Parts

comK

Under conditions of nutrient deprivation (energy starvation), B. subtilis can be made to uptake foreign DNA. However, transformation through starvation is not the most ideal process, as outcomes can be uncontrollable, time-consuming, and labour-intensive. Fortunately, we can bypass the need for energy starvation by using cells derived from a specific strain of B. subtilis (SCK6) with the pAX01-comK plasmid constructed by Zhang & Zhang (2010).

The key feature conferred by this plasmid is the overexpression of comK, which is the master regulator of competence in B. subtilis. It is known as such because it encodes a transcription factor which upregulates the expression of these competence genes (comC, comE, comF, comG, nucA). All these genes contribute to the DNA uptake mechanism in B. subtilis.

In this “supercompetent” plasmid, the overexpression of these genes is enabled by the master regulator comK, the control of which is placed under the xylose-inducible promoter P_xylA. This means that if we add xylose to the cells, they will activate P_xylA and subsequently comK, resulting in the “turning on” of competence genes. The cells can then uptake foreign DNA without the need for energy starvation. The figure below depicts the pAX01-comK plasmid (Zhang & Zhang, 2010).

Figure 1. pAX01-comK vector map (Zhang & Zhang, 2010). Constructed plasmid for B. subtilis wherein the master regulator comK competence gene is placed under the control of P_xylA, a xylose-inducible promoter.

The part submitted by iGEM Calgary to allow future teams to take advantage of this xylose-inducible system includes the P_xylA, RBS, and comK gene. We have taken the functional units of Zhang & Zhang's constructed plasmid and placed it between the standard BioBrick prefix and suffix.

Composite Regulatory Parts

At the core of our DNA detection strategy is our genetic circuit. We designed a straightforward circuit consisting of two interacting genes; i) a constitutively expressed repressor, flanked by sequences homologous to the target pathogen DNA allowing our circuit to be triggered and ii) a reporter gene under the control of our custom composite regulatory parts. The composite regulatory sequence consisted of a constitutive promoter, a repressible promoter (i.e. operator), and an RBS compatible with B. subtilis. Twenty variants of the composite regulator were designed and synthesized, with the intention of characterizing and developing an optimal circuit. The sequences varied in the type of repressible promoter, the number of copies of the repressible promoter, and with the type of RBS. The constitutive promoter P_veg remained constant for all variants. Triple copies of the repressible promoter were designed to allow a stronger and more reliable repression in an effort to reduce 'leakiness' of expression.

Figure 2: Examples of the composite regulatory parts that we designed. Arrows represent the constitutive promoter P_veg; orange rectangles represent the repressible promoter, also referred to as the operator; blue and green semicircles represent the ‘weak’ or ‘consensus’ variants a RBS B. subtilis. Structures (i) and (ii) contain a single operator, whereas structures (iii) and (iv) contain a triplicate of operators adjacent to each other. In the interest of characterizing and developing an optimal system, a total of five different operators were chosen, for a total of 20 unique composite regulatory parts.

lacZ (β-galactosidase)

The gene lacZ encodes β-galactosidase (EC 3.2.1.23), a classic enzyme most commonly used in blue-white colony screening when used in specialized vectors. This part consists of the full length beta-galactosidase sequence which can be inserted downstream of a promoter to produce the tetrameric, 47.4kDa enzyme.

β-galactosidase cleaves the β-glycosidic bond of a galactose bound to an organic moiety; the physiological function is to break down β-galactosides, such as lactose, into its respective monnosaccharides. β-galactosidase can act as a reporter when provided substrates such as X-gal. The cleavage of X-gal releases galactose and the chromophoric, subtsituted indole moiety. The most common form of X-gal is 5-Bromo-3-indolyl β-D-galactopyranoside, which produces an intense blue pigment. Other versions of X-gal may be used to produce other color pigments.

Typically, 40-60uL of a 20ug/mL X-gal solution (dissolved in dimethyl sulfoxide or dimethyl formamide) is spread on a plate and allowed to dry before plating the bacteria expressing β-galactosidase. X-gal may also be added to liquid cultures or incorporated into solid agar media.