Part:BBa_R0010:Experience

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Applications of BBa_R0010

User Reviews

Promoter Activity in Varying Concentrations of Glucose and IPTG

For assay, GFP generator BBa_E0240 was placed under the control of BBa_R0010.

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Antiquity

This review comes from the old result system and indicates that this part worked in some test.

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UNIPV-Pavia iGEM 2010

BBa_R0011 hybrid lac promoter and the BBa_R0010 wild type lac promoter were characterized at different copy number in TOP10 E. coli strain. This strain contains a lacI expression system in the genome.

Induction static transfer function (computed in Relative Promoter Units), dynamics and metabolic burden were evaluated as a function of different IPTG concentrations in M9 supplemented with glycerol growth medium.

A RFP generator (BBa_I13507) was used as a reporter gene. In particular, these measurement systems were used:

At first, BBa_J107010 and BBa_J04450 inducibility was tested in a high copy number vector (pSB1A2 or pSB1A3). The results are shown here as the relative RFP synthesis rate per cell.

Relative RFP synthesis rate per cell in pSB1A2-BBa_J107010 and pSB1A3-BBa_J04450. The error bars represent the standard errors of three independent measurements.

Results show that in this condition BBa_R0010 is about 2-fold stronger than BBa_R0011, but induced and uninduced cultures did not show differences in the RFP signal.

This result is expected because the vectors are propagated at about 200 copies per cell, while the lacI repressor is present at single copy in the genome and thus it is not able to repress the lac promoters in such high copy.

BBa_J107010 and BBa_J04450 were then tested in the low copy (~5 copies per cell) vector pSB4C5 in order to test their inducibility. The results are shown here as the RPU values at the steady state (constant RFP sysnthesis rate per cell) at different IPTG concentrations.

RPU of pSB4C5-BBa_J107010 and pSB4C5-BBa_J04450 as a function of IPTG concentration. The error bars represent the standard errors of three independent measurements.

Results show that in this condition both BBa_R0010 and BBa_R0011 produce different amounts of RFP as a function of the IPTG concentration. The amplitude of the two curves show that the promoters are very strong when induced with IPTG >= 10 uM. Although the experiments were carried out in the same conditions, the variability between experiments was high, especially for BBa_R0010 (mean coefficient of variaton of about 37% for BBa_R0010 and 15% for BBa_R0011), while the RPU variability between three wells in the same experiment is much lower (mean coefficient of variaton of bout 3.5% for both promoters).

The above figure shows that BBa_R0011 is stronger than the BBa_R0010 wild type promoter in low copy plasmid. This result is unexpected because the same promoters in high copy vectors behaved differently (BBa_R0010 was stronger than the BBa_R0011, see above).

In the uninduced state, BBa_R0011 has about the same strength as the BBa_J23101 reference standard promoter. This static characteristic shows that the promoters are both leaky and a very low IPTG concentration (10 uM) is sufficient to trigger gene expression at *very* high levels.

These results demonstrate that the genomic lacI is partially able to repress the two promoters, but very low IPTG concentrations are sufficient to bind the repressor and trigger the promoters transcription.

Dynamic characterization in low copy vector: The figure below shows a typical relative RFP synthesis rate per cell time series for BBa_J107010 and BBa_J04450 induced with 1 mM of IPTG and uninduced. These time series show that the full induction can be reached after about 50 min from the induction.

Mean Scell signal as a function of time for pSB4C5-BBa_J107010 and pSB4C5-BBa_J04450. Induced (with 1 mM of IPTG) and uninduced cultures are shown. Induction occurs at t=0. The shown graph is relative to one of the three experiments performed in different days.

Conclusion: the characterization of two IPTG-inducible promoters has been performed and the performance of these two promoters have been compared in terms of transcriptional strength. The reported results are easily sharable in different laboratories thanks to the used standard RPU approach.

Methods:

A of long term storage glycerol stock was streaked on a LB plate with suitable antibiotic. Tha plate was incubated overnight at 37°C.
A single colony was inoculated in 1 ml of M9 + suitable antibiotic in a 15 ml tube and incubated at 37°C, 220 rpm for about 16 hours.
The grown cultures were then diluted 1:100 in 2-5 ml of M9 supplemented medium and incubated in the same conditions as before for about 4-5 hours.
For each desired IPTG concentration to be tested, three 200 ul aliquots of the cultures were aliquoted in a flat-bottom 96-well microplate, avoiding to perform dynamic experiments in the microplate frame (in order to prevent evaporation effects in the frame).
2 ul of properly diluted IPTG (Sigma Aldrich) were added to the three wells for each desired concentration.
The microplate was incubated in the Tecan Infinite F200 microplate reader and fluorescence and absorbance were measured with this automatic protocol:
- 37°C constant for all the experiment;
- sampling time of 5 minutes;
- fluorescence gain of 50 or 80;
- O.D. filter at 600 nm;
- RFP filters at 535nm (ex) / 620nm (em);
- 15 seconds of linear shaking (3mm amplitude) followed by 10 seconds of waiting before the measurements in order to make a homogeneous culture.
- Experiment duration time: about 6 hours.
This experiment was performed three times in different days.

Data analysis: Relative Promoter Units (RPUs) were estimated as described by [Kelly JR et al. (2009), J Biol Eng 3:4].

Briefly:

Absorbance and fluorescence time series were normalized by subtracting the absorbance of the media and the fluorescence of a negative control (a non fluorescent TOP10 culture) respectively, thus yielding O.D.600 and RFP time series.
RFP synthesis rate per cell (called Scell) was computed as (1/O.D.600)*dGFP/dt. (this signal is not actually the RFP synthesis rate, but is proportional to it).
The RFP synthesis rate per cell was averaged at the steady state during the exponential growth phase (validated by identifying the linear region of the ln(O.D.600)).
The RPU of the promoter of interest in a specific condition was computed as mean_Scell,phi/mean_Scell,J23101 where phi is the promoter of interest, J23101 is the reference standard and mean_Scell is the mean Scell signal value, computed as explained above.

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