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

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

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

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 Timer Module 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 PoPS (Encapsulation) AB B Inducible Constitutive

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

! 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

! 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

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

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

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

Dry Lab Preparation

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

BioBricks Remaining… Promoter = Repressible (lambda cI) M3 Part BBa_K098995 [Harvard ‘08] Part Obtain by… Diagram CI (temperature sensitive) Synthesise? / PCR? Promoter = Repressible (lambda cI) Synthesise (different sequence) Registry

Wet Lab Parts, 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 Protocols Cloning Strategies Finalise genetic circuit Order BioBricks Primer design Wet 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) PtetR Bba_K145300 J23109 Lactonase LVA BBa_K145102 Lock + Key PLux J23114 LuxI M2 Lux R + I PoPS (Encapsulation) J23114 LuxR