Dual Light Controlled Arabinose Biosensor
Aguilar, Mónica; Cano, Nelson ; Colunga, Indira I. ; Díaz, Aldo A. ; Guerrero, Israel ; Machado, Rodrigo ; Maycotte, David ; Morales, Cintli C. ; Nieto, Mariana ; Taveras, Rossel ; Vásquez Jorge A. ; Villarreal,
Antonio. Mishra Prashant K. (pmishra@itesm.mx); Vázquez-Flores, Sonia (svazquef@itesm.mx )
Abstract
How does it work?
Integrating the work of many other previous iGEM teams (Tokyo
NoKoGen 2010, Chiba 2009, 2010, British Columbia 2009, Cambridge
2010, UNAM-Genomics México 2010, ITESM Monterrey 2010), the
aim of this project is to develop a way of giving a cell the command to
perform a function at user’s will, improving current lock-and-key
designs. A novel mechanism based on an E. coli chassis, was designed
with two main objectives: to sense arabinose reporting its
concentration and to use light receptors to trigger the expression of
the required pathways. The first receptor enables E. coli activity,
expressing the arabinose sensing mechanism; whereas the second
receptor activates a quick deactivation (degradation) of the sensing
mechanism, depriving the cell of that capability.
Assembly and construct description
The construct combining three plasmids, the green receptor activates the expression
of the recA final product, this RecA protein binds into the operators, allowing the
expression of pBAD´s, in presence of arabinose, depending on the arabinose
concentration; if there is a high concentration, the low concentration plasmid will be
inactivated by the iTa-st. If there is a low concentration, the plasmid will activate the
pBAD´s and along with keys and anti-keys to avoid the expression of both the
fluorescent proteins.
Fig. 1. Plasmid assembly
First, we need to have our
transformed cells with the
entire construct and apply
green some light to
activate the mechanism
Results and discussion
Once the green light and a
small amount of arabinose
is present I express GFP
With some red light all my
fluorescent protein will
start to degrade and the
mechanism will be reset
The following composite parts were
cloned into DH5α and BW27783
competent cells using the standard CaCl2
transformation protocol:
a. Constitutive promoter + Crx-st + RBS
+ GFP + Terminator
b. Constitutive promoter + Ta-st +
Terminator
c. Constitutive promoter + iTa-st +
Terminator
d. a + b + c
1 % gel, V Ligated pieces with lambda phage T4, 4µl of
Fermentas O’Gene RulerTM 1kb ; digested DNA 10 µl in 0. 5µl of
6x Orange DNA Loading Dye .
The gel was run at 100 V for 30 minutes.
2% gel with PCR product of the constructed
Backbone BBAJ_04450 with a 4µl of Fermentas
O’Gene RulerTM 100 kb.
Lane
Sample
Expected size
Digestion
enzymes
1
3
4
5
6
7
8
9
10
11
12
Ladder 1 kb
Ta-wk
Rec A
CR12+DNAx
iTa-st
Vector pUC57: 2710 bp Insert: 122 bp
Vector pUC57: 2710 bp Insert: 1103 bp
Vector pUC57: 2710 bp Insert: 78 bp
Vector pUC57: 2710 bp Insert: 124 bp
EcoR1-Pst1
EcoR1-Pst1
EcoR1-Pst1
EcoR1-Pst1
Lane
Sample
Molecular weight
of expected bands
10
3139 bp
DH5α
Ta-st’
Ta-wk’
DNA competent cell
Vector pUC57: 2710 bp Insert: 123 bp
Vector pUC57: 2710 bp Insert: 122 bp
EcoR1-Pst1
EcoR1-Pst1
EcoR1-Pst1
11
pSB1C3
PCR
pSB1C3
PCR
RecA’
iTa-st’
Crxst’
Vector pUC57: 2710 bp Insert: 1103 bp
Vector pUC57: 2710 bp Insert: 124 bp
Vector pUC57: 2710 bp Insert: 78 bp
EcoR1-Pst1
EcoR1-Pst1
EcoR1-Pst1
3139 bp
Conclusions
If I detect higher concentration of
arabinose I can produce the Cyan
Fluorescent Protein, but first I
have to block the GFP expression
We have some evidence that the construct fully assembled. Bacteria were inoculated into
several Petri dishes with LB agar and the appropriate antibiotic (Chloramphenicol) to screen for
transformants. After a 24 hour incubation at 37°C, there was growth in the dishes that had
bacteria transformed with all the composite parts. These dishes were submitted to UV light to
see if they produced fluorescence, although there was evidence of reaction, it was not
conclusive, nor uniform in all the cultures. The levels of GFP expression might not have been
high enough to directly observe fluorescence.
The GFP fluorescent mechanism was successfully designed, incorporated and tested in a
bacterial system.
The system has yet to be tested for low and high arabinose quantities, and quantify the
lowest detection level of fluorescence in a fluorometer. Nonetheless, this study opens a new
window for further experimentation for concentration dependent detection mechanisms for
other metabolites.
Fig. 2. Construct conformation and part assembly
Modelling
Photoreceptor mechanism
Inspired by the Tokio-Nokogen iGEM team 2009. This mechanism was modified by adding only the green light receptor instead of the whole
mechanism, including also the red light receptor mechanism. The most important modification of this system was the inclusion of the protein RecA in
our construct to make it compatible to the regulation system of lambda phage incorporated in the concentration scheme. The green receptor, used
to initiate the entire mechanism, is composed by eight parts, in a sequence of twelve . RecA is a protein used for the cleavage of protein lambda. It has
shown that it has a cleavage activity when a lambda repressor is bonded. This is an essential part of the project, because these interactions are the
link between the photoreceptor and the concentration mechanisms by the lambda repressor and the lambda operators.
Figure 3. Scheme of the photoreceptor mechanism
Concentration mechanism
Based on the experiments and mechanisms developed by British Columbia University iGEM team in 2009. We re-designed certain pieces to make
them more specific, modifying the lock and key mechanism, and adding more parts, one lock and key specific for the high concentration and other
lock and key designed for low concentrations. Also the inclusion of one new Biobrick® that regulates one of the keys by inactivating it. This operators
can only be free once the RecA protein cleaves the lambda repressor, so the expression can continue. To assure that only the high concentration
mechanism is enabled , there is the need to turn down the low concentration, this is achieved by expressing an antisense sequence key (iTa-st) that
inhibits the production of the low concentration key (Ta-st) sequence, thus the low-concentration lock (crx-st) will activate and will inhibit the
expression of GFP.
Figure 4. Scheme of the concentration mechanism
PCR Reaction of the
Backbone BBAJ_04450
Future research
Millions of numbers. There are a wide variety of biosensors in the World, responsible for
detecting a specific factors but not all of them can tell exactly the amount of such factor.
We propose a biosensor that can be capable of detecting a specific analyte by glowing
according to the detected concentration.
Switch off. Just as electrical energy, “if you are not using it…turn it off”, our mechanism is
designed to be useful just when in need, when it matters. As it had been set to express
two kind of fluorescent proteins, this biosensor can be easily interpreted, by anyone with
no previous trainning.
Applications. The mechanism of the bacteria can be set for different analytes, giving the
opportunity to expand the market of the biosensor to practically any industry.
Examples:
Contaminants
Intelligent medicines
Domestic care