Experiments
From 2006.igem.org
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===Results=== | ===Results=== | ||
| + | After having adjusted the samples to OD600=1, we used the Pr-GFP sample to set the highest expected fluorescence(501). Relative fluorescence of the Pr-GFP was 474, whereas for the two control it was 80. | ||
| + | That results in a Pr/Prm activity ratio of 1.05. | ||
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===Discussion=== | ===Discussion=== | ||
Revision as of 13:02, 27 October 2005
NOTE: this page above all is currently under construction.
Contents
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General Remarks
While putting together the Counter-system there are multiple experiments to carry out. One aspect is of course to test and prove the overall functionality. However, although the system we plan to implement consists largely of registry parts it will be new as a whole and it may well be very fragile due to its high complexity of interdependent parts and states. Thus it is far from assured whether the whole device will work in the end. For this reason it is sensible to test every single intermediate on the way to assure that a flaw is detected right away and not propagated through weeks of hard work - we will refer to this as testing and debugging, just like in software projects.
Another aspect - especially within the framework of Synthetic Biology - is the characterization of intermediate parts. One has to distinguish different aspects of the system: PoPS at the part interface tell you how much mRNA is produced per second and thus the efficiency of the promoter that is used (and the influence of possible additional factors like the roadblocks we will implement). However, PoPS might be different from the concentration of the actual proteins present in the cytoplasm due to variation in the translation efficiency. Another important detail is the stability and degradation rate of these proteins - especially for the rather fragile system dynamics of our device.
Protein Properties of Interest
The system behavior obviously depends on the interdependent sequence of fluctuating protein concentrations. While for the mathematical modeling of our system and for Synthetic Biology as a whole low level parameters are important - such as production and degradation rates, binding affinity, cooperativity of dimers, etc. - the thing that we typically can observe are reporter protein concentrations. Some things we can deduce from these observations, others we can't. However, to keep track of this delicate interdependence of fluctuations we put a whishlist of what we would like to know and what we do know - strictly in the context of our system, that is.
| Protein | Production Rate | Degradation Rate | Max. Conc. | Min. Conc. |
| IPTG | adjustable: test | ? | adjustable: test | 0 |
| LacI+LVA | ? | ? | ? | ? |
| cI | ? | ? | ? | ? |
| cI+LVA | ? | ht = 4 min | ?: test | ?: test |
| GFP @Pr | ? | ? | ?: test | ?: test |
| GFP @Prm | ? | ? | ?: test | ?: test |
| RFP @Prm | ? | ? | ?: test | ?: test |
| YFP+LVA @? | ? | ? | ?: test | ?: test |
| CFP+LVA @? | ? | ? | ?: test | ?: test |
| XFP @Pr+ZFBSn (ZF i,k) | ? | ? | ?: test | ?: test |
| XFP @Prm+ZFBSn (ZF i,k) | ? | ? | ?: test | ?: test |
It is indeed a tedious task to measure these parameters, since most aspects of a biological system have to be observed indirectly over markers, e.g. fluorescent proteins. We are determined to carry out as many experiments to test and quantify as we can reasonably afford - but of course, this is mostly grey theory within the framework of this competition, since we are running out of both time and resources. The actual concentration and dynamics of proteins is the crucial factor for the system to work and thus the aspect we ultimately care about - as opposed to PoPS at the part level.
Focus of Experiments
Since Blue Heron is hopelessly delayed with the synthesis of the parts of the 4-State Device and does not seem to be able to deliver the samples, all we can do for now is to characterize parts of the Event Processing Device as they become available.
Quantification of Relative Pr and Prm Activity
There are two external states: normal activity (no IPTG present) and the simulation of an external event (IPTG present). An important aspect is the quantification of the relative activity of the key promoters, Pr and Prm, in these two states.
Ground State
We can easily compare the basal activity of the constitutively active Pr and the low basal activity of the inactive Prm by adding GFP (Green Fluorescent Protein) to both, parts S03335 and S03336, and measuring the statistical distribution of GFP intensity over a sample, i.e. a cell population, with FACS (Fluorescent-activated Cell Sorting). The instrument is the BD FACSAria Cell-Sorting System. We will also quantify Prm activity with RFP (Red Fluorescent Protein), i.e. part S03337, in order to have comparison when carrying out measurements on the mixed system, see below.
External Event
Since we chose a very stable GFP to make sure it is not degrading faster than it can be measured it is more difficult to quantify the repressed activity of Pr and the induced activity of Prm in presence of IPTG. We will try to inverse the states in certain cultures, i.e. parts J05503 and J05504, by adding IPTG already during transformation (when the cells take in the plasmids) and thus inducing high cI production from the very beginning so that the stable GFP is not produced in the first place. Then we can observe the dynamics of the system when IPTG is slowly degrading and Pr becomes active again and Prm is repressed. In any case we are cloning other GFP with degradation tags in parallel to Pr and Prm. Also we are cloning ECFP (cyan) and EYFP (yellow) with degradation tags and which can both be found in the current Registry 7.05 package we are using.
