Part:BBa_K4491007
pBAD_AP
| araBAD promoter | |
|---|---|
| Function | Inducible promoter |
| Use in | Bacterial |
| Chassis Tested | Escherichia coli |
| Abstraction Hierarchy | Part |
| Related Device | BBa_K2442101 |
| Backbone | pSC101 |
| Submitted by | Cambridge iGEM 2022 |
This gene encodes an improved version of the wild-type araBAD promoter (nicknamed pBAD), an inducible promoter controlled by araC protein. The native pBAD is part of the araBAD operon, which is responsible for regulating arabinose metabolism in E.coli.
Contents
Usage and Biology
The exact definition of the araBAD promoter varies ambiguously between sources. Historically, pBAD only refers to a short segment upstream of the +1 transcription start site (referred to as the core promoter), containing the -35 and -10 boxes. However, as a complete promoter Part, the regulatory region further upstream is also included. In the following discussion, our definition of pBAD refers to the whole sequence consisting of both the regulatory sequence and the core promoter.
The araBAD promoter regulates the araBAD operon and is controlled by araC - a regulatory protein known for its “love-hate” mechanism of action. The upstream regulatory region consists of various protein binding sites - araI1, araI2, araO1 and araO2 can be occupied by araC. Between araI1 and araO1 also lies a CAP binding site, which recruits Catabolite receptor protein for transcription activation. The spacing between araO1 and O2 is noticeably large, reaching nearly 210 bp, which, unsurprisingly, is responsible for the overall bulkiness of the promoter. Interestingly, a region within this spacer contains a promoter of araC gene, which runs in the opposite direction to the araBAD operon. This promoter is regulated by araO1 just downstream. The araC protein therefore not only regulates the araBAD operon but also controls its own production by binding to araO1 region (negative autoregulation).
In the absence of L-arabinose, transcription is repressed by the action of two araC molecules binding to the structure. One araC binds to araI1 and the other binds to araO2 further upstream. The dimerization domain confers high affinity, bringing the two protein molecules closer to dimerize, essentially creating a DNA loop as a result. This looping mechanism prevents any sigma factors, RNA Polymerase or CRP from being recruited, thus repressing transcription.
In the presence of L-arabinose, binding of the sugar molecule to the arabinose-binding domain triggers a conformational change in the DNA-binding domain of araC, which reduces its affinity to distal araO2 site. This makes the protein more favourable to bind to araI2, just downstream of araI1. Therefore, the dimerized complex is formed at araI1-araI2 site instead of araO2-araI1, which breaks the loop and hence activates transcription. The dimer araC also “nudges” the RNA Polymerase for enhanced transcription.
pBAD Entries in the Registry
TBA
Preamble from Cambridge team
The Cambridge 2022 Team had intended to use the araBAD promoter to build an antithetic feedback circuit that demonstrates robust perfect adaptation (RPA). However, we were concerned that wild-type PBAD is relatively large, topping at around 300 bp, which increases the bulkiness of our Level-2 construct and reduces efficiency. Also, pBAD, by nature, is a relatively leaky and medium-strength promoter, therefore, lots of araC is needed to fully reach maximal activation. However, overexpression of araC is toxic to cells, and thus can drastically impede performance of the circuit. we therefore thought it would be an interesting Part Improvement project to redesign the existing wild-type promoter. Our initial goal was to engineer a PBAD with minimal length, but shows both lower leakiness and higher maximal activity. To achieve this, we did intensive literature search for prior optimization strategies. To understand the rationales, we also gathered information on the regulatory mechanism of the promoter, which was presumably only prevalent in papers from the 1970s.
Design rationales
Understanding the overall mechanism of araBAD promoter allowed us to pinpoint several aspects of the structure that can be rationally improved.
Rationale 1: Spacing between araI1 - araO2
To minimise the length of pBAD, we investigated the presence of araO2 site, as well as the 211 bp spacer between araI1 and araO2 (this region includes CAP binding sites and araO1). Previous literature suggested that complete or even partial deletions of araO2 would increase leaky expression of pBAD, emphasising that the existence of this region is essential for normal repression. Strangely, most commercially available sequences for pBAD (in CIDAR MoClo kit, for example) omitted the araO2 site, so its significance is still uncertain.
