•As the various factors were altered, the predicted

Chitin generation rate changed accordingly as

hypothesized

(ex) As the Chitin Synthase promoter part was

changed to a more or less effective part, the chitin

level changed accordingly as shown in Figure 4.

•Model is effective in predicting general trends

•Model could be fit with empirical data to generate

a semi-empirical model for empirical accuracy

Results & ConclusionsChitin Synthase (CHS3) with pMAL ends

successfully obtained from S. cerevisiae through

PCR

•Unforeseen restriction sites within the gene

complicated basis of chitin production in project

chiA and tqsA knockouts obtained from Keio

Collection

•chiA knockout competent cells.

IPTG inducible Lac promoter cassette

•Three of our six attempted constructs were

confirmed successful via sequencing analysis

•Hoping to characterize parts by charting

fluorescence and absorbance against time, but were

unable to attach them to green fluorescent protein.

Apoptosis cassette thought to be successfully

assembled

•Placed into chiA knockout cells but did not induce

any visible apoptosis in cells following 16-hour

incubation period

•Sequencing analysis returned negative results from

the desired sequence

BackgroundChitin

•Biopolymer primarily found in exoskeletons of

arthropods (ie insects and crustaceans)

•Provides structural support and protective surface

layer

•UDP-N-acetylglucosamine monomers (Figure 1)

•UDP-N-acetylglucosamine is present in E.Coli as a

precursor of endotoxin A

•Medical, industrial and commercial applications

Biofilms

•Bacteria adhere to surfaces and to each other

•Adherent cells in a matrix of extracellular

polymeric substance (EPS) of DNA, proteins and

polysaccharides

Future ConsiderationsInduction system

•Determine expression dynamics of each

permutation of the induction system collection

•Placing a fluorescent reporter after each

permutation and recording its expression over time

over a range of IPTG induction concentrations.

•Determine appropriate combination for fast chitin

synthase production and slow cell lysis

Characterization of Chitin Expression

•Determine where chitin synthase is localized after

translation; in S. Cerevisiae, chitin synthase is an

integral membrane protein

•To localize chitin synthase to the inner bacterial

membrane, we fused chitin synthase with p5x signal

sequence and maltose binding protein (MBP) at the

N-terminal end.

•Remains unclear if the chitin synthase-p5x-MBP

fusion folds properly within the inner membrane

and is subsequently functional

•Follow up with a fractionation assay to determine

if the fusion protein actually localizes to inner

membrane and use anti-maltose binding protein

antibodies conjugated with horseradish peroxidase/

fluorophor to judge whether chitin synthase-MBP

has successfully inserted into the membrane.

Assembly

•Verify that subparts are compatible in a common

system

•Assemble subparts to form self-regenerating chitin

induction machine

ReferencesAnderson MS, Bull HG, Galloway SM, Kelly TM, Mohan S, Radika K, et al. UDP-N-

acetylglucosamine acyltransferase of Escherichia coli. The first step of endotoxin

biosynthesis is thermodynamically unfavorable. Journal of Biological Chemistry

1993;268(26):19858-65.

Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, et al. Construction of

Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol

Syst Biol 2006;2.

Bulik DA, Olczak M, Lucero HA, Osmond BC, Robbins PW, Specht CA. Chitin Synthesis

in Saccharomyces cerevisiae in Response to Supplementation of Growth Medium with

Glucosamine and Cell Wall Stress. Eukaryotic Cell 2003;2(5):886-900.

Herzberg M, Kaye IK, Peti W, Wood TK. YdgG (TqsA) Controls Biofilm Formation in

Escherichia coli K-12 through Autoinducer 2 Transport. J. Bacteriol. 2006;188(2):587-98.

Khor E. Chitin : fulfilling a biomaterials promise. Amsterdam: Elsevier Science Ltd., 2001.

Uragami T, Tokura S. Material Science of Chitin and Chitosan. Tokyo: Springer, 2006.

Wang HH, Isaacs FJ, Carr PA, Sun ZZ, Xu G, Forest CR, et al. Programming cells by

multiplex genome engineering and accelerated evolution. Nature 2009;460(7257):894-98.

Zoloth L. Jewish Perspectives on Oncofertility: The Complexities of Tradition. In:

Woodruff TK, Zoloth L, Campo-Engelstein L, Rodriguez S, editors. Oncofertility: Springer

US, 2010:307-17.

AbstractChitin is one of the most abundant substances in

nature. Like keratin in skin, it comprises the

protective outer layer of these animals. Our chitin

expression platform involves generating a layer of

chitin from a lawn of bacteria in response to an

external molecular cue. The external molecular cue

induces chitin synthesis (fast) and cell lysis (slow).

This system allows for a build-up of chitin followed

by cell lysis and subsequent release into the top

layer of the lawn. Abrasions expose cells to the

external cue for self-repair. This system establishes

a regenerative chitin biolayer with potential medical

and industrial applications.

