Difference between revisions of "Part:BBa F2620:Experience"

Endy Lab Characterization

We (Barry Canton, Anna Labno, Drew Endy) have measured four characteristics of BBa_F2620. Protocols and results for the four characteristics are linked below. For the purposes of characterization, we connected BBa_E0240 downstream of BBa_F2620 to make the composite part, BBa_T9002. This allowed us to measure the PoPS output from BBa_F2620 indirectly via the fluorescence output of BBa_E0240. Data processing is described here. The data resulting from these experiments is summarized on the paper datasheet.

1. Transfer function
2. Response time
3. Input compatibility
4. Reliability

Leakiness

See Part:BBa_I13556 for more characterization of F2620 upstream of mCherry instead of GFP. In particular, it was found that when grown in LB, there was strong leaky expression of mCherry in the absence of AHL. There was no leaky expression when grown in EZ rich medium. --Austin 17:11, 6 September 2007 (EDT)

F2620 in front of GFP and F2620 in front of lacZa were also both grown up in LB to stationary phase (without AHL) and both of these in addition to the mCherry also showed leaky expression not present when grown in EZ media. --Austin 17:38, 14 April 2008 (EDT)

Quality control

This part was inconsistent in 2009 DNA Distribution. The UNIPV-Pavia iGEM 2009 team requested this part to iGEM Headquarters, sequenced it and found that sequence was correct.

BBa_F2620 - 3OC6HSL receiver device - UNIPV-Pavia TEAM (Test performed by L. Pasotti, S. Zucca)

Description

This device gives PoPS as output and can be induced with 3OC6-HSL autoinducer molecule: it binds luxR protein (encoded by BBa_C0062), which is constitutively expressed by tetR promoter (BBa_R0040). LuxR-HSL complex can work as a transcriptional activator for lux promoter (BBa_R0062).

Several studies have been performed on this BioBrick. Here we provide the experimental characterization we performed during this summer. The tests have been performed through BBa_T9002 measurement system, which has a GFP protein generator downstream.

Characterization

Compatibility: E. coli TOP10 in pSB1A2.

BBa_T9002 Growth curves for 3OC6HSL in M9

BBa_T9002 Growth curves for 3OC6HSL in LB

BBa_T9002(dGFP/dt)/O.D. in LB and M9

BBa_T9002 Characterization of part in M9

BBa_T9002 Characterization of part in LB

Conclusions

The induction curve of the receiver device, reported in BBa_F2620 page (M9 supplemented medium), was represented in PoPS units, while ours is reported in RPUs and has been obtained through a very similar protocol (see Growth conditions section). Anyway, the experiments we performed in M9 supplemented medium confirmed the induction curve shape of this device, with a switch point between 1nM and 10nM.

We also estimated the transfer function of this device in LB medium, for which no data were reported in the Registry from previous characterization. More experiments sholud be perfomed on the behaviour of this device in M9 because the one performed show a great variation in RPU range.

Growth conditions

Microplate reader experiments

• 8 ul of long term storage glycerol stock were inoculated in 5 ml of LB + suitable antibiotic in a 15 ml falcon tube and incubated at 37°C, 220 rpm for about 16 hours.
• The grown cultures were then diluted 1:100 in 5 ml of LB or M9 supplemented medium and incubated in the same conditions as before for about 4 hours.
• These new cultures were diluted to an O.D.600 of 0.02 (measured with a TECAN F200 microplate reader on a 200 ul of volume per well; it is not comparable with the 1 cm pathlength cuvette) in a sufficient amount of medium to fill all the desired microplate wells.
• These new dilutions were aliquoted in a flat-bottom 96-well microplate, avoiding to perform dynamic experiments in the microplate frame (see [http://2009.igem.org/Team:UNIPV-Pavia/Methods_Materials/Evaporation Frame effect section] for details). All the wells were filled with a 200 ul volume.
• If required, 2 ul of inducer were added to each single well.
• The microplate was incubated in the Tecan Infinite F200 microplate reader and fluorescence (when required) and absorbance were measured with this automatic protocol:
  • 37°C constant for all the experiment;
  • sampling time of 5 minutes;
  • fluorescence gain of 50;
  • O.D. filter was 600 nm;
  • GFP filters were 485nm (ex) / 540nm (em);
  • 15 seconds of linear shaking (3mm amplitude) followed by 10 seconds of waiting before the measurements in order to make a homogeneous culture.
  • Variable experiment duration time (from 3 to 24 hours).

Data analysis

Growth curves

All our growth curves have been obtained subtracting for each time sample the broth O.D.600 measurement from that of the culture; broth was considered in the same conditions of the culture (e.g. induced with the same inducer concentration).

