Difference between revisions of "Part:BBa K625003"

Revision as of 09:22, 1 November 2011

Pu promoter short version

Contains a shortened version of BBa_K625002 not containing the RBS and first 81bp of XylU but all necessary elements of the promoter.

Characterization

Experimental setup

Figure 1: Experimental setup of XylR experiment.

E. coli strain JM101 was transformed with two plasmids containing the transcriptional regulator XylR, the degradation cassette xylMABN and a GFP reporter coupled to BBa_K625002 respectively BBa_K625003. For the first two of those we used the plasmid pCK04AxylR according to [1]. The reporter plasmid we constructed ourselves by using BBa_K625005 as a backbone.

We inoculated 50 mL of LB medium with an overnight culture of the co-transformed strains and set the OD₆₀₀ to 0.1. Upon reaching the exponential growth phase, the cultures were induced with m-xylene. Therefore we put a sterile test tube with m-xylene into the flask and sealed it with parafilm in order to get an air-induced response.

The samples were taken 3 hours after induction. OD₆₀₀ and GFP fluorescence was measured then in a 96-well plate. Afterwards the data was normalized in order to get more consistent results.

Results

Comparison with BBa_K625002

Figure 2: Characterization of BBa_K625002 and BBa_K625003 m-xylene was added by air-induction.

We could observe a clear increase in GFP fluorescence when m-xylene was added in a test tube. Thus GFP production can be induced by m-xylene present from the air. The results of the experiments can be seen in fig. 2. By comparing BBa_K625002 and BBa_K625003, we can see that the leaky expression of GFP is higher in the shortened promoter BBa_K625003 compared to the longer one. There are several possible reasons for this observation. Most likely it is due to the drop of the natural part downstream of the promoter, which is usually also included in P_u. Nevertheless, we could also see a higher level of induction in the setup with BBa_K625003 (around 7-fold) compared to the BBa_K625002 setup (around 5-fold). Thus both of the provided promoters can be used for transcriptional regulation with XylR.

Characterization of BBa_K625003

The short version of the P_u promoter was characterized in more detail. These experiments were performed in M9 medium containing 0.5 % glucose, in the hope of less leaky transcription through more defined medium. The extent of xylene induction was modulated by performing the induction over air not only with pure xylene, but also with mixtures of xylene and paraffin oil, which gives lower xylene concentration within the medium due to a lower partial pressure. Therefore a 50 ml shaking flask culture of JM101 containing the sensor plasmid PcK04AxylR and a medium copy plasmid with a sfGFP reporter under BBa_K625003 control was induced in exponential phase with different dilutions of xylene in paraffin oil. The specific fluorescence was measured in a platereader and dose-responsiveness could be shown for all timepoints (Figure 3), with the overnight one having the lowest detection limit.

These promising results made us [http://2011.igem.org/Team:ETH_Zurich/xylene calculate] the xylene distribution in our thermodynamically closed system, to get a proper dose response for XylR. (The induction with 25 % xylene in paraffin oil in excess would for example correspond to a concentration in the medium of 775 μM, which means a responsiveness in the micromolar range, which is expected)

In another experiment (some shaking flasks containing the test tubes for induction shown in Figure 4), the dose response curve for the same cells was collected in the same way after overnight growth (Figure 5). Xylene was already detected in the lower micromolar range. For the highest induction rates in the low millimolar range, a certain toxicity was already observed, as can be seen by a decreased OD₆₀₀ (Figure 6). The promoter was found to be rather strong, as it showed expression rates of about a quarter of a Tac promoter induced with 100μM IPTG (positive control) and its leakiness was found to be negligible (negative control). (see Figure 7)

• ETH Columns for xylene.png

  Figure 5: Characterization of BBa_K625003 different concentration of m-xylene were added by air-induction. sfGFP expression could be induced by lower micromolar concentrations of xylene.

• 

  Figure 6: Influence of xylene on the cell growth of E.coli. Xylene shows toxic effects on E.coli in the lower millimolar range.

• ETH Induction level comparison.png

  Figure 7: Positive and negative control in comparison to fully xylene-induced (+) and non induced (-) GFP expression by BBa_K625003. (Positive control being fully induced Lac promoter, negative promoter the same strain without GFP)

Dose response curve

We fitted the activation data in dependency of the xylene concentration to a Hill function. Since for a xylene concentration of 3100 μM the measured OD₆₀₀ value was significantly reduced compared to the other xylene concentrations, this data point was removed from the analysis (this concentration can be considered to be toxic for the cells). For a xylene concentration of 1550 μM the fluorescence signal was considered to have reached steady state (see Figure 5). Thus, we defined the relative activation for a given xylene concentration as the ratio between a given fluorescence signal and the fluorescence signal for a xylene concentration of 1550 μM (see Figure 8).

To this data (see Figure 8) we fitted the Hill function

with K_D the dissociation constant and n the Hill coefficient. We obtained optimal values of

K_D=144.1 μM≈144 μM;

n=1.055≈1

Since the Hill coefficient is close to 1, we can assume non-cooperative activation of XylR. This data was subsequently used to redefine the corresponding parameters for the xylene model .

Sequence and Features

References

[1] [http://aem.asm.org/cgi/content/abstract/64/2/748 S. Panke, J. M. Sanchez Romero, V. de Lorenzo: Engineering of Quasi-Natural Pseudomonas putida Strains for Toluene Metabolism through an ortho-Cleavage Degradation Pathway , Applied and Environmental Microbiology, 1998, Vol. 64, No. 2]