From 2006.igem.org

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Contents 1 General Remarks 2 Protein Properties of Interest 3 Focus of Experiments 3.1 Quantification of Relative Pr and Prm Activity 3.1.1 Ground State 3.1.2 External Event 3.2 Estimate of Minimal IPTG Concentration/Degradation 3.3 Quantification of Lac-Input System 3.4 System Dynamics 4 Measurements 4.1 Experiment 2005/10/07: OM-01 / FM-01 4.1.1 Purpose 4.1.2 Samples 4.1.3 Expectations 4.1.4 Results 4.1.5 Discussion 4.2 Experiment 2005/10/25: FACS-01 4.2.1 Purpose 4.2.2 Samples 4.2.3 Expectations 4.2.4 Results 4.2.5 Discussion 4.3 Experiment 2005/10/27: FM-02 4.3.1 Purpose 4.3.2 Samples 4.3.3 Expectations 4.3.4 Results 4.3.5 Discussion 4.4 Experiment 2005/10/27: OM-01 4.4.1 Purpose 4.4.2 Samples 4.4.3 Expectations 4.4.4 Results 4.4.5 Discussion 4.5 Experiment 2005/10/28: FACS-02 4.5.1 Purpose 4.5.2 Samples 4.5.3 Expectations 4.5.4 Results 4.5.5 Discussion 4.6 Experiment 2005/10/31: FACS-03 4.6.1 Purpose 4.6.2 Samples 4.6.3 Expectations 4.6.4 Results 4.6.5 Discussion

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 [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 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 [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 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) 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 [http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6133| 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 [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 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 and to have a slower cell cycle. 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

• 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 good results, i.e. an approximate ration of the two promoters of

Pr(active)/Prm(basal) = 3 to 6

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 FACS. However, so far things look good, since the difference between active Pr and basal Prm is bigger than 2.

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

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

• Prm+RFP (tbd) -> comparison (rel. intensity GFP vs. RFP)

• lac-system only

• lac-system, 0.00 mM IPTG ([http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6133| J05505]) -> control @ no induction
• lac-system, 0.25 mM IPTG ([http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6133| J05505]) -> control @ intermediate induction
• lac-system, 2.50 mM IPTG ([http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6133| J05505]) -> control @ high induction

• lac-system+GFP, 0.00 mM IPTG ([http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6133| J05505]) -> leakiness @ no induction
• lac-system+GFP, 0.25 mM IPTG ([http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6133| J05505]) -> activity @ intermediate induction
• lac-system+GFP, 2.50 mM IPTG ([http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6133| J05505]) -> activity @ high induction

Expectations

The goal is to confirm the outcome of previous experiments, i.e. the Pr/Prm-ratio from FM01. Also, we want to put the intensity of GFP and RFP into relation for the same promoter (Prm). Finally we would like to estimate the leakiness of the two alternative lac-input systems (cI degradation does not matter at this point) - which is known to be leaky - at different levels of IPTG-induction.

Results

Unfortunately, we could not confirm the Pr/Prm intensity ratio of 3 to 6. The ratio measured in this experiment is around 1.6.

Discussion

The different Pr/Prm ratio clearly contradicts previous experiments. So either the samples have changed or one of the experiments is invalid. Thus we will have to carry out more experiments to narrow this down.

Experiment 2005/10/27: FM-02

Purpose

Repetition: Quantify relative activity of Pr and Prm with the fluorometer to confirm / falsify first measurement.

Samples

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

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

The difference in the two measurements could be due to a measurement error (the wrong sample was measured), but possibly it could be due to a saturation of GFP production in both systems. Since the plates are 2 weeks old, and since GFP is very stable, it can be supposed that after a while the concentration of GFP reaches a maximum level, independently of the promoter activity. Therefore new transformations should be performed with both plasmids and measurements should be then started ASAP.

Experiment 2005/10/27: OM-01

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

Very weak GFP signal (Pr) in the ground state of the system, i.e. without IPTG. Very high RFP signal (Prm).

Discussion

If there is nothing wrong with the samples, then the lac-input system is indeed very leaky, too leaky. The good news would be that Pr is properly repressed by cI. We have to check this with more FACS measurments and with the single cell system Pr+GFP+Prm+RFP.

