Team:Uppsala-SwedenWeek4

From 2010.igem.org

Week-4

We studied the below articles for getting an understanding of how to achieve our goals. Having studied each of the articles we had extensive discussions on "Google Wave" and during our off-line group meetings.

Initially the idea was along the lines of obtaining band detects as done by Basu etal(See below for further details). However, we realised that the approach used by Basu etal, would only work for AHL. If we or another iGEM team wanted to used some other sensor they would have to again produce mutants of the sensory promoter. These mutants would respond to different concentrations of the input signal. Moreover, we realised that few of these AHL detection sensor mutants where not readily available in the registry.

The brilliant approach used by the iGEM team from Cambridge last year(2009) seemed to hold the answer for our problems. Instead of depending on the sensor for measuring the signal, a separate set of elements would do the part of measurement. However, a linear relationship between the sensor and the measuring element would be necessary. We decided to combine the idea used by Basu etal and the 2009 iGEM team from Cambridge using the parts available to us in the registry.

An inverter was readily available for us to invert the signal. This inverter is used in combination with a measuring element which gets activated at a lower level of sensory input. As soon as the input signal is strong enough to activate the inverter, the reporter inhibitor is deactivated. The higher level of the sensory input directly controls another reporter inhibitor.

An article about the design of toggle switches and an oscillator, later used for pattern generation

1) Danino etal 2010 (http://www.ncbi.nlm.nih.gov/pubmed/20090747)

A general paper about synthetic biology for better understanding of what we were getting into

2) Mukherji etal 2009 (http://www.ncbi.nlm.nih.gov/pubmed/19898500)

The papers about quorum sensing using AHL for pattern generation. These two papers played a major role in driving our project.

3) Basu etal (http://www.ncbi.nlm.nih.gov/pubmed/15858574 and http://www.ncbi.nlm.nih.gov/sites/pubmed/15096621)

A synthetic multicellular system for programmed pattern formation

The article discusses about pattern formation which is a typical characteristic of both single and multicellular organism. Pattern formation involves cell-cell communication and intracellular signal processing. In the article the authors have showed a multicellular system in which genetically engineered ‘receiver’ cells respond to AHL (acyl-homoserine lactone) chemical stimulus which is sent by the ‘sender’ cells and form ring-like patters. The pattern that forms accounts to the ‘band-detect’ gene network which the receiver cells have, that respond to specific AHL concentration. By having a reporter gene which produces a fluorescent protein on induction, a lawn of receiver cells around a sender colony can give rise to bullseye pattern. Depending on the location of the sender colony different patterns can be formed as per the article. Such experiment is done to improve our quantitative understanding of naturally occurring developmental processes.

The multicellular system was constructed with the principle that the receivers at intermediate distances only express the fluorescent protein. This is explained with Figure 1 (a). This kind of band detection is achieved by having a high detection component (pHD plasmid) and a low detection component (pLD plasmid) in the engineered construct of receiver cells (Figure 1(c), (d)).

Figure 1: Cell-cell communication system of E.coli. (a) Band detect receiver cells are engineered to respond only to intermediate concentrations of AHL as shown by the graph where the concentration differs with distance from sender cell. (b) Sender cell- plasmid map (c) pHD plasmid (d) pLD plasmid (figure adapted from Basu S et. al . 2005)

Cell-Cell communication from the sender cell is triggered by the expression of LuxI enzyme which catalyzes the synthesis of AHL. The AHL which is thus synthesized diffuses through the cell membrane and gives rise to a chemical gradient around the sender colony. AHL in the receiver cells activates the expression of lambda repressor (CI) and Lac I repressor (Lac IM1). Receiver cells which are nearer to the sender colony are exposed to a high level of AHL thereby has a high cytoplasmic CI and Lac IM1 and hence the GFP gets repressed. On the flip side, when the receiver cells are farther from the sender colony, they are only exposed to a minimal amount of AHL thereby expresses only basal amount of CI and LacIM1 which triggers the cells to express wild type Lac I to compensate and this also represses GFP expression. Cells which are exposed to intermediate amount of AHL expresses moderate amount of CI and Lac IM1 but since CI is more efficient than Lac IM1, it effectively shuts down the expression of Lac I and Lac IM1 concentration is below the threshold to repress GFP. Thus the system expresses GFP only in the presence of intermediate dosage of AHL. The article also discusses about the mathematical model, spatiotemporal simulations and dynamic behaviour of the multicellular system. The concept has been adapted with some modifications in our current project.

4) Original paper about toggle switches (http://www.ncbi.nlm.nih.gov/pubmed/10659857)

5) For degrdation of color proteins -ex:carotenoids (http://www.jbc.org/content/281/15/9845.full). We planned to degrade the colors generated by the clock display unit after displaying the time for a short duration.

Having studied the articles we decided to make use of the band detect concept introduced in Basu etal to send and detect the signals between adjacent colonies without being affected by the noise from other colonies.