourite
Tunability
PBAD
PLux
LuxI M2
Lux R + I
PoPS
(Encapsulation) X
LuxR
Start

11. ! PAH or Cellulase LuxR LuxI Encapsulation (M2) genes PBAD PluxR X M1
Conflict between:
Start of experiment
Threshold setting X
PBAD
LuxI
LuxR
Timer M2
PluxR
Encapsulation (M2) genes

12. ! PAH or Cellulase LuxR LuxI Encapsulation (M2) genes PBAD PluxR
Conflict between:
Start of experiment
Threshold setting
PBAD
LuxI
J23114
LuxR
Timer
PluxR
Encapsulation (M2) genes M2

13. X Anti-LuxI LuxI M2 Lux R + I PoPS LuxR Timer – v2 PBAD PLux J23114
Bba_K145013
Anti-LuxI
PBAD
PLux
LuxI M2
Lux R + I
PoPS
(Encapsulation)
J23114
LuxR

14. Tunability of IPTG in lacY-strains
Induction of lac-regulated promoter in lacY- and lacY+ strains, from Jensen et al. Eur.J.Biochem, 211, (1993)

15. PluxR Output

16. PTET Lactonase luxI tetR M2 PoPS LuxR Timer – v4 PLAC PLux Lux R + I
Td (lactonase) =4.7hrs
PTET
Lactonase
Bba_C0060
Ts (TetR)
PLAC
PLux
luxI
tetR M2
Ts (LuxI)
Lux R + I
PoPS
(Encapsulation)
J23114
LuxR

17. Dry Lab Preparation

18. Dry Lab So far… Designed & ordered primers
Designed TaqI, DpnII & cI BBs
Testing construct design
Clones ordered (Kirsten)
Started step-by-step protocols & shopping list
Next week…
Place MrGene order
SLIC preparation
Complete step-by-step protocols & shopping list

19. BioBricks Remaining… Promoter = Repressible (lambda cI) M3 Part
BBa_K [Harvard ‘08]
Part
Obtain by…
Diagram
CI (temperature sensitive)
Synthesise? / PCR?
Promoter = Repressible (lambda cI)
Synthesise (different sequence)
Registry

20. Wet Lab Parts, Testing & Progress

21. So Far… Competent cells – 4 x 10^6 cells/ug DNA 0 contaminants
No native resistance

22. So Far…
Registry - 8 taken out

23. Next week… Registry – extract timer BBs
Start PCR – M1 (2), M2 (5) & M3 (1)
Start to characterise promoters (Jason Kelly)
Organise teams (1 bioscientist, 1 bioengineer)
- team 1 = PCR
- team 2 = promoter testing

24. Summary - Checklist Weekly 5 Aims: Trade name Assay Protocols
Cloning Strategies
Finalise genetic circuit
Order BioBricks
Primer design
Wet lab plan

25. Summary - Checklist Week 6 Aims: Place MrGene Order (Wednesday)
Assay Protocols (Monday)
Cloning Strategies (Monday)
Wet lab
– PCR & test promoters & extract BBs
Dry lab tutorial

26. Any questions?

27. Timer – v3 (Lock/Key system)
PtetR
Bba_K145300
J23109
Lactonase
LVA
BBa_K145102
Lock + Key
PLux
J23114
LuxI M2
Lux R + I
PoPS
(Encapsulation)
J23114
LuxR