From 2012.igem.org

(Difference between revisions)

Revision as of 16:59, 26 September 2012

NOVA

Project Biowave You have never seen something like this.

Background

A group of rough-toothed dolphins surfacing synchronously
Origin Link

One of the most interesting and complex types of group behaviors in animals is that in which several organisms simultaneously repeat the same activity at regular intervals of time.¹ In ordinary usage, this kind of behavior is called “synchronous”. It has been observed in Thailand that male Pteroptyx malaccae fireflies, congregated in trees, flash in rhythmic synchrony with a period of about 560 ± 6 msec (at 28°C). Males of a China’s special species of firefly called Qiongyuying flash synchronously during mating season. And we also observed that bees crawling in the same small area display a synchrony of wings fluttering in order to scare off intruder，and we filmed a video clip to demonstrate this phenomenon. Moreover, synchrony has been described in a range of mammal groups, including odontocete cetaceans (a kind of toothed whale).² The odontocete cetaceans behave synchronously when they breathe on the surface of water.

Synchronous fireflies (Pteroptyx Tener) flashing on the mangrove tree (Sonneratia Caseolaris) at Kuala Selangor, Malaysia (2008). Courtesy of Photo Researchers, Inc.
Origin Link

In the microcosm, a synthetic gene oscillator whose oscillatory cycle can be tuned by altering inducer levels, temperature and the media source has been designed by I and his cooperators to accomplish the synchronous oscillation. It uses both E.coli and yeast as component cells. In this oscillator system, chemicals (IPTG, arabinose) are used as a series of signals to initiate synchronous oscillations within single cells of the colony.³

Oscillations in the dual-feedback circuit.
a, Network diagram of the dual-feedback oscillator.
b, Single-cell fluorescence trajectories induced with 0.7% arabinose and 2mM IPTG.
Origin Link

Compared with chemical molecules, optical signal is more efficient for both the macrocosm and the microcosm. We wonder whether we can change the induced signal into light, which is visible and easy to control. It propagates much faster and has countless channels, for each wavelength is a different channel.

Introduction

Recently, there has been increased interest in visible light controlled system. Optogenetics, a seven-year-old field branching out from molecular biology and neuroscience, is leading the trend of creating novel synthesized proteins as “tools”

helping people understand the brain mechanism. These “opsins”, which light-activated domain borrowed from microbes or plants, were transferred into model animal organisms. These controllers are acting in the center part of light system. Unlike the drug treatment, light can be delivered by lasers with extremely high spatial precision; therefore, one can manipulate only target cells.

Our Biowave project is more than performing “Optogenetics can solve controversies that have been going on for many, many years.”light control. By establishing the light depending feedback system within the cell we have realized a light communication system. This is the very first time that putting the intracellular and intercellular light communication in the biological system. Using the fundamental gene circuit and brand new parts we created, each cell becomes a communication node.

The first Biowave circuit we have created is the Negative Feedback Biowave. The circuit comprising light sensor and light generator established the negative feedback between them. The properties of time delay, caused by gene expression or the accumulation of the element’s concentration, and negative feedback could form an oscillation of light output within a single cell or the neighbouring cells. By placing the bacteria on a Petri dish and letting them evenly distributed, each cell could sense the light of others equally. With the certain condition and observation analysis, the whole dish could form a detectable synchronized oscillation. In a macroscopic view, this kind of synchronization could be distracted by the attenuation of light. So we could expect a wave like pattern on this level.

The Positive Feedback Biowave is another attempt for the intracellular and intercellular light communication. Thus the positive feedback under this situation makes the system a bistable circuit; each cell can remain in either a light state or a dark state. But the light irradiation across the colony makes things different. The bacteria would form a static strip or other bistable system caused patterns within or beyond our expected.

	LIGHT	CHEMICAL
manipulate	controllable	Hard to control
elimination	light induction could be changed or erased	hard to wipe out
transfer speed	Signal travels fast(as a speed of light) Non-time-delay	Speed is limited by chemical diffusion System has a minimum delay
crosstalk	never has the crosstalk with constitutive pathway	may cross the constitutive pathway
measurement	Visible and detectable easy to measure	hard to detect or measure
noise immunity	sensitive could be easily disturbed by the nature light	only the same molecule or the analogue could disturb the signal pathway
channel	has limited channels(only 390nm to 700nm)	countless channels(each kind of chemical analogues group is a channel)

Design Process

Establish feedback system

Feedback exists between two parts when each affects the others. In our system, these two parts are the light sensor and the concentration of the light generator protein. By involving the light sensor in the loop, we can establish the feedback.

