Team:CU Boulder/Description

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Project Description

The goal of our project is to create a biosensor to detect naphthalene. This could be achieved simply by placing a reporter behind iGEM part BBa_J61051. When naphthalene is present, the reporter is produced so one could be alerted to the exposure. However, this design presents a few major limitations. It signals that naphthalene is present only in the moment when the cell is exposed. This provides no real advantage over taking a water sample and testing for naphthalene without a biological system. In our design, we aimed to create a recording aspect within our device to show if there has at any point been naphthalene exposure.

There are two key features of our design to achieve a recording aspect:

  1. Genetic Switch: On plasmid #2, there is a genetic logic gate (see black arrows) in the DNA sequence, which the Bxb1 integrase can invert the DNA, creating a sort of genetic switch. Between the two Bxb1 recognition sites we have placed a terminator so that in the absence of naphthalene, the switch is “off” as no transcription is occurring at this locus. Once the integrase is expressed, the DNA will be inverted, turning the genetic switch “on”. This creates a genetic and therefore permanent change that is recorded in future progeny as well.
  2. Signal Amplification: Our design amplifies a small signal via the Quorum Sensing system from Vibrio Fischeri. In our design, we use the LuxR, LuxI, and LuxPr promoter components, which allows us to employ cell to cell signaling to create a device that will visibly turn a single color across the entire device, even if it was just one single cell that was exposed to naphthalene.

Plasmid #1

NahR (BBa_J61051): The NahR-pSal-sfGFP-Terminator part designed by Peking iGEM 2013 is a sequence of genes that promotes transcription via a metabolic pathway that converts naphthalene to salicylic acid.

Integrase: This gene produces a recombinase enzyme isolated from mycobacteriophage Bxb1. Bxb1 Integrase recognizes attP and attB sequences from the phage genome. When these sequences are orientated anti parallel, whatever region they flank will be inverted by the recombinase.

Plasmid #2

Genetic Switch

Genetic Switch: Located downstream of a constitutive Anderson promoter are attp and attb Bxb1 integrase recombination sites (represented by black arrows). By being placed into the DNA sequence in an antiparallel manner, the integrase will invert the segment between the two sites. In our design, a terminator is flanked by these recombination sites so once the integrase is expressed, the terminator sequence will be flipped, preventing the terminator from halting transcription. In this scenario, Lux I and GFP will be expressed.

LuxI: The LuxI gene produces an enzyme that converts the small molecule SAM (S-adinosylmethionine) into AHL (N-(B-ketocaproyl)-L-homoserine lactone. AHL molecules will diffuse through the cell membrane and can enter the neighboring cells, binding to those cells’ LuxR transcription factor.

GFP: Green Fluorescent Protein serves as our primary reporter. Only cells that have been exposed to naphthalene will express the integrase, which will invert the terminator. Then GFP will be produced, which will allow the differentiation between cells that turned red due to cell-to-cell signaling and those that turned red but also were exposed to naphthalene.

Signal Amplification

LuxPr Promoter: The LuxPr promoter is an ineducable promoter induced by the LuxR transcription factor when bound to AHL. In our construct, it will only be activated once naphthalene is present in at least one cell. AHL produced from that original cell exposed to naphthalene will diffuse throughout the media to neighboring cells where it will bind to the neighboring cells’ LuxR transcription factor. This will allow expression of RFP and is the final part of the signal amplification.

LuxR: Expression of the LuxR gene produces the LuxR transcription factor, which in our design is being constitutively promoted.

RFP: Red Fluorescent Protein serves as out secondary reporter. Once AHL has bound to the LuxR transcription factor, the LuxPr promoter will be activated and RFP will be expressed, and the cell will turn red.

Team:CU-Boulder -