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E.Chiver – Creating a “Bacterial Filing Cabinet”

Jones W., Chang A., Chen D., Pepping J., Chen A., Brown M., Murzyn K., Weber J., Sun J., Khanna M.

Rao C., Jin Y., Bhalerao K., Fuentes Evans C., Price N., Manaster J.

Introduction

We are the iGEM team of the University of Illinois at Urbana-

Champaign. This is our fourth year competing since our original

team began in 2008. We are composed of a diverse and talented

group of students from a wide array of majors including

Agricultural/Bioengineering, Biochemistry, Psychology, and

Electrical Engineering. Having this diverse group allowed us to pull

from each member’s talent when the

right occasion arose. Our

project, E. chiver-a bacterial

filing cabinet, parallels this team

structure.

The goal of E. chiver is to create a system that allows a construct

to be expressed at a high copy number when induced, but then file

back into the chromosome as a stable integrant for energetically

favorable storage. The workplace analogy of a filing cabinet is

made:

While the first step in the E. chiver design, our Lambda system,

was meant to allow a single file to shuttle in and out of the

chromosome, the second step was to allow multiple files to coexist.

The P21 system addresses the second step.

Background: CRIM System Components

Lambda System Overview

Parts Submitted

Human Practices Project

Summary

We Would Like to Thank Our Diamond Sponsors!

Basic machinery for E. chiver design already exists in the

CRIM system:

attP, attB

These two elements

undergo Site-Specific

Recombination

Excisionase (Xis), Integrase (Int)

Sole expression of Integrase

causes an integration event

between attP and attB. Co-

expression of both leads to

excision of the attP plasmid.

R6K origin, pir

R6K is only functional when

the product of pir is present.

Chromosome Shuttle (CRIM vector) Helper Plasmid

Xis Int

pir

attP attB

R6K

The CRIM system allows a user to integrate, excise, and replicate a

construct at will. However, each of these steps requires several

transformation, selections, and a phage transfer. E. chiver seeks to

place the process under a chemical inducer rather than manual

labor in order to use the system outside the laboratory environment.

Multiple Files: λ and P21 Chemical Inducers for Multiple Files Easy Integration and Excision

FILE 1 FILE 2

The idea is to be able to express high amounts of File 1, integrate it

back into the chromosome, and then express high amounts of File 2,

and integrate that back into the chromosome as well. Putting each

file under the control of a unique chemical inducer helps to achieve

this task!

Step 1: Integration of “File”

attB

attP

PcI Int

• PcI is active

• Integrase is expressed

• Shuttle plasmid integrates at attB site

• Integrase is turned off after integration event

R

Step 3: Replicating the “File”

PcI

Int attB

attP

Plac Pir

cI repressor

R6K • Chemical inducer still present

• Plac is active on Shuttle, causing

pir and cI repressor to be expressed

• pir-116 gene allows high-copy

replication at R6K origin

Step 4: “Filing” it Back In

attB

attP

PcI Int

Plac

• Remove chemical inducer

• Inactive Plac now downregulates

expression of pir and cI repressor

• Without cI repressor, PcI is now

active

• This causes Int to be expressed

once again

• Integration of Shuttle occurs at

attB site

• Shuttle is now back where it

started!

Step 2: Removing the “File”

R6K

Plac

Int Xis

Pconst

cI

PcI

Int L

• Chemical inducer is introduced

(IPTG in this case)

• Plac is active, Xis and Int are now

expressed and both work together

to excise out Shuttle at “L” and “R”

• Before excision, cI repressor is

under control of Pconst , and down-

regulates expression of first Int

gene

The P21 system is, essentially, the machinery required for a second file. All the parts are

analogous to the λ system. Only the repressor of Integrase, the origin, and the origin

trans- factor need be changed. For example, instead of the R6K origin, we use oriV,

instead of pir, we use trfA, and instead of cI repressor, we use cII repressor.

R6K Kan

R GFP

CI

Repressor

pir

E

X

λ

Shuttle

Vector LacZ

Plac Pconst

Pconst

λ

Xis

λ

Int λ

attB

CI

repressor

λ

Int PCI

λ attP

oriV CmR CFP

CII

Repressor

TrfA

E

X

P21

Shuttle

Vector TetR

Ptet Pconst

Pconst

P21

Xis

P21

Int attB

(P21)

CII

repressor

P21

Int PCII

P21 attP

Name Type Description Designer Length

BBa_K617000 Plasmid Biobrick Compatible

Lambda Chromosomal

Insertion Plasmid

Will Jones 2440

BBa_K617003 DNA R6K origin of replication Will Jones 392

BBa_K617004 DNA Lambda attP P'OP Will Jones 396

K617003

R6K ori

K617004

Λ attP

Conversion of these CRIM

elements into Biobrick parts

will allow teams to further

rearrange and improve the

filing-cabinet design.

This is a modified λ CRIM

Vector that will allow teams

to integrate their designs.

The plates below show the

Plasmid only replicates in a

pir+ strain (shown to the

right).

K617000

Λ a

ttP

R6K

kanR

BioBrick site

Synthetic Biology College Course

Synthetic Biology for Educators

Our team collaborated with faculty from the U of I to outline three

ways in which we can address Human Practices in the future!

Surveying perceived error-

proneness in constructing

synthetic biology circuits

We surveyed laboratory workers of various skill levels to determine which

protocols were the most error-prone in order to tell us which ones to look into

automating. The graph above shows the degree of error-proness in each

protocol.

In collaboration with Professor Ting Lu from

Bioengineering department in University of

Illinois, we designed a synthetic biology class for

college students. This proposal will be further

developed and the proposed class will come to

reality in the 2012 Fall semester.

1. Teachers want to present cutting edge science

such as synthetic biology to their students in the

classroom, but there are currently few simple

lesson plans and kits available.

2. Teachers are inexperienced with the concepts

and materials in Synthetic Biology.

We suggest two solutions to these problems:

1. iGEM students create kits and protocols with

background information.

2. iGEMers will be available via Skype to provide

remote support to interact with students and

educators.

• Overall, our project focuses heavily on the design aspect of

engineering. We have designed a system which would allow for the

integration, excision, and high copy expression of multiple plasmids.

We have submitted 3 BioBricks™ to the Parts Registry: K617000,

K617003, and K617004.

• We have also outlined new ways to approach Human Practices,

from addressing the issue of standardization within the field of

Synthetic Biology, to designing courses for secondary educators as

well as at the college level.

• Future work will include collecting more survey results for Human

Practices, preparing for the 2012 UIUC-Illinois team, as well as further

characterizing our E. chiver system.