Difference between revisions of "Part:BBa K1316015"

Revision as of 17:15, 14 October 2014

pRhamnose regulating csgB and csgA curli proteins

This part is meant to express the csgB and csgA curli genes, which respectively code for the production of the curli protein that is anchored to the cell membrane (CsgB); and for the major curli protein (CsgA), which forms long fibrils with other CsgA monomers in a self-assembly process. These CsgA long fibrils bind to the anchored CsgB protein forming the amyloid-like curli structure.

The expression of these two genes will, then, occur at the same time and under the same conditions (in the presence of Rhamnose in this case)

This construct was designed as a negative control for BBa_K1316013 and BBa_K1316014, to see if expressing CsgA constitutively has a positive impact on the speed of curli formation

Characterization

Different type of experiments were conducted to characterize this BioBrick:

Plate Reader

A plate reader was used to detect cell concentration (OD600) and fluorescence of cells carrying the curli-forming BioBricks (BBa_K1316013-BBa_K1316015), which were at the same time also carrying the BBa_K1316016 construct, constitutively expressing eGFP. By doing this assay, cells were observed to be more attached to the walls of a 96-well plate when they had curli-forming BioBricks induced with Rhamnose (cruli genes induced) (figure 1).

Figure 1. OD after washing out the cells twice as a fraction of initial OD observed on 96-well plates, with (+) and without (-) induction of the curli-formation genes. Induced cells are induced with 1% rhamnose solution.

Confocal Microscopy

Confocal microscpoy technology was used to observe the deposition of cells at the bottom of the microscope slide. Figures 2 and 3 show how after induction with Rhamnose the cells forming curli are attached faster to the surface (bottom) of the microscope slide than when they are not induced.

Figure 2. Fluorescent images taken using the Confocal Microscope of the cells carrying the CC constructs BBa_K1316015 and BBa_K1316016, induced (top) and non-induced (bottom).

Sequence and Features