Doubling time

The natural logarithm of the growth curves (processed according to the above section) was computed and the linear phase (corresponding to the bacterial exponential growth phase) was isolated by visual inspection. Then the linear regression was performed in order to estimate the slope of the line m. Finally the doubling time was estimated as d=ln(2)/m [minutes].

In the case of multiple growth curves for a strain, the mean value of the processed curves was computed for each time sample before applying the above described procedure.

Relative Promoter Units (RPUs)

The RPUs are standard units proposed by Kelly J. et al., 2008, in which the transcriptional strength of a promoter can be measured using a reference standard, just like the ground in electric circuits.

RPUs have been computed as:

in which:

• phi is the considered promoter and J23101 is the reference standard promoter (taken from Anderson Promoter Collection);
• F is the blanked fluorescence of the culture, computed subtracting for each time sample fluorescence measure for negative control from that of culture, where the negative control is a non-fluorescent strain (in our experiment it is usually used TOP10 strain bearing BBa_B0032 or BBa_B0033, which are symmply RBSs do not have expression systems for reporter genes);
• ABS is the blanked absorbance (O.D.600) of the culture, computed as described in "Growth curves" section.

RPU measurement has the following advantages (under suitable conditions)

• it is proportional to PoPS (Polymerase Per Second), a very important parameter that expresses the transcription rate of a promoter;
• it uses a reference standard and so measurements can be compared between different laboratories.

The hypotheses on which RPU theory is based can be found in Kelly J. et al., 2008, as well as all the mathematical steps. From our point of view, the main hypotheses that have to be satisfied are the following:

• the reporter protein must have a half life higher than the experiment duration (we use GFPmut3, BBa_E0240, which has an estimated half life of at least 24 hours, and the experiments duration is always less than 7 hours);
• strain, plasmid copy number, antibiotic, growth medium, growth conditions, protein generator assembled downstream of the promoter must be the same in the promoter of interest and in J23101 reference standard.
• steady state must be valid, so (dF/dt)/ABS (proportional to the GFP synthesis rate per cell) must be constant.

Inducible systems

Every experiment is performed on the following cultures:

• the culture of interest (system studied expressing GFP)
• the benchmarck used to evaluate R.P.U. (BBa_K173001 measurement part, that is BBa_J23101 with BBa_E0240 downstream)
• a negative control (generally, BBa_B0033 RBS)

For inducible systems several plots are reported. The first plot is a panel containing 4 subplots, numerated this way:

Plot (1) contains growth curves of the cultures, after blank value has been removed. Every curve is calculated averaging on three replicates of the same culture and subtracting the blank for each time sample. Blank is calculated averaging the replicates of blank wells.

Plot (2) shows the logarithm of absorbance in exponential phase of bacterial growth, determined by a visual inspection of log-plots. These values are used to evaluate doubling time and R.P.U..

Plot (3) contains (dGFP/dt)/O.D., the value named S_cell in Kelly J. et al., 2008 procedure for RPU evaluation.

In these plots are reported black veritcal lines that define the range of values used to evaluate RPU. It is important to underline, as explained in next paragraph, that RPU are calculated on cultures at the same O.D. level, not at the same time.

The second graphic shows S_cell VS O.D.. This plot allows the conparison of S_cell values between different cultures, that are supposed to reach the same level of growth not at the same time, but at the same O.D. value.

The third graphic shows the induction curve. The RPU value is calculated on S_cell values corresponding to O.D. values in exponential phase (typically, from 0.05 to 0.16). The curve is obtained averaging in time S_cell values corresponding to exponential phase.

Error bars rapresent the minimum and maximum value of R.P.U. belonging to the range of O.D. in exponential phase.

Materials

• Long term glycerol stocks were stored at -80°C with a final glycerol concentration of 20%
• Antibiotics were: Ampicillin (Amp) 100 ug/ml stored at -20°C in 1000x stocks. Amp was dissolved in water.
• LB medium was prepare with: 1% NaCl, 1% bactotryptone, 0.5% yeast extract. The medium was not buffered with NaOH.
• M9 supplemented medium was prepared according to: [http://openwetware.org/wiki/Knight:M9_supplemented_media Openwetware protocol].
• 3OC6-HSL (Sigma) was dissolved in water and stored at -20°C in a 2mM stock.

User Reviews

UNIQf8090ea6d9152fbe-partinfo-00000019-QINU

••••• UNIPV-Pavia iGEM 2010

The measurement system of this part (BBa_T9002) was successfully used as a biosensor to estimate the concentration of HSL present in the growth media of cultures expressing luxI (BBa_C0061). Additional information and details are available in BBa_K300009 Experience page. BBa_T9002 calibration curve for detection of [HSL] produced by autoinducer generators in high copy vector. BBa_T9002 calibration curve for detection of [HSL] produced by the autoinducer generators in low copy vector.