StrategiesInduction

Our induction system consists of a combinatorial

set of constitutive promoters (BBa_J23100,

BBa_J23104, BBa_J23105) constructed in

sequence with a pair of Lac induction cassettes

(BBa_Q01121 and BBa_Q04121). Each Lac

induction cassette consists of a ribosome binding

site, coding region of Lac repressor protein DNA, a

terminator and a promoter; in that order. From the

six possible combinations of constitutive promoters

and Lac cassettes, a fast-acting version provides a

means of rapid chitin synthesis, while a slow-acting

version activates the apoptotic system.

Chitin Synthase

Chitin synthase 3 (CHS3), derived from S.

cerevisiae cDNA, does not require post-

translational processing or additional cofactors to

function, making it an ideal candidate for chitin

synthesis in a foreign E. coli host. Since CHS3 is a

multipass trans-membrane protein, it is fused with

p5x signal sequence and maltose binding protein

(MBP) at the N-terminal end using pMAL-p5x from

New England Biolabs to localize chitin synthase to

the inner bacterial membrane and induce proper

folding. In addition, PstI restriction sites are

removed using site-directed mutagenesis. Fast

induction, under the control of Lac induction

cassettes (BBa_Q01121 or BBa_Q04121), provides

a mechanism for rapid chitin production.

Apoptosis

The T4 bacteriophage lysis cassette

(BBa_K124017), designed by Brown ‘08, contains

coding regions for holin, endolysin, and Rz protein.

Slow induction prevents premature cell lysis and

permits each apoptotic component to buildup

gradually, allowing sufficient time for chitin

synthesis.

Modeling

In order to optimize the time delay between

activation of the expression vectors, we developed a

semi-empirical model that allows us to investigate

how changing each system component affects the

expression dynamics of chitin.

Chassis

A chitinase knockout (∆chiA) that prevents chitin

degradation and a ∆tqsA (∆ydgG) knockout that

grows that a thick bacterial lawn are available from

the Keio Collection. Multiplex automated genome

engineering (MAGE) generates a double knockout

strain (∆chiA/∆tqsA).

Verification

To ensure successful chitin production, rhodamine

chitin-binding probe is used to stain chitin in vivo.

The Live/Dead Baclight Bacterial Viability KitTM

from Invitrogen provides a means for studying cell

lysis efficiency. When used in tandem with confocal

microscopy, these stains allow two-dimensional

imaging of the bacterial lawn and a way to monitor

the progression of chitin production and cell death.

Mechanism

A lawn of ∆chiA/∆tqsA bacteria transformed with

both CHS3 and apoptosis induction plasmid is

sprayed with a solution of Isopropyl β-D-1-

thiogalactopyranoside (IPTG). Layers of cells

within the biofilm produce chitin and lyse after a

delay, depositing a layer of chitin in the top-most

layer of the bacterial lawn. Likewise, disruptions in

the bacterial lawn and any surface abrasions are

filled in by a chitinous media.

ModelingIPTG diffusion model

•To mathematically characterize the process and

assist in experimentation with MATLAB

•Kinetics model and the diffusion model employed

to explore effect of altering various experimental

factors:

•IPTG concentration and diffusion

•lacI concentration (determined by lacI

expression construct)

•lac-operon, ribosome binding site

•copy number of the plasmid

on the concentrations and reaction rates of all

species involved, especially Chitin Synthase and

Chitin

•Diffusion of IPTG down through the biofilm

modeled using Fick's Law with a finite time

differential model

•Another finite time differential kinetics model and

the IPTG diffusion model used to simulate system

in Figure 3

Self-regenerating Chitin INduction

Timi Chu, Ragan Pitner, Kevin Rogacki, Matthew Tong, Sean Yu, Benjamin ZhangDr. Michael Jewett, Dr. Joshua Leonard, Dr. John Mordacq

Northwestern University

iGEM Jamboree 2010

Figure 3: Kinetic Topography of CHS3 Synthesis

Figure 4: Chitin Production Overtime

Figure 5: Confocal Microscopy of a Bacterial Lawn

Figure 2: Project Overview

Acknowledgements: Debora Boetcher (McCormick ChemE), Paul Brannon (BIF), Jamie Bufkin (BIF), Jackie Ciardo (Qiagen), Sara Fernandez Dunne (High Throughput), Sara Mann (Promega), John Marko (NU Prof. of Biochem.), Linda Mattice (Yale), William Russin (BIF), Margaret Saks (NU Prof. of Biochem.), Lori Tonello (NEB), Amy Vanarsdale (VWR), Wei Zhang (Civil and Environment Engineering), XueNong Zhang (Epoch Life Sciences), Laurie Zoloth (NU Prof. Medical Humanities & Bioethics)

Figure 1: Molecular Structure of Chitin