This year our new team has been developing the project established last year which you can see on the McGill 2006 wiki. We have made many new discoveries and have run into some problems hindering our progress along the way; nevertheless, we were able to make some new developments as outlined below:
Reproducing Last Year's SystemClick here for methods
From these experiments, we were able to come up with some interesting results.
1. The more concentrated the cells, the worse the apparent oscillations.
2. The presence of Dox seemed to stabilize the oscillations.
3. The greater the amount of AHL, the worse the apparent oscillations, as predicted with the simulations.
4. Using an optical density assay, it was found that the cells were in fact dying (or clumping) thus lowering the optical density throughout the testing period.
5. Oscillations occured with the J40001 (pLux-LacI-ECFP) alone. The reason for this could be some interaction with the expression of endogenous Lac in the BL21 cell line we used.
We replaced the BL21 cells with MC4100 cells, the lac-, tet- cells used by Elowitz himself.
Results: no oscillations which led us to the next point, the malfunctioning I15004 brick needs to be replaced.
Production of a new I15004 biobrick
This sequence contains the following:
1. pLac, ribosome binding site, LuxI These three components are present from the I15004 and are required for the oscillating network.
2. RFP The RFP is our own addition. It allows us to track the expression of both the oscillating I15004 brick and the J40001 brick allowing us to evaluate oscillations in more than one way (note, a third method of reporting is included with the repressillator).
3. pConstitutive, ribosome binding site, LuxR Inculsion of this constitutive promoter was another one of our design ideas. When coupled with the repressilator, this eliminates one of the crossing links that would make the system more connected and create more variables. By eliminating the tetracycline promotor, our system is much cleaner.
As ideal and wonderful as this system sounds, we were unable to put it into action. We ordered the sequence from GenArt in early August and it has still not arrived in time for the Jamboree due to ligation problems at the company.
Coupling of the Oscillator with the Repressilator
Modelling using deterministic rate equations predicted beautiful curves that we have attempted to reproduce in the lab. Again, we ran into some problems. Repeated restriction digests produced anomalous gel results and when attempting to view the reporting gene in the repressilator, we were unable to see the fluorescence.
Our conclusion is that the I5610 is not what is appears to be. So, onto our next section : creating a new represillator. Thus, coupling was not yet possible in practice.
Synthesizing a new Repressilator
We placed a new degradation tag on the I5611 to make a functional Elowitz repressilator. Another biobrick, E0434, contains the same YFP reporter with a functional degradation tag. Using BsrG1 and SpeI to cleave the YFP at an identical point in both the I5611 and the E0434, we plan to preform a ligation of these two pieces to develop the functional unit.
Unfortunately, though in progress, this could not be completed for the Jamboree due to ligation difficulty. Due to the low copy number of the plasmids to be amplified and the fidelity of the ligation proceedure, time did not permit the completion of this vital component of our project thus there are no represillator oscillations to report.
Synthesizing an AI (AHL) Inhibitor into our J40001 biobrick
We are currently still working on incorporating this construct into J40001 to create I724001 (LacI + ECFP under luxpR, with autoinducer inactivation).
Sadly we were unable to complete the ligation in time to submit the part to the registry by the deadline.