“The whole is more than the sum of its parts” - Aristotle
All parts used for IBDetection to be successful can be found on these pages. Part Overview gives a brief overview of all parts used in the sensor-reporter system and the Part Collection explains our used and designed collection in more detail, which is separated in the Basic Parts page and the Composite Parts page. Furthermore, the button Improvement brings you to the page of our improved part.
Our designed composite parts combine the two-component TtrS/R (BBa_K3972000 & BBa_K3972001) sensing part and the ARG1 (BBa_K3972002) reporter part, to form a sensor-reporter cascade inside E. coli bacteria. The parts are optimized for the expression of ARG1 in E. coli cells. The cascade functions as a tetrathionate sensor and in response to the binding of tetrathionate, the bacteria produce gas vesicles, which can be detected using ultrasound equipment. Two DNA constructs were designed, namely BBa_K3972003 and BBa_K3972004, and incorporated in design A and design B respectively, as described below.
Design A: Tetrathionate sensor with pTtrB activated ARG1 (BBa_K3972003)
The idea for this first design was based on keeping the length of the plasmids as low as possible, for a higher chance of proper co-transformation in bacteria. The design A plasmid consists of the TtrS/R promoter pTtrB185-269 (BBa_K2507019) and ARG1. The insert required to make this composite part, was ligated into the ordered and restricted pET28a plasmid, of which the T7 promoter was removed. An overview of the constructed plasmid can be seen in Figure 1 and more details about restriction and ligation of the insert and plasmid can be found in Experiments segment Restriction & Ligation and in Notebook.
The ARG1 gene is controlled by the TttrB185-269 promoter. This promoter is activated by TtrR in the presence of TtrS and tetrathionate. The TtrR kinase protein binds to the pTtrB185-269 promoter and induces expression of gas vesicle proteins (Gvps). Figure 3 shows a brief overview of the sensor-reporter cascade. A more detailed description of this mechanism can be found in Proof of Concept segment Sensor.
Figure 1: Overview plasmids design A.
Design B: Tetrathionate sensor with transferred TtrR and pTtrB activated ARG1 (BBa_K3972004)
Design A has the drawback of possessing two plasmids, sharing the same origin of replication. This can lead to unstable replication of the plasmid in the bacteria by the competition between the two plasmids [1]. As can be read on Human Practices, this was supported by several experts, and an alternative design should therefore be considered. Design B was constructed, consisting of only two plasmids.
This DNA system consists of the TtrS plasmid and a plasmid containing a combination of the gene coding for TtrR and ARG1. Additionally, functional parts are added to the construct, namely a TetR protein (BBa_K1475003), a pLtetO-1 promoter (BBa_K3332034) and the TtrB185-269 promoter. An overview of the constructed composite part can be seen in Figure 2. The combined parts were ligated into the pET28a plasmid and co-transformed into E. coli cells with the p15A TtrS plasmid.
The system from tetrathionate sensing to gas vesicle formation functions the same as design A, however, only two plasmids are required and the system lacks the constitutive mCherry or competing pTtrB185-269-GFP. The ARG1 gene is controlled by the pTtrB185-269 promoter and this promoter is activated by TtrR in the presence of TtrS and tetrathionate. The composite part system is visualized in Figure 3.
This composite part is a combination of the part BBa_K3972001 and the part BBa_K3972002. Additionally, functional parts are added to the construct, BBa_K1475003, BBa_K3332034, and BBa_K2507019.