e.chiver creating a bacterial filing cabinet -...
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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.