Integrating the work of many other previous iGEM teams (Tokyo - PowerPoint Presentation

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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.

Abstract

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 )

Dual Light Controlled Arabinose Biosensor

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.

2. Construct conformation and part assembly

Assembly and construct description

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.

Concentration mechanismBased 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.

How does it work?

Future research

Conclusions

Results and discussion

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: ContaminantsIntelligent medicines Domestic care

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.

The following composite parts were cloned into DH5α and BW27783 competent cells using the standard CaCl2 transformation protocol:

Constitutive promoter +

Crx-st + RBS + GFP + TerminatorConstitutive promoter + Ta-st + TerminatorConstitutive promoter + iTa-st + Terminatora + b + c

Fig.

1. Plasmid assembly

Figure 3. Scheme of the photoreceptor mechanism

Figure

4.

Scheme of the

concentration mechanism

Lane

Sample

Expected

sizeDigestion enzymes1Ladder 1 kb 3Ta-wkVector pUC57: 2710 bp Insert: 122 bp EcoR1-Pst14Rec AVector pUC57: 2710 bp Insert: 1103 bpEcoR1-Pst15CR12+DNAxVector pUC57: 2710 bp Insert: 78 bpEcoR1-Pst16iTa-stVector pUC57: 2710 bp Insert: 124 bpEcoR1-Pst17DH5αDNA competent cellEcoR1-Pst18Ta-st’Vector pUC57: 2710 bp Insert: 123 bpEcoR1-Pst19Ta-wk’Vector pUC57: 2710 bp Insert: 122 bp EcoR1-Pst110RecA’Vector pUC57: 2710 bp Insert: 1103 bpEcoR1-Pst111iTa-st’Vector pUC57: 2710 bp Insert: 124 bpEcoR1-Pst112Crxst’Vector pUC57: 2710 bp Insert: 78 bpEcoR1-Pst1

1 % gel, V

L

igated

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.

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.

Lane

SampleMolecular weight of expected bands10pSB1C3PCR3139 bp11pSB1C3PCR3139 bp

PCR

Reaction of the Backbone BBAJ_04450

2% gel

with

PCR

product

of

the

constructed

Backbone

BBAJ_04450

w

ith

a

4µl of Fermentas

O’Gene

RulerTM

100 kb.