The spacing will dictate the size of the loop, which also controls the level of repression under the absence of arabinose and determines the promoter’s leakiness. Here, the spacing is defined to be between position -59 within araI1 and -270 within araO2 (underlined and asterisked). It was shown that there are no lower bounds for loop size - a functional araBAD promoter was designed with a 34-bp loop, after deleting a significant portion of the spacer []. While it maintained similar leakiness to that of the wild-type counterpart, the loss of CAP binding site drastically reduced the maximal strength. Still, the finding demonstrated the great flexibility of bulky araC proteins in mediating small loop formation.
Another important observation was that as the spacing varied, the promoter activity oscillated with a 11.1 bp periodicity. Specifically, any insertion or deletion of integer multiples of 5bp noticeably increased leakiness, while integer multiples of 11.1 bp retained wild-type’s full repression [8]. This value was determined to be approximately equivalent to one helical repeat of DNA (10.5 bp). It was further explained that insertion of 5 bp between araO2 and araI1 would rotate one site halfway around the DNA double helix with respect to the other and impede repression. Despite araC’s flexibility, the torsional stress of DNA makes such looping much more energetically unfavourable.
Taking into account these results, our first design strategy is to reduce the spacer region down to 56 bp while still maintaining the CAP binding site downstream. We removed the araO1 site completely due to its minor role on P_BAD activity. The schematic of our rationale is depicted below.
Rationale 2: araI1 and araI2
We then investigated araI1 and araI2 17-bp regions, both containing two unique sites called the A- and B-box, which serve as specific binding sites for araC. In previous literature, Niland et al (1996) showed that any single base-pair substitution occurring in these two sites would drastically reduce binding of araC. Flanked between the two boxes are seven invariant nucleotides that, upon selected single substitution, demonstrated higher binding affinity to araC by somewhat 140% compared to that of wild-type araI1 []. We therefore modified the araI1 site to contain all the different substitutions which initially yielded tighter binding, while keeping the A- and B-boxes unchanged.
In another paper, Reeder (1993) found that the B-box of araI2 overlaps with four base pairs of the -35 consensus sequence [9]. Thus, any substitution in this box will negatively impact P_BAD’s activity, either resulting in very high leaky expression or lowered inducibility. However, the author remarked that araI1 has much higher affinity to araC than araI2 does, especially when no arabinose is present. This makes sense, as araC prefers binding to distal araO2 and araI1 than the nearby araI2. From this insight, we questioned whether a duplicate araI1-I1 may confer higher maximal activity than wild-type araI1-I2. We then sought to change the last nucleotide of the A-box and the interbox sequence of araI2 to that of wild-type araI1, but still leaving the araI2 B-box untouched. In a sense, we created a chimeric half-araI1-half-araI2 in place of wild-type araI2. We did not duplicate the entire araI1 as this would affect the overlapping -35 consensus sequence and make the promoter extremely leaky.
We group the modifications for both araI1 and araI2 as our second strategy.
Rationale 3: -35 and -10
Our final design input comes from the work of the 2013 DTU iGEM team (see <a href="https://parts.igem.org/PBAD_SPL">here</a>). They managed to create a synthetic promoter library (SPL) for araBAD promoter by randomly mutating different base-pairs between the -35 and -10 boxes, right downstream of araI2. We decided to use the Col15 sequence, which showed promising low level of leakiness and high induced strength.
Combinatorial design
We finally opted for a combinatorial approach for the three strategies, and thus have initially designed 23 = 8 different pBADs, with or without the preceding optimizations. After some thoughts, we introduced another design with a slightly larger spacing between araO2 and araI1 (PB5). These designs were tested against the wild-type, full-length araBAD promoter, corresponding to part BBa_K2442101 in the registry (see here). We were aware that while an individual strategy may yield noticeable improvements, this might not be true when combining them together. In fact, some can result in antagonistic effects rather than the desired synergy. Still, we hoped that within the different permutations, some good designs may emerge. We also thought that, given this variety of promoter expression, we should not restrict our aim to only creating a single better pBAD, but also making a “family” of the promoter with different strengths, such as weak, medium and strong, for various purposes (in some cases, lower maximal activity might be necessary).
| dissociation_constant | 31.1 |
| hill_coeficient | 0.56 |