Estimate of Minimal IPTG Concentration/Degradation
For the efficient execution of later experiments it is useful to know the minimal concentration of IPTG that is needed to reach saturation of the cI production rate. Also, IPTG is by definition very stable. We are using it because we could not be sure if we would see anything at all. Now that we know we have to estimate the degradation time of IPTG or rather find alternatives, such as Lactose (which degrades much faster).
Quantification of Lac-Input System
We use the LacI system in the registry as an interface to make the production of cI which in turn controls Pr and Prm dependent on an external event, i.e. the presence of IPTG. This input system is inactive in absence of IPTG. With part J05505 we will test over the reporter protein GFP the basic functionality: we hope to roughly quantify the basal activity and thus the minimal concentration of cI (due to leaking) compared to the active state in presence of IPTG and thus the maxium concentration of cI.
System Dynamics
If we have time, we will observe the dynamics of the input system with parts J05503 and J05504 with FACS (quantification, statistics) and under an optical microscope (tracking of specific single cells). The impact of different concentration levels of IPTG will be tested as well. If the inversion of intial states as described above does not work, we will use other parts with fast degrading GFP (which will hopefully be ready by then).
Measurements
To assure basic functionality and to characterize the intermediate parts, multiple additional parts, or "debugging" parts, have to be designed. We will try to maintain 25° C during growth and preparation of the cell cultures in order to have consistent conditions on different measurement systems. However, E. coli's niche is obviously very constant 37° C - so we will have to switch to that temperature if we encounter unexpected behavior.
Experiment 2005/10/07: OM-01 / FM-01
Purpose
Verify basic functionality / success of cloning under the optical microscope and fluorometer. Get a first rough idea of the relative activity of Pr and Prm.
Samples
Expectations
Results
Discussion
Experiment 2005/10/25: FACS-01
Purpose
More precise measurement of basal activity of Pr and Prm and statistical properties of the population. Relative intensities. Basal (leakiness) and IPTG-induced activity of lac-input system and thus cI production.
Samples
Note: the promoters have been prepared at 37° C, the lac-systems at 25 ° C
- Prm+RFP (tbd) -> comparison (rel. intensity GFP vs. RFP)
- lac-system, 0.00 mM IPTG (J05505) -> control @ no induction
- lac-system, 0.25 mM IPTG (J05505) -> control @ intermediate induction
- lac-system, 2.50 mM IPTG (J05505) -> control @ high induction
- lac-system+GFP, 0.00 mM IPTG (J05505) -> leakiness @ no induction
- lac-system+GFP, 0.25 mM IPTG (J05505) -> activity @ intermediate induction
- lac-system+GFP, 2.50 mM IPTG (J05505) -> activity @ high induction
Expectations
Results
Discussion
Experiment 2005/10/27: FM-02
Purpose
Repetition: Quantify relative activity of Pr and Prm with the fluorometer to confirm / falsify first measurement.
Samples
Expectations
We expected to reconfirm the results from the first analysis, i.e. to assess a 3-6 times higher activity of Pr (which should be fully active in this status) than Pr (which should just have basal activity).
Results
After having adjusted the samples to OD600=1, we used the Pr-GFP sample to set the highest expected fluorescence(501). Relative fluorescence of the Pr-GFP was 474, whereas for the two control it was 80. That results in a Pr/Prm activity ratio of 1.05.
Discussion
Experiment 2005/10/27: OM-02
Purpose
Observe single cells under the optical microscope. Try to get an idea of the dynamics of what we have right now by making time series. Learning how to prepare the samples properly for on-instrument induction. Live induction of IPTG, hopefully increasing RFP intensity (current GFP does not degrade and will thus probably remain constant).
Samples
E.coli fixed on Agarose between glass object holder and ...
Expectations
Results
Discussion
Experiment 2005/10/28: FACS-02
Purpose
Repeat Pr/Prm comparison with samples prepared at 25° C to confirm / falsify results from FACS-01. Test relative activity when Pr and Prm are active in the same cell. Test whole EPD performance in ground state and IPTG-induced state. Not testing dynamics.
Samples
- Pr+GFP+Prm+RFP (S03338) -> individual cell comparison (intensity Pr+GFP vs. Prm+RFP)
- lac-system+Pr+GFP+Prm+RFP, 0.00 mM IPTG (J05503) -> control @ no induction
- lac-system+Pr+GFP+Prm+RFP, 0.25 mM IPTG (J05503) -> control @ no induction
- lac-system+Pr+GFP+Prm+RFP, 2.50 mM IPTG (J05503) -> activity @ high induction
- lac-system+Pr+GFP+Prm+RFP (cI+LVA), 0.00 mM IPTG (J05504) -> control @ no induction
- lac-system+Pr+GFP+Prm+RFP (cI+LVA), 0.25 mM IPTG (J05504) -> control @ no induction
- lac-system+Pr+GFP+Prm+RFP (cI+LVA), 2.50 mM IPTG (J05504) -> activity @ high induction
Expectations
Results
Discussion
Experiment 2005/10/31: FACS-03
Purpose
Time series to test activity at different induction levels. Test suitable IPTG range. Inverse case: cell preparation with IPTG present from transformation on (IPTG won't degrade?!).
