Part:BBa_K1316014
pRhamnose regulating csgB + J23110 constitutive Anderson promoter regulating csgA with a His tag
This part is meant to constitutively express the curli gene csgB which codes for the production of the curli protein that is anchored to the cell membrane (CsgB). The csgA gene codes 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.
As many more CsgA than CsgB subunits are required for curli formation, this part intends to have an already high amount of CsgA present in the cell (constitutively expressed) and, once the cell is induced (with Rhamnose in this case), only CsgB protein is required to be expressed for curli formation, thus leading to a faster curli generation process.
The His tag present in the CsgA protein has two functions: on one hand it allows easy protein purification of the CsgA protein; on the other hand, it enables the formation of conductive curli by adding gold nanoparticles (AuNP) with Nickel-Nitrilotriacetic-acid (NTA-Ni) attached to them. This last part containing Nickel would bind to the Histidines of the curli fibril, whereas the gold attached to it will make the environment more conductive.
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 constructs BBa_K1316014 and BBa_K1316016, induced (top) and non-induced (bottom).
For more information about the characterisation of this construct visit the Conductive Curli module characterisation on our wiki page! (http://2014.igem.org/Team:TU_Delft-Leiden/Project/Life_science/curli/characterisation)
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 516
Illegal NheI site found at 551 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |