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

310

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