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 ([http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=189| R0051]) -> control
• Pr+GFP ([http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6125| S03335]) -> max. activity
• Prm+GFP ([http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6126| S03336]) -> basal activity

• Pr+GFP+Prm+RFP ([http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6128| S03338]) -> individual cell comparison (intensity Pr+GFP vs. Prm+RFP)

• lac-system+Pr+GFP+Prm+RFP, 0.00 mM IPTG ([http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6131| J05503]) -> control @ no induction
• lac-system+Pr+GFP+Prm+RFP, 0.25 mM IPTG ([http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6131| J05503]) -> control @ no induction
• lac-system+Pr+GFP+Prm+RFP, 2.50 mM IPTG ([http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6131| J05503]) -> activity @ high induction

• lac-system+Pr+GFP+Prm+RFP (cI+LVA), 0.00 mM IPTG ([http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6132| J05504]) -> control @ no induction
• lac-system+Pr+GFP+Prm+RFP (cI+LVA), 0.25 mM IPTG ([http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6132| J05504]) -> control @ no induction
• lac-system+Pr+GFP+Prm+RFP (cI+LVA), 2.50 mM IPTG ([http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6132| 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?!).

Samples

Incubation over night at 37° C. 225rpm. Volume: 3mL LB. Selection: Amp75/Km30.

• Pr ([http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=189| R0051]) -> control
• Pr+GFP ([http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6125| S03335]) -> max. activity
• Prm+GFP ([http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6126| S03336]) -> basal activity
• Prm+RFP

Transformation without IPTG

• lac-system+GFP (0.000 mM IPTG)
• lac-system+GFP (0.050 mM IPTG)
• lac-system+GFP (0.250 mM IPTG)
• lac-system+GFP (2.500 mM IPTG)

Transformation with 0.250 mM IPTG

• lac-system+GFP (transferred to 0.000 mM IPTG)
• lac-system+GFP (maintained at 0.250 mM IPTG)

• Pr+GFP+Prm+RFP ([http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6128| S03338]) -> individual cell comparison (intensity Pr+GFP vs. Prm+RFP)

-> printout missing

• lac-system+Pr+GFP+Prm+RFP (cI+LVA), 0.00 mM IPTG ([http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6132| J05504]) -> control @ no induction
• lac-system+Pr+GFP+Prm+RFP (cI+LVA), 0.25 mM IPTG ([http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6132| J05504]) -> control @ no induction
• lac-system+Pr+GFP+Prm+RFP (cI+LVA), 2.50 mM IPTG ([http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6132| J05504]) -> activity @ high induction

(measurement: basal RFP activity )

• lac-system+Pr+GFP+Prm+RFP, 0.00 mM IPTG ([http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6131| J05503]) -> control @ no induction
• lac-system+Pr+GFP+Prm+RFP, 0.25 mM IPTG ([http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6131| J05503]) -> control @ no induction
• lac-system+Pr+GFP+Prm+RFP, 2.50 mM IPTG ([http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6131| J05503]) -> activity @ high induction

Expectations

Pr in active state is stronger than Prm in basal state. RFP intensity is weaker than GFP intensity for same promoter in FACS system.

Lac-input system with initial induction of IPTG will show maximum activity of Plac and stay at this level as long as the IPTG concentration is maintained, but will go back to basal activity if transferred to a medium without IPTG.

Lac-input is going to be leaky. Different induction levels of IPTG lead to different activity levels. Identify minimal concentration leading to sufficient activity.

Estimate of leakiness of lac-input system based on a comparison of the -0/+0 samples.

Degradation/dilution of GFP can be estimated based on a comparison of -0/-250 samples (for the whole system dilution might overcome the lack of degradation to some degree).

Results

Pr is stronger than Prm by a factor of 2 according to the GFP comparison.

RFP intensity is around 15% of GFP intensity.

Lac-input: set maximum activity as reference (001-j5+2500 - or higher in future experiments). put everything into relation to this reference.

compute relation of j5+0 and j5+250.

compute this

Discussion