K.U.Leuven Essencia coli: The Fragrance Factory Essencia coli2009.igem.org/files/presentation/KULeuven.pdf · Five step conversion to vanillin Essencia coli 12 Follow-up on previous

K.U.LeuvenEssencia coliEssencia coli : The Fragrance Factory

The multidisciplinary team from the University of Leuven Belgium

1

University of Leuven, Belgium

K.U.LeuvenEssencia coli

2

K.U.LeuvenEssencia coliThe Fragrance Factory

Aim:Aim:Design and build a bacterium that sets and controls the vanillin concentration in the environment

Results:

vanillin concentration in the environment

Results:

• fully integrated modelfully integrated model• 19 parts and devices made• 7 new parts!7 new parts!

3

K.U.LeuvenEssencia coliSubsystems of Essencia coli

Input = blue light receptor

Production = tyrosine to vanillin conversion

Sensing = vanillin receptorSensing = vanillin receptor

Controlling = key/antisense key/lock4

Controlling key/antisense key/lock

K.U.LeuvenEssencia coliIntegrated system of Essencia coli

5

K.U.LeuvenEssencia coliInput: the Blue light receptor

6

K.U.LeuvenEssencia coliBlue light drives the vanillin production

7N. Tschowri, S. Busse and R. Hengge. Genes Dev 23: 522-534 (2009)

K.U.LeuvenEssencia coliBlue light drives the vanillin production

8N. Tschowri, S. Busse and R. Hengge. Genes Dev 23: 522-534 (2009)

K.U.LeuvenEssencia coliThe Blue light responsive promoter is weak

9

K.U.LeuvenEssencia coliBlue light increases expression

10

K.U.LeuvenEssencia coliProduction: converting tyrosine to vanillin

11

K.U.LeuvenEssencia coliFive step conversion to vanillin

12Follow-up on previous work done by Prof. C. French and the iGEM team 2007 of the University of Edinburgh

K.U.LeuvenEssencia coliSensing: the Vanillin receptor

13

K.U.LeuvenEssencia coliSensing drives antisense key expression

PvirB

14Natural BioBricks made from Agrobacterium tumefaciens

K.U.LeuvenEssencia coliControlling: the key/antisense key system

15

K.U.LeuvenEssencia coliControl by key/antisense key balance

1500

Vanillin sensing

Blue light100

molecules

Production of vanillin

Vanillin sensing

50

16Follow-up on previous work done by the 2006 iGEM team from the University of Berkeley

0                            2000                         4000                          6000                         8000     time (s)

K.U.LeuvenEssencia coliSummary animation

17

K.U.LeuvenEssencia coliUsing a model to realise the concept

Solution 

ConceptSimplified 

Conceptmodel

18

K.U.LeuvenEssencia coliSimplified model

Disturbance

+‐

[Vanillin][Key] [Key] ++‐ +

[Antikey][Antikey]

19

K.U.LeuvenEssencia coliFully integrated kinetic model

20

K.U.LeuvenEssencia coliTwo operational modes

Deficiency Abundance Deficiency Abundance

+ + +[L k]+‐

++ ‐

++

[Leak]

Speed of feedback ∆t Speed of feedback

Effect of copy number

∆t

21

Effect of copy number

K.U.LeuvenEssencia coliTagging reduces feedback delay

Half life determines delay

KEY

tration

Production enzymes

T i V illi tial con

cen

τ=10τ=22

Tyrosine Vanillin

% init

key

τ=2τ=4

τ 10

Time [au]Use tag to reduce the feedback delay

Half lifeTAG

No TAG

COMT

22

0 500 1000Half life [min]

K.U.LeuvenEssencia coliThe effect of copy number

45 copies

25

15

25

0

23

K.U.LeuvenEssencia coliThe effect of copy number

45 copies

25

15

25

0

24

K.U.LeuvenEssencia coliWhat in the case of abundance?

‐+[Leak]

+

Evaporation rate determines feedback!

But vanillin is not very volatile

25

K.U.LeuvenEssencia coliVirA: internal sensing is not an issue

Phenol sensor senses intracellular vanillin

OM

Cout Dif

PP

ffu

τ ≈ 5 [ms]k = 1/ τ ≈ 200 [1/s]

VirA IM

Cin

sioCin

nVanillin

But fast diffusion over cell membrane!

(C C ) 1/k S26

(Cin – Cout) = 1/k Svanillin

K.U.LeuvenEssencia coliAccomplishments

made sequenced

parts

Fully integrated model

made sequenced

3 3

Pitfalls in system tackled2 2

Characterized new natural BioBrick: Blue light receptor11 7

3 1

OtherEthics & human practice Helped other teams

Wiki Had a great summer

27Poster follow our team members

K.U.LeuvenEssencia coliThanks to

Gold Sponsors

BioSCENTer l f

CMPG

Faculty of Bioscience EngineeringFaculty of EngineeringFaculty of ScienceFaculty of ScienceFaculty of Biomedical Sciences

Silver Sponsors

28

K.U.Leuven sympathizer

K.U.LeuvenEssencia coliThanks for your attention!

29