Part:BBa_K4633006

paraE - improved promoter for the araE gene from Bacillus subtilis 168

Usage and Biology

This paraE sequence represents an enhanced version of the promoter for the araE gene, which encodes the araE protein, an arabinose transporter vital to the L-arabinose operon in Bacillus subtilis. araE functions as a proton symporter, facilitating the transport of arabinose into bacterial cells for utilization as a carbon source [1]. This promoter is subject to inhibition by the protein araR (BBa_K4633003), which detaches and allows expression in the presence of L-arabinose.

This promoter was an integral component of our kill-switch system in B. subtilis 168. Subsequently, following the development of this promoter, we aimed to express the toxin MazF to enable the controlled elimination of our engineered bacteria.

Optimization and Design

The original sequence was derived from Sá-Nogueira et al. [1] but was characterized by excessive A-T richness, rendering it impractical for production as a gBlock by IDT or Twist Bioscience. To rectify this, optimization measures were undertaken, including the removal of a Cre binding site (which would inhibit the promoter in the presence of glucose) and the replacement of bases within the undefined areas of the promoter (between the araR binding sites, likely acting as spacers) to ensure sufficient GC content for production. Detailed information is available on our wiki. Following this sequence, an RBS should be inserted, with the native RBS available at BBa_K4633007. Testing and characterization of this part were conducted in B. subtilis 168 and in E. coli TOP10 using composite part BBa_K4633103.

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

According to Mota et al. [2], the araR protein binds to the araE promoter at two distinct sites, enabling the protein to induce DNA bending and achieve effective repression. In the presence of L-arabinose, araR dissociates from the promoter, leading to an approximately 66-fold increase in the expression levels of the native paraE [2]. To assess the functionality of this part, we inserted the gene for the mCherry protein (BBa_J06504 ) downstream of it. Expression of the protein was subsequently measured via fluorescence using a plate reader at 560 nm for excitation and 610nm for emission. Testing was conducted in both the presence and absence of arabinose to evaluate the influence of the sugar on the system.

Testing protocol:

1. Transform gene into bacteria. Verify via colony PCR (for B. subtilis plasmid used for transformation was extracted from positive E. coli colonies).
2. Grow two starters from each colony, with appropriate amount of antibiotic. Make starters without bacteria, for blank, and with wild type bacteria (no antibiotics) for control. Another control is bacteria containing a plasmid without insert.
3. Add L-arabinose solution to one of the starters. For E. coli a concentration of 0.4% (w/v) was used, while for B. subtilis a concentration of 0.8% (w/v) was used.
4. Grow in shaker at 37 degrees Celsius for 24 hours.
5. Prepare a black 96 well plate. Load 75 microliters of clean LB into each well.
6. From each sample, load 25 microliters. Repeat this step for four adjacent wells. Since starters had 24 hours to grow it is necessary to dilute the bacteria to stay within the range of the plate-reader device.
7. Measure plate for 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. Average OD values for every four adjacent wells, according to loading map.
2. Average fluorescence values for every four adjacent wells, according to loading map.
3. Subtract average blank OD value from the average OD value of each treatment. Pay attention to subtract the correct blank - with or without L-arabinose.
4. Subtract average blank fluorescence value from the average fluorescence value of each treatment. Pay attention to subtract the correct blank - with or without L-arabinose.
5. Divide fluorescence by OD.
6. Calculate standard deviation.
7. Exclude treatments as necessary, present results in a graph.

Behavior in E. coli

We performed the test and the data analysis as explained above. Unfortunately, all treatments failed but one, and the results are presented in the following graph (an average of four technical repeats).

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

There is a clear trend of reduced expression in the presence of L-arabinose, the inducer. This of course is contrary to what was expected based on the mechanism of the system as explained in the Usage and Biology section. We believe that this discrepancy is due to the fact that E. coli is not the natural host for this system.

E. coli has an equivalent L-arabinose utilization system, with a notable difference being the repressor of the system, E. coli's repressor is called araC and it is slightly different than araR [1]. When arabinose enters the bacteria it binds to the repressor protein and induces a conformational change meant to detach the repressor from the DNA. Because araR and araC have different binding sequences it is possible that araC has difficulties detaching from the araR binding site present in the sequence of this part [3–5]. In addition to that, araC is self-regulated, meaning that in the presence of arabinose its levels of expression should rise, since araC would not have a problem detaching from its own natural binding sequence [5]. The combination of these could lead to a situation where there is more araC present in the E. coli cell, that has difficulties detaching from paraE, thus leading to lower level of mCherry expression.

Since this experiment only included one biological repeat, it would need to be repeated for statistical significance.

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.
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 the paraE starter and wild type that were induced with arabinose since the experiment was cut short due to the war breaking out.

The following results are only for the paraE starter and wild type that were 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. Top half is paraE starter induced with arabinose, bottom half is WT starter induced with arabinose. Each starter was imaged twice.

As can be seen in the figure, despite the low fluorescence reading and the fact that the starters didn't turn pink we can see that some of the bacteria did produce mCherry, and there is no fluorescence in WT.

Behavior in B. subtilis

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

Figure 3: Normalized fluorescence level of mCherry expressed in B. Subtilis under the control of paraE.

Despite the relatively large standard deviation, there is a clear trend of higher levels of expression in the presence of arabinose, as expected [2]. We received about a 2.6 fold increase, much lower than the reported 66-fold increase [2]. This could be due to the fact that we used a different measurement method than the one found in literature (β-Galactosidase assay).

Fluorescence microscopy pictures of B. Subtilis were supposed to be taken as well, but as mentioned, the experiment was cut short.

References

• [1] 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).
• [2] 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).
• [3] 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).
• [4] Brunelle, A. & Schleif, R. Determining residue-base interactions between AraC protein and araI DNA. J. Mol. Biol. 209, 607–622 (1989).
• [5] Bustos, S. A. & Schleif, R. F. Functional domains of the AraC protein. Proc. Natl. Acad. Sci. 90, 5638–5642 (1993).