Light generator

Approach
Next issue we placed our focus on the properties of the candidate parts. At the beginning we laid eyes on the luciferin illumination system. But the engineering bacteria we were using do not have the luciferin producing enzyme system or the luciferin cycle. Fortunately the 2010iGEM Cambridge has successfully deliver this cycling system into the E.coli. But the wave length does

not fit the light sensor which we will discuss later. So we picked another well-known bio illuminator, the Lux operon. It is also presented by 2010 Cambridge.

Lux operon
The Lux operon is a set of genes active in bacterial luminescence. The genes encoding the luciferase and a fatty acid reductase complex which synthesizes the substrate are contained in a single operon. When cloned into a recipient host, the lux operon facilitates the generation of a blue-green light with a maximum intensity at 490 nm without the requirement of adding exogenous substrate, which was the prime reason why we chose it to be our light source in this study.

In our project, we using PCR extracted the DNA sequence from the BBa_K325909, and cloned it under the controllable promoter. The coding device relieved from the original promoter is also available as the BBa_K785003. The growth curve of the Lux transformed dH5α strain is measured. See the summery at the result page.

Light sensor

ycgF-ycgE-ycgZ endogenous light sensing system

Light sensor that directly regulates the expression at the transcription level is rarely seen. Bacterial opsins usually appeal as light activated kinases or phosphodiesterases. BBa_K238013, an exact promoter of the blue light receptor, is a portion of an E.coli constitutive light sensing system. This system consists of a receptor, ycgF, which is responsive to blue light. When blue light strikes, the When blue light strikes, the receptor changes conformation and dimerizes driven by the BLUF-domain. This allows it to bind to the ycgE repressor through its EAL-domain, releasing the repressor from the promoter region. In summary, the irradiance of blue light will cause a positive induction, expression downstream Light sensing feedback using ycgF-YcgE-YcgZ sensing system.

MG1655 strain
The PCR process results that dH5α,the strain we used as normal competent cell, does not contain such endogenous system. The MG1655 strain as a wild type of E.coli K12 was used for the construction of our light sensing design. See MG1655 genotype.

Light Sensor

Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum tortor quam, feugiat vitae, ultricies eget, tempor sit amet, ante. Donec eu libero sit amet quam egestas semper. Aenean ultricies mi vitae est. Mauris placerat eleifend leo. Quisque sit amet est et sapien ullamcorper pharetra. Vestibulum erat wisi, condimentum sed, commodo vitae, ornare sit amet, wisi. Aenean fermentum, elit eget tincidunt condimentum, eros ipsum rutrum orci, sagittis tempus lacus enim ac dui. Donec non enim in turpis pulvinar facilisis. Ut felis. Praesent dapibus, neque id cursus faucibus, tortor neque egestas augue, eu vulputate magna eros eu erat. Aliquam erat volutpat. Nam dui mi, tincidunt quis, accumsan porttitor, facilisis luctus, metus

Pellentesque habitant morbi tristique senectus et netus et malesuada fames, Pellentesque habitant morbi tristique senectus et netus et malesuada fames Pellentesque habitant morbi tristique senectus et netus et malesuada fames.

Application

Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum tortor quam, feugiat vitae, ultricies eget, tempor sit amet, ante. Donec eu libero sit amet quam egestas semper. Aenean ultricies mi vitae est. Mauris placerat eleifend leo. Quisque sit amet est et sapien ullamcorper pharetra. Vestibulum erat wisi, condimentum sed, commodo vitae, ornare sit amet, wisi. Aenean fermentum, elit eget tincidunt condimentum, eros ipsum rutrum orci, sagittis tempus lacus enim ac dui. Donec non enim in turpis pulvinar facilisis. Ut felis. Praesent dapibus, neque id cursus faucibus, tortor neque egestas augue, eu vulputate magna eros eu erat. Aliquam erat volutpat. Nam dui mi, tincidunt quis, accumsan porttitor, facilisis luctus, metus