A toggle switch is an important design motif in synthetic biology as it allows for the use of different constructs dependent on the active chemicals and gene sequences involved. This allows for multiple constructs to be activated at different times dependent on the wants of the synthetic biologist who created the BioBrick. Expression levels of different gene sequences can be controlled by various conditions such as the presence of ligand molecules in culture media. We improved upon the genetic toggle switch by designing a toggle mechanism that is initiated in the presence of two different wavelengths of light (green and blue light). This allows us to control the rate of reaction of these constructs simply by imploring specific wavelengths that are read by a gene sequence sensitive to that wavelength. The control of this construct is given to the individual performing the experiment as they can induce a gene of interest by simply shining a specific wavelength of blue light onto the construct. In the case of this period of the process, the sequence simply glows red when it is being induced, however, this can be changed at any time to induce nearly any gene of interest. When the construct is completed and all data is collected, the sequence can be terminated with the use of a green light which induces specific aspects of the construct to be activated, and eliminating previous processes.
PROOF OF CONCEPT DESIGN
New Natural Part: Why are you using this part? How did you find this part? What database/ resources did you use? What primers will you use to isolate it and turn it into a BioBrick?
Key Pre-existing Part: If you didn't need to use a new natural part, describe one important pre-existing BioBrick from the parts registry.org that you will use. Why are you using it? Describe how well-documented the part is. Do you trust it?
Assembly Scheme
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Our construct is a light-responsive genetic toggle switch. The construct’s design was inspired by the toggle switch introduced during Unit 1. Our version improves upon that design by exchanged light for ligand molecules as a triggering mechanism for the switch between “ON” and “OFF” states. Light is advantageous over substrate-coupling mechanisms in applied systems because of enhanced control and efficiency in signal exposure.
Our construct consists of three key open reading frames (ORF 1, 2, 3). ORF 1 is regulated by a strong constitutive promoter, coding regions for two light responsive proteins and the Lac I repressor, and a terminator sequence. ORF 2 contains a Lac promoter, a Tet operator, a reporter gene, BLRP, a Tet operator, and a coding region for Lac permease followed by a terminator site. ORF 3 contains a PcpcG2 promoter and a coding region for tetracycline repressor followed by a terminator. In the initial state, LacI inhibits the lac promoter and the reporter gene is silent. Exposure to blue light induces Lac permease production mediated by BLRP activation. E. coli tranformants cultured in lactose will initiate lactose uptake, increasing intracellular lactose concentration. Intracellular lactose inactivates LacI, which de-represses the lac promoter and initiates reporter gene expression. At this time point, the blue light signal is terminated. Because there is not terminator site separating the reporter gene and the BLRP, LacY is continually expressed (thereby propagating the “ON” state despite lac permease half-life). The chassis will remain in the “ON” state as long as sufficient lactose is present in the culture media.
To toggle the “OFF” state, the culture is exposed to green light. The regulator and receptor proteins whose genes are located in ORF 1 respond to green light, initiating a phosphorylation mechanism that activates the PcpcG2 promoter. This in turn induces TetR expression. The tet repressor targets the tet operators in ORF 2, thereby physically blocking RNA polymerases from transcribing the reporter gene and LacY. Lac permease is no longer replenished, and lactose concentration declines at a rate directly proportional to the rate of lac permease degradation. In the absence of intracellular lactose, the lac promoter is repressed and reporter gene expression ceases.
We use a naturally-occurring blue light responsive promoter (BLRP) derived from Arabidopsis thaliana. In A. thaliana and many other plant systems, the BLRP is used to regulate psbD expression. psbD is a coding gene that yields machinery involved in photosystem II. The sequence for PLRB in A. thaliana and other plant systems was found in literature (Hoffer and Christopher 1997). PLRB from A. thaliana was chosen specifically because it has been reportedly cloned into E. coli with success. The sequence was queried in NEBCutter to confirm that it is compatible with the BioBrick assembly standards. Forward and reverse primers were designed that would incorporate the appropriate BioBrick prefix (E-N-X) and suffix (S-N-P). Using these primers and a PLRB template, a PLRB BioBrick could be successfully amplified.
Your NameYour area of study/ academic program/ major, why you are taking BME494, and something interesting about yourself. You may add a link to your personal OWW page.
Your NameYour area of study/ academic program/ major, why you are taking BME494, and something interesting about yourself. You may add a link to your personal OWW page.
Your NameYour area of study/ academic program/ major, why you are taking BME494, and something interesting about yourself. You may add a link to your personal OWW page.
Your NameYour area of study/ academic program/ major, why you are taking BME494, and something interesting about yourself. You may add a link to your personal OWW page.
