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 possibly 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 injection	no degradation	adjustable: test	0
LacI+LVA	?	?	?	?
cI	?	?	?	?
cI+LVA	?	ht = 4 min	?: test	?: test
GFP @Pr	?	?	?: test	?: test
GFP @Prm	?	?	?: test	?: test
RFP @Prm	?	no degradation (20 years)	?: 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 in Different States

There are two external states (simulated with IPTG in this prototype): normal activity in ground state (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 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 [http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6125| S03335] and [http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6126| S03336], and measuring the statistical distribution of GFP intensity over a sample, i.e. a cell population, with [http://en.wikipedia.org/wiki/Fluorescent-activated_cell_sorting FACS] (Fluorescent-activated Cell Sorting). The instrument is the [http://www.bdbiosciences.com/immunocytometry_systems/products/display_product.php?keyID=53| BD FACSAria Cell-Sorting System]. We will also quantify Prm activity with RFP (Red Fluorescent Protein), i.e. part [http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6127| S03337], in order to have a comparison of relative intensities when carrying out measurements on the mixed system, see below.

External Event

Since we chose a very stable GFP for the initial prototype 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 [http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6131| J05503] and [http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6132| J05504], by adding IPTG already during [http://en.wikipedia.org/wiki/Transformation_(genetics)| 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. Possibly (but unlikely) we can observe the dynamics of the system when IPTG is slowly degrading (it does't really) or is diluted (cell division, change of substrate) 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 planning to clone 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 for the initial prototype 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 (the regulator 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 [http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6133| J05505] we will test the basic functionality with the reporter protein GFP: 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 [http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6131| J05503] and [http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6132| J05504] with [http://en.wikipedia.org/wiki/Spectrofluorometer| spectrofluorometers], the FACS (quantification, statistics), and optical microscopes (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 fluorophores.

Measurements

To assure basic functionality and to characterize the intermediate parts, multiple additional parts, or "debugging" parts, have to be designed.

We have carried out multiple measurements while learning on the job about instruments, preparation procedures, and cell behavior. Only measurements with sensible results will be logged on this page.

In a first series of measurements we maintained 25° C during growth and preparation of the cell cultures in order to have consistent conditions on different measurement systems and to have a slower cell cycle. However, the preparation at these temperatures was very slow and we can not be sure that we see "real" production rates, since E. coli's niche is obviously some very constant 37° C. Thus we switched to 37° C for later experiments.

Experiment 2005/10/07: OM-01 / FM-01

Purpose

Verify basic functionality / success of cloning under the optical microscope and [http://en.wikipedia.org/wiki/Spectrofluorometer| spectrofluorometer]. Get a first rough idea of the relative activity of Pr and Prm.

Samples

Debugging part S03335: Pr+GFP

Debugging part S03336: Prm+GFP

Pr ([http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=189| R0051]) -> control
Prm ([http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=4799| I12007]) -> control

Pr+GFP ([http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6125| S03335]) -> basal activity
Prm+GFP ([http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6126| S03336]) -> basal activity (leakiness)

Expectations

We expect to see a clear difference in the activity of Pr in maximum activation and Prm in basal state.

Results

This preliminary experiment showed promising results, i.e. an approximate ratio of the two promoters of Pr(active) / Prm(basal) = 3 to 6

	Fluorescence	Visual
Pr
Prm

Discussion

This was only a quick and dirty measurement for testing the basic functionality. We will have to confirm these results with the more precise measurements. However, so far things look promising, since the difference between active Pr and basal Prm is a factor between 3 and 6.