Encapsulation for Drug Delivery
description
Transcript of Encapsulation for Drug Delivery
Encapsulation for
Drug Delivery7th AugustiGEM Team 2009
CharlesDaveDinekaJames
KunNuriRoyahTianyi
The Project
“Auto-encapsulation of protein drugs for release in the small intestine.”
Gantt ChartACTIVITIES Week 4 Week 5 Week 6 Week 7 Week 8 Week 9 Week 10
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
Modules 1 & 2:Update
M1 - Hunt for Cellulase
No clone readily available.
Professor input?
M2 - Sodium Acetate
• Food grade• RAPID inducible crystallisation• Exothermic• Dissolves under acidic conditions
The TimerModule
Integration
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.
Timer - Logic
A
B
PoPS(Encapsulation)
AB
Inducible Constitutive
Timer – v1
LuxI
LuxRLux R + I
X
PBAD
PLux
PoPS(Encapsulation)
M2
Start
Tunability
Kirsten’s
favourite
PAH or CellulaseM1
LuxR
Encapsulation (M2) genes
LuxIPBAD
PluxR
Timer
!
XConflict between:• Start of
experiment• Threshold setting
M2
PAH or CellulaseM1
LuxR
Encapsulation (M2) genes
LuxIPBAD
PluxR
!Conflict between:• Start of
experiment• Threshold setting
J231
1
4
M2
Timer
Timer – v2
LuxI
LuxRLux R + I
J231
1
4
PBAD
PLux
PoPS(Encapsulation)
M2
Anti-LuxI
XBba_K145013
Tunability of IPTG in lacY-strains
Induction of lac-regulated promoter in lacY- and lacY+ strains, from Jensen et al. Eur.J.Biochem, 211, 181-191 (1993)
PluxR Output
Timer – v4
luxI
LuxRLux R + I
J231
1
4
PLAC
PLux
PoPS(Encapsulation)
M2
PTET
Bba_C0060
tetR
Td (lactonase) =4.7hrs
Ts (TetR)
Ts
(LuxI)
Lactonase
Dry LabPreparation
Dry LabSo 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
Part Obtain by… Diagram
CI (temperature sensitive)
Synthesise? / PCR?
Promoter = Repressible (lambda cI)
Synthesise (different sequence)
Promoter = Repressible (lambda cI)
Registry
BBa_K098995 [Harvard ‘08]
M3
BioBricks Remaining…
Wet LabParts, Testing &
Progress
So Far…
• Competent cells – 4 x 10^6 cells/ug DNA
• 0 contaminants
• No native resistance
So Far…• Registry - 8 taken out
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
Summary - Checklist
Weekly 5 Aims:
Trade name Assay ProtocolsCloning StrategiesFinalise genetic circuitOrder BioBricksPrimer designWet lab plan
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
Any questions?
Timer – v3 (Lock/Key system)
LuxI
LuxRLux R + I
J231
1
4
J231
1
4PLu
x
PoPS(Encapsulation)
M2
Lactonase
PtetR
Lock + Key
Bba_K145300
BBa_K145102
LVAJ2
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