Difference between revisions of "Part:BBa K4375019"

Revision as of 10:07, 10 October 2022

Blue light inducible production of Cytolysin A (BLADE)

This device is meant for the light-inducible production of Cytolysin A (BBa_K811000), a pore-forming toxin. To achieve this, we used a modified arabinose operator system, where the transcription factor, AraC, is engineered to homodimerize upon blue-light illumination instead of arabinose binding. This system is called BLADE (BBa_K4375003), and it is produced under the strong constitutive promoter J23101*.

Characteristics

Fig 1. Blue-light induced Cytolysin A production and characterisation of the BLADE Expression System; A) Blue-light induced Cytolysin A production characterisation via SDS-PAGE; B) Blue-light induced Cytolysin A production characterisation via SDS-PAGE

We aimed to characterise the Cytolysin A production BLADE Expression System using SDS-PAGE, and study its hemolytic activity on blood agar plates. (Fig 1.)

For the Cytolysine production characterisation, we obtained the supernatant of the bacterial culture after 4 h of blue light illumination and precipitated its protein content. We observed a band indicating Cytolysin A production, where we expected, which was absent in the case of the dark control sample. (Fig. 1/B) We implemented the protocol of Chiang et al. for this aim, but it should be noted, that this experiment needs to be repeated with a construct to which an affinity tag, such as 6xHis-Tag, is added. [3] In this way, the protein of our interest could be purified lessening the number of aspecific bands.

We also meant to assess the hemolytic activity of Cytolysin A, using the BLADE Expression System. We spread the overnight cultures on Blood Agar plates, grew them for 8 h in dark at 37°C, which was followed by an 4 h long illumination with blue light at 25 °C. (The dark control plate was kept in dark at 25 °C for that time.) For additional negative control, we also spread BLADE-mCherry sample on the plates.) As we expected, no hemolytic activity was observed in the case of mCherry. Regarding the BLADE-ClyA samples, the biological activity of Cytolysine A was undoubtful since the blood agar discoloured where the samples were plated. Unfortunately, we could not observe a significant difference between dark and illuminated plates. This could also be explained by the considerations we beforehand mentioned. (Temperature, frozen bacterial culture)

Usage and Biology

This device is a blue light-activable system, which is based on the BLADE protein. Upon illumination, the BLADE binds to the AraBAD promoter and Cytolysin-A (ClyA) is expressed. ClyA is a protein native to E. coli and Salmonella typhi that is capable of forming pore complexes (BBa_K811000). ClyA is a 34 kDa protein and has alpha-helical-based monomers which oligomerize and form the pore. The picture shows the construction scheme

Figure 2 ClyA after BLADE promoter construct scheme.

Sequence and Features