Part:BBa_K4633004

paraR - promoter for the araR gene from Bacillus subtilis 168

Usage and Biology

The paraR promoter is responsible for regulating the expression of the araR gene, which encodes the araR repressor protein in Bacillus subtilis. The primary role of the arabinose operon is to facilitate the utilization of L-arabinose as the sole carbon source. This operon is common among various bacterial species, including B. subtilis and E. coli [1]. The araR protein inhibits expression in the operon by binding to specific sequences in the B. subtilis genome, including the paraR promoter, in the absence of L-arabinose. In the presence of L-arabinose, the araR protein dissociates from the DNA, allowing for transcriptional activation.n the presence of L-arabinose, the araR protein dissociates from the DNA, allowing for transcriptional activation.

It is noteworthy that the paraR promoter exhibits inducible characteristics, although its responsiveness to L-arabinose is relatively modest compared to other L-arabinose-induced promoters such as araE and araA. This lower fold change suggests that paraR may function as a leaky promoter, exhibiting almost constitutive activity even in the absence of the inducer molecule [1].

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization and Measurements

To assess the functionality of the paraR promoter, we conducted experiments using E. Coli TOP 10 with the commercial plasmid pBE-S from Takara. As a reporter protein, mCherry (BBa_J06504) was inserted downstream of the promoter. The expression levels of mCherry were quantified using fluorescence measurements.

Our characterization experiments involved measuring mCherry fluorescence both in the presence and absence of L-arabinose. This approach allowed us to evaluate the impact of L-arabinose on the regulatory behavior of the paraR promoter. Testing was done via composite part BBa_K4633102.

Testing protocol

1. Transform gene into bacteria. Verify via colony PCR (for B. subtilis, the plasmid used for transformation was extracted from positive E. coli colonies in which it was amplified).
2. Grow two starter cultures from each colony with the appropriate antibiotic concentration. Blank samples and control samples with wild-type bacteria (no antibiotics) were also prepared. An additional control included bacteria containing a plasmid without the insert.
3. Add the inducer, L-arabinose solution, into one of the starter cultures. Concentrations of 0.4% (w/v) for E. coli and 0.8% (w/v) for B. subtilis were employed.
4. Put the starters in a shaker incubation at 37 degrees Celsius for 24 hours.
5. Prepare a 96-well plate for measurements, loading 75 microliters of LB into each well.
6. Load 25 microliters from each sample into four adjacent wells. Given the 24-hour growth period, bacteria were diluted appropriately to fall within the plate-reader device's range.
7. Measure OD (600nm) and fluorescence (excitation 560 nm, emission 610nm).
8. Choose appropriate gain for fluorescence (in our case, gain 50 was optimal).

Results analysis

1. Calculate the average OD and fluorescence values for four adjacent wells based on the loading map.
2. Subtract the average blank OD and blank fluorescence values from the corresponding treatment values. Pay attention to subtract the correct blank - with or without L-arabinose.
3. Divide the fluorescence by OD value.
4. Analyze the standard deviation.
5. Presenting results through graphical representation.

Behavior in E. coli

We performed the test and the data analysis as explained above. The results are from four different colonies from the same transformation plate (overall four biological repeats, each with four technical repeats).

Figure 1: Normalized fluorescence level of mCherry expressed in E. coli under the control of paraR.

Our experiments revealed a trend of reduced expression in the presence of L-arabinose, the inducer. However, this trend was not definitively clear due to overlapping error bars, raising the possibility that there was no significant change in expression levels upon L-arabinose induction. As explained in the Usage and Biology section, paraR was expected to exhibit minimal changes in expression levels, considering its leaky nature, which was evident in literature as a fourfold increase in expression levels compared to other L-arabinose-regulated promoters that experienced approximately a 50-fold increase [1]. This reduced responsiveness results from the unique mechanism of araR, which, in B. subtilis, binds to DNA and bends it. Unlike other promoters, paraR possesses only one araR binding site, rendering it comparatively leaky, since effective bending and silencing can't be achieved [1]. The decrease in expression levels upon L-arabinose induction may be attributed to host incompatibility, which was also observed in our paraE part (BBa_K4633006).

E. coli naturally possesses an L-arabinose utilization operon, with an araC repressor that differs slightly from araR [2]. Similar to araR, araC is self-regulated, meaning that in the presence of L-arabinose there are higher levels of araC [3]. When L-arabinose induces a conformational change in araC, it may not detach properly from the araR binding sequence, leading to higher levels of repressor that remains bound to the DNA [3–5].

In addition to the plate reader measurement, we also imaged our bacteria in a fluorescent microscope, according to the following protocol:

1. Prepare microscopy agarose gel.
2. Place five microliters of bacteria from starter onto gel. It is possible to dilute the bacteria before placing onto the gel.
3. Wait 15 minutes for bacteria to fix.
4. Image twice with a fluorescent microscope at 550nm (for mCherry) and CID (white light).

The following results are only for paraR starters and wild type that was induced with arabinose since the experiment was cut short due to the war breaking out in our country.

Figure 2: from left to right: overlay of 550nm reading and CID, reading at 550nm only, CID only. From top to bottom: paraR starter without arabinose, paraR starter induced with arabinose, WT starter induced with arabinose. Each starter was imaged twice.

According to figure 2, it can be seen that mCherry is expressed both with arabinose and without it. However, quantification of red fluorescence by omage analysis also supports the conclusion that there is higher expression in the absence of arabinose.

References

• [1] Mota, L. J., Morais Sarmento, L. & De Sá-Nogueira, I. Control of the Arabinose Regulon in Bacillus subtilis by AraR In Vivo: Crucial Roles of Operators, Cooperativity, and DNA Looping. J. Bacteriol. 183, 4190 (2001).
• Sá-Nogueira, I., Nogueira, T. V., Soares, S. & De Lencastre, H. The Bacillus subtilis L-arabinose (ara) operon: Nucleotide sequence, genetic organization and expression. Microbiology 143, 957–969 (1997).
• Bustos, S. A. & Schleif, R. F. Functional domains of the AraC protein. Proc. Natl. Acad. Sci. 90, 5638–5642 (1993).
• Brunelle, A. & Schleif, R. Determining residue-base interactions between AraC protein and araI DNA. J. Mol. Biol. 209, 607–622 (1989).
• Sá-Nogueira, I. & Mota, L. J. Negative regulation of L-arabinose metabolism in Bacillus subtilis: characterization of the araR (araC) gene. J. Bacteriol. 179, 1598–1